MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION … · ... 60131 Ancona, Italy - r.papa@ ......

Proceedings of the 50th Italian Society of Agricultural Genetics Annual CongressIschia, Italy – 10/14 September, 2006ISBN 88-900622-7-4

Poster Abstract – A.01

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF THENEWLY DISCOVERED NICOTIANA WUTTKEI CLARKSON & SYMON

L. DEL PIANO*, C. SORRENTINO*, M. ABET*, V. SPAGNUOLO**, L. BARBATO*,M. SICIGNANO*, M. CRIMALDI *, A. CUCINIELLO*.

*) CRA-Istituto Sperimentale per il Tabacco, Via P. Vitiello n.108, 84018 Scafati (SA), Italy –[email protected]**) Facoltà di Scienze Matematiche, Fisiche e Naturali - Dipartimento di Biologia strutturale efunzionale - Università di Napoli “Federico II”

Nicotiana wuttkei, Nicotiana genus, ISSR, SEM

The genus Nicotiana, with 76 naturally occurred species, classified in 14 sections and 3subgenera, is the sixth largest in the family Solanaceae. Species of Nicotiana occur largely in theAmericas (75%) and Australia (25%). Exceptions include N.fragrans, which is restricted to islandof South Pacific Ocean, and N.africana which is found in Africa. In 1991 Clarkson & Symondescribed for the first time a new Nicotiana species, named Nicotiana wuttkei Clarkson & Symon.According to this authors it should be included in the section Suaveolentes for its morphologicalcharacteristics.

At present there is little and disagreeing information about morphology and citology of thisspecies. Furthermore its sistematic position is not yet clearly defined.

A study was performed in order to assess, at morphological and molecular level, therelationships among the newly discovered Nicotiana wuttkei and other Nicotiana species, belongingto the section Suaveolentes.

Nicotiana species were grown in pots in greenhouse. During the vegetative cyclemorphobiometrical data were collected, in particulary as regards the flower parameters. Moreoverobservations were carried out on seed and pollen morphology, respectively by Microscopy andScanning Electron Microscopy. Cytological investigations were also performed on root tip ofN.wuttkei to determine the chromosome number. Further, ISSR (inter simple sequences repeats)analysis was used to reveal the genetic polymorphism among the Nicotiana species examined.

Cytological studies on N.wuttkei revealed a chromosome number of 2n =32, as N. exigua, N.maritima, N. suaveolens and N. velutina. From morphological observations the flower of N.wuttkeiis similar to N. exigua, while the seed, as well as the pollen, resembles N. velutina. As concerngenetic relationship, revealed by ISSR analysis, N. wuttkei resulted groupped together with three ofthe four 32 chromosome members of the examined Suaveolentes.



UTILIZATION OF WILD GERMPLASM FOR PLANT BREEDING INCOMMON BEAN (PHASEOLUS VULGARIS L.)

A. GIARDINI*, E. BELLUCCI*, M. ROSSI*, L. NANNI*, S.A. ANGIOI**, F. DESIDERIO*,D. RAU*, V. FERRARI***, A. CARBONI****, P. GEPTS*****, G. ATTENE**, R. PAPA*

*) Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, Via BrecceBianche, 60131 Ancona, Italy - [email protected]**) Dipartimento di Scienze Agronomiche e Genetica Vegetale Agraria, Università di Sassari,Via De Nicola, 07100 Sassari, Italy***) CRA Research Institute for Vegetable Crops, Section of Ascoli Piceno, Via Salaria 1,63030 Monsampolo del Tronto (AP), Italy****) CRA Institute for Research on Industrial Crops Plant Breeding and Biotechnology SectionVia di Corticella, 133 - 40128 Bologna, ITALY*****) Department of Plant Sciences, Section of Crop and Ecosystem Sciences, UC Davis, 1Shields Avenue, Davis, CA 95616-8780, USA

breeding, diversity, domestication, Phaseolus vulgaris, wild relatives

Random genetic drift and selection during domestication and plant breeding have stronglyreduced the level of genetic variation in modern crops. Recently, the work conducted in tomato andrice has shown that the use of wild relatives associated with the use of molecular tools could have atremendous impact on crop improvement. However, several problems are associated with theintroduction of exotic germplasm such as the occurrence of deleterious traits that tend to maskuseful variants, the presence of various level of incompatibility between domesticated crops andtheir wild relatives, and the need to develop an efficient identification system of the most promisingwild genotypes to build segregating populations. For autogamous species, in particular, the regionsof the genome surrounding the major domestication genes appear to be particularly interesting totag the introgression from wild relatives into modern cultivars. Because of the combined action ofselection and recombination, these “domestication regions” have probably experienced a higherlevel of isolation between domesticated and wild forms. Indeed, farmers and breeders selecting fordomesticated alleles, have probably selected also against tightly linked genes. As shown recently inP. vulgaris, the domestication regions of the genome appear to harbour a much higher level ofgenetic variation in the wild populations in comparison with other regions not involved indomestication. To develop breeding strategies aimed at exploiting wild germplasm, the followingelements are needed: a deep understanding of the population history and structure of bothdomesticated and wild populations, the identification of the loci that have been the target ofselection during domestication and breeding episodes, and knowledge of the level and structure ofdiversity along the genome. Here we present an ongoing project aimed at utilizing the geneticdiversity of wild P. vulgaris. This project includes the analysis of the genetic diversity using variousmolecular tools, the identification of genomic regions related to the domestication process, theidentification of a set of wild genotypes to be used as donors parents and the analysis of segregatingpopulations for QTL analysis, in the short term, and breeding purposes, in the longer term.



ISOLATION OF LOW PHYTIC ACID IN BEAN

E. DORIA*, B. CAMPION**, M. FILEPPI**, I. GALASSO***, F. SPARVOLI***,R. BOLLINI***, E. NIELSEN*

*) Dipartimento di Genetica e Microbiologia Università di Pavia, Pavia, Italy**) Istituto Sperimentale per l’Orticoltura, Monatanaso Lombardo, Lodi, Italy***) Istituto di Biologia e Biotecnologia Agraria, CNR, Milan, Italy

Phaseolus vulgaris, EMS, lpa, seed quality

Phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6) is the storage form ofphosphorus in seeds and acts as an antinutrient for humans and nonruminant animals because itreduces the bioavailability of phosphate and of cations such as zinc and iron (Sandberg, 2002).Phytic acid was considered essential for seed germination or seedling growth until a few years ago.A number of lpa (low phytic acid) mutant lines with greatly reduced levels of phytic acid andconcomitant high phosphate level in the seed (HIP phenotype) have been generated by randommutagenesis in various crops (Raboy,2000, Hitz et al. 2002, Pilu et al. 2003). Although some ofthese mutations appear to cause negative pleiotropic effects (Raboy 2001, Pilu et al 2003), themodulation of phytic acid content in the seed can be regarded as a major goal in seed crop geneticimprovement by mutant selection. Due to the importance of bean (Phaseolus vulgaris) for humannutrition as a protein-rich source, the isolation of lpa mutants in this species is a very attractivegoal because it may lead to decrease iron and zinc deficiencies, particularly widespread in someAfrican and South / Central America populations feeding on seed-based food.

Therefore, we have treated with EMS (ethyl methane sulphonate) 6200 seeds and obtained1774 M1 fertile plants, corresponding to 1774 pools of M2 seeds, which were screened for HIPphenotype. For each of the 8 HIP lines discovered, 16 single seeds were re-analyzed for phosphatecontent: among line 280 seeds, one revealed to contain about ten fold more free phosphate than wildtype.

Among 280-M2 plants, three out of 80 produced seeds all showing HIP phenotype andyielding plants all producing HIP M4 seeds, thus confirming to be endowed with a lpa mutation.

Further analyses and verifications of phytic acid, raffinose and iron content of lpa M4 seeds,as well as of their germination rate and of growth- and yield-related features of M4 plants are underway.



AN IMPROVED CROSSING TECHNIQUE OFFERING BETTEROPPORTUNITIES TO LENTIL BREEDING

D. CHIARETTI*, F. FELICI*, M. IANNETTA*, A. BOZZINI**

*) ENEA, C.R. Casaccia, Santa Maria di Galeria 301, 00060 Roma**) EUROGEN Srl, Piazza Armerina, Enna.

lentil, crossing, breeding

Lentil breeding has been till now mainly limited to selection of lines derived from localpopulations coming from different cultivation areas.

During the last years, our lentil germplasm analysis and breeding have identified several linesshowing characters of potential high value for semi-arid or good temperate areas: high earliness orlateness, small or large plant and seed size, different seed colour, resistance to warm or cold areasetc.

However, lentil breeding suffers of two major difficulties: the very small flower size, makingrather difficult the success of manual crossing and the production of only 1-2 seeds for each crossedflower. Facing this problem, one of us (C.D.) has developed a crossing method leading to about80% of cross success.

This technology provided the opportunity of performing high number of crosses, in the attemptto accumulate several selected characters in relation to specific cultivation needs.

This contribution deals with the surprising high variability showed by the F2 plants and the F3seeds for the seeds shape and size, cotyledon and esosperm colour, number of seeds per pod etc.coming from different parents. In the next generations we intend to obtain lentil selections withaccumulated characters favouring the crop adaptation to cultivation in semi arid conditions, typicalof large Mediterranean and Middle East areas, or to more fertile and humid temperate areas typicalof Central Europe, North America, Australia.



MALE-STERILE TOMATO MUTANTS FOR POSSIBLE USE IN HYBRIDSEED PRODUCTION

A. MAZZUCATO*, I. OLIMPIERI*, F. RUIU*, E. OVIDI**, A. TIEZZI**, V.K. SAWHNEY***

*) Department of Agrobiology and Agrochemistry, University of Tuscia of Viterbo, Via S. CamilloDe Lellis s.n.c., 01100 Viterbo, Italy - [email protected]**) Department of Environmental Sciences, University of Tuscia of Viterbo, Largo dell’Università,s.n.c. - Blocco D, 01100 Viterbo, Italy - [email protected]***) Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK. S7N5E2, Canada - [email protected]

gene mapping, hybrid seed, male sterility, Solanum lycopersicum, tomato

Notwithstanding the autogamous pollination system, hybrid varieties are presently the mostwidely adopted commercial seed stocks for tomato (Solanum lycopersicum L.). While a number ofmale-sterile mutants have been described, genic male sterility (GMS) has not been the choicematerial of plant breeders because GMS lines retain the major disadvantage that male-fertilesegregants must be rogued out of hybrid seed fields. Therefore, the production of tomato hybridseed still relies on manual emasculation. One workable solution to harness GMS is the selection ofconditional sterility sources, where sterile anthers could be restored to fertility by permissive growthconditions or by the application of appropriate growth regulators. To this end, mutant alleles mustbe characterized that ensure maximum expressivity in the prohibitive conditions and a good fertilityin permissive environments. The molecular characterization of mutants candidate to be used asconditional male-steriles will be extremely advantageous, for developing tools for assistedbackcrossing, for discovering new alleles by screening saturated mutagenized populations and forunderstanding the molecular basis of ‘conditionality’ in order to modulate it with conventional orbiotechnological approaches.

In this research, five tomato mutants showing staminal [pistillate (pi), stamenless-2 (sl-2), 7B-1, variable male sterile (vms)] or functional [positional sterile-2 (ps-2)] sterility have been studiedat the phenotypic and molecular levels. The expressivity of these mutations in spring conditions hasbeen high, because very scarce or no seed was produced under open pollination. Differently, all themutants except pi produced seed when pollinated with wild-type pollen. For this reason, pi wasdiscarded as a possible source of male sterility for hybrid seed production. Mapping studies on thefive genes integrated all the loci into the tomato molecular map. Comparison with the map locationof the sequences corresponding to tomato class B MADS-box genes indicated vms as a candidatefor being involved in the SlGLO locus, whereas Sl-2 and 7B-1 were candidate to correspond to theSlDEF gene. These results provide the basis for unraveling the genetic and molecular mechanismscontrolling male sterility/fertility in the studied genotypes and to pursue their exploitation in hybridseed production.



A TOMATO HOMEOTIC MUTANT PUTATIVELY INVOLVED IN THEFUNCTION OF SEPALLATA MADS-BOX GENES

I. OLIMPIERI, M.E. PICARELLA, A. MAZZUCATO

Department of Agrobiology and Agrochemistry, University of Tuscia of Viterbo, Via S. Camillo DeLellis s.n.c., 01100 Viterbo, Italy – [email protected]

flower development, homeotic mutants, male sterility, Solanum lycopersicum, tomato

Despite the importance of tomato as a model and crop species, studies on its reproductivebiology have lagged behind those that have elucidated the molecular control of flower developmentin other taxa. Indeed, many floral phenotypes have been described in decades of tomato genetics,but for very few of them the underlying genes have been identified so far. Because the increasingavailability of genome sequence data will greatly facilitate forward genetics in tomato, noveldescriptive and mapping information will help the attribution of genes to phenotypes. In thiscontribution, we present our work on pistillate (pi), a mutant forgotten after the first description ofC.M. Rick and J. Robinson in the middle of the last century, that directly recalls mutations affectingB class MADS-box genes.

Plants homozygous for the pi allele appear with Mendelian proportions in segregatingpopulations. Compared to wild-type (WT), mutant plants show higher frequency of compoundinflorescences, reversion of inflorescence meristem to vegetative identity and frequently a modifiedsympodial segment. The most striking aberration in p i mutant flowers is the homeotictransformation of stamens into carpels. More rarely, homeotic conversions are reported also in thesecond floral whorl, with staminoid and carpelloid petals. Ultrastructural analysis reveals more orless subtle sepaloid features in the three inner floral whorls, mainly based on the presence,distribution and amount of glandular and non glandular trichomes. In the ovary, a ‘flower withinflower’ phenotype was seldom observed; in one instance such phenotype was coupled with thesetting of a parthenocarpic fruit, that contained a new flower inside. Mapping information indicatedthat none of the tomato class B MADS-box genes can be candidate for the Pi locus. Conversely, thecomplex pi phenotype parallels those described in natural or engineered mutants affected in thefunction of SEPALLATA (class E) genes.



ANEUPLOID HYBRIDS FROM 3X X 4X CROSSES IN POTATO:CHROMOSOME NUMBER, FERTILITY AND RESISTANCE TRAITS

I. CARUSO, L. CASTALDI, G. CARUSO, L. FRUSCIANTE, D. CARPUTO

Department of Soil, Plant and Environmental Sciences, University of Naples “Federico II”,Via Università 100, 80055 Portici, Italy

Solanum tuberosum, Solanum commersonii, Ralstonia solanacearum, pentaploid

The aim of this study was to characterize twenty-nine S. commersonii – S. tuberosumprogenies, deriving from 3x X 4x crosses to provide evidence that they can be used in potatobreeding. The chromosome number of hybrids analyzed ranged from hypo-pentaploid (2n=5x-10=50) to hyper-pentaploid (2n=5x+7=67), with (2n=5x=60) class predominant. Despite beinganeuploid, the hybrids did not generally show phenotypic aberrations or vigor reduction common toaneuploids of other species. Most genotypes resembled S. tuberosum in growth habit, except eyedepth and stolon lengh. Variability was found for tuber production. Interestingly, a number ofhybrids (21%) displayed introgression from S. commersonii of resistance to Ralstoniasolanacearum, the casual agent of bacterial wilt. Although aneuploidy has often been associatedwith reduced fertility, many hybrids were fertile as female parents with S. tuberosum. Indeed, theaverage berry set and number of seeds/berry were 37.1 and 31.5 respectively. In particular, valuesof seeds/berry decreased as the aneuploid level of female parents increased. The relationship wasprincipally linear. No significant quadratic relationships were noted. The correlations betweenaneuploid chromosome number and fertility/agronomic parameters are discussed.



EVIDENCE OF CYTOMIXIS IN POA PRATENSIS L.

G. MARCONI, E. ALBERTINI, L. RAGGI, ANNA MOOSBRUGER, A. MARIANI,M. FALCINELLI

Department of Plant Biology and Agro-environmental and Animal Biotechnology,University of Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy

cytomixis, polyploid, apomixis, Kentucky bluegrass

Cell to cell migration of chromatin, nucleus, nucleolus, individual chromosome, group(s) ofchromosomes, whole spindles from one cell to another that is close by, which has been reported andstudied in several plants belonging to diverse families (Bell 1964; Maheshwari 1950) is referred ascytomixis. This phenomenon was first observed in the pollen mother cells (PMCs) of Crocussativus by Kornicke in 1901 (Brown and Bertke 1969) and later in Oenothera gigas and O. biennis(Gates 1911). In addtion to meiocytes, cytomixis has also been reported in apical meristems,epidermal cells of scales and leaves, tapetal cells and cells of nucellus and integuments.

Polyploid species seem to exhibit cytomixis more than their diploid counterparts as shown bystudies in triploids and tetraploids of sugarbeet (Semyarkhina and Kuptsou 1974). Moreover,cytologically, physiologically and biochemically imbalanced plants like haploids, triploids,aneuploids and apomicts show cytomixis more often than normal cytogenetically balanced andestablished plants (Percival 1930; Nandi 1937; Sapre and Deshpande 1987).

So far, cytomixis has been detected in many plant species but never in Kentucky bluegrass(Poa pratensis L.) which is a hardy, persistent, attractive forage and turf grass adapted to a widerange of soils and climate (van Wijk 1997). The mode of reproduction of P. pratensis is extremelyversatile and ranges from naturally obligate apomixis to complete sexuality. The high polyploid andcontrasting mode of reproduction of P. pratensis should make it a model species for investigatingapomixis and cytomixis phenomenon.

Our preliminary results demonstrate that cytomixis and abnormal meiosis are present in PMCsof apomictic (aposporic and parthenogenetic) and recombinant (aposporic and non-parthenogenetic)plants while it is not present in sexual (non-aposporic and non-parthenogenetic) genotypes.Cytomixis phenomenon was seen occurring exclusively at the prophase of first meiotic division,while meiotic abnormalities were present overall the entire meiotic process. We also observedunivalents in diakinesis and metaphases I while metaphases and ana-telophases II showed laggards.

Results will be used to try to understand: i) how much cytomixis can inflence pollen fertility inP. pratensis; ii) if there is a correlation between cytomixis and apomixis and the possible role thatcytomixis could have played in the development of apomictic reproduction; iii) to study candidategenes involved in cytomixis and meiotic abnormalities.



A CONTRIBUTION TO ALFALFA GENETIC IMPROVEMENT. TWO NEWCVS: KATANA (WITH WHITE FLOWERS) AND LUCREZIA (WITHYELLOW FLOWERS)

A. BOZZINI, F. CALCAGNO, T. SOARE, G. SOARE, F. CALCAGNO

EUROGEN S.r.l., C.P.Aperta, 94010 Pergusa (En) - [email protected]

alfalfa, genetics, breeding, new cultivars

Alfalfa (Medicago sativa L.)is a forage crop among the most cultivated in the world, for itshigh quality and productivity. In Italy is the most important forage crop, covering 66% of theseeded forage area. Its cultivation is particularly diffused in north Italy.

In 1996 the EUROGEN research team started a new breeding programme, obtaining finally twocultivars of alfalfa with peculiar flower colors: Katana, with white flowers and Lucrezia, withyellow flowers, both of particular interest regarding productivity, longevity, quality of forage andfield resistance to Fusarium.

The basic material was provided by the Senior Author, who identified and produced the twobasic populations: the first one coming from Sannio (Province of Benevento) and the second onederived from Latium’s Maremma (Province of Viterbo).

In the paper are described the breeding methods utilized, the genetic basis of flower colors andthe results of the agronomic evaluation of characters connected with green forage quantity andquality and the analysis of phenotypic correlations of characters related to seed production.



CONTAMINATION IN CONTROLLED CROSSES IN SUNFLOWER(HELIANTHUS ANNUUS L.)

D. LAURETI, A. DEL GATTO, S. PIERI, E. HABYARIMANA

C.R.A. Istituto Sperimentale Colture Industriali, Via Cagiata 90, 60027 Osimo (AN), Italy –[email protected]

Out crossing might inadvertently interfere with controlled crosses owing to either the practicaldifficulty to achieve complete emasculation or the unavoidable wind transported pollen during theprocess of emasculation. Crossing in sunflower requires emasculation many times because thehundreds of flowers are arranged in concentric circles radiating from the periphery to the centre ofthe head and flowering proceeds from the periphery innerwards at the rate of one to four rows perday. To verify the amount of cross contamination that may occur in crossing sunflower anappropriate experiment was undertaken in 2004.

In Autumn 2004, plants deriving from one hybrid and two maintainer lines susceptible to theimidazolinone family, were emasculated and crossed with a hybrid resistant to the herbicides. Thisseason was particularly poor in free pollen since only parental lines to cross were in the field and 17plants per genotype were allowed to freely release pollen in the environment.

The imidazolinone-resistance is semi dominant in nature (Miller and Khatib, 2000). Thehybrid between two resistant strains resists to the herbicide at a dose 10 fold higher than the normal.The F1 plants from a cross between a resistant and a susceptible genotype can withstand a dose ofimazamox as high as 3 fold the normal, whereas a casual cross between two common plants woulddie at normal application rate of the product.

In spring 2005, resistant x susceptible crosses and parental genotypes were planted in thenursery and sprayed with Altorex at a dose as high as three times that recommended. After 20 days,treated plants were classified as survived or dead. One cross did not yield vital seeds, whereas theprogeny from the other crosses were destroyed at 11 percent, which could be considered as theamount of out crossing. However, since the out crossing depended upon the amount of pollenpresent in the air, and considering that in the field there were three susceptible and one resistantpollen parent, the value of the uncontrolled crosses was underestimated because also the resistantplants contributed the allopollen albeit their contribution could not be phenotyped, their progenybeing herbicide resistant: the 11 percent dead plants were therefore only three quarters of total outcrossing. It can be inferred that the amount of out crossing during controlled hybridization canreach important values that can not be overlooked while managing the progeny.

LiteratureMiller J. F., Al Khatib, K. 2000. 15th International sunflower Conference. O-37-41



RESPONSES TO DIVERGENT SELECTION FOR COB COLOUR IN AMAIZE POPULATION

M. A. CANÈ, E. FRASCAROLI, S.VECCHI, P. LANDI

Department of Agro-environmental Science and Technology (DiSTA), University of Bologna,Viale Fanin 44, I-40128 Bologna, Italy

Cob colour, P1 gene, marker assisted selection, correlated traits, leaf number

In a previous study, we carried out four cycles of recurrent selection for grain yield in the F2

population of the maize single cross A632 (red cob) x Mu195 (white cob). Significant gains wereachieved for grain yield and other agronomic traits; moreover, an increase of the frequency of thered cob allele was observed throughout the selection process, suggesting an association betweengrain yield and cob colour (determined by P1 gene in chromosome 1). In a subsequent study, near-isogenic lines with red or white cob were developed by backcross to both recurrent parents A632(using Mu195 as donor of the white allele) and Mu195 (using A632 as donor of the red allele).These near-isogenic lines were evaluated both per se and in hybrid combination and the lines withred cob (R) yielded more than the corresponding lines with white cob (W) in both A632 and Mu195backgrounds, thus confirming the association between grain yield and cob colour. These findingsprompted us to undertake a divergent selection for cob colour, i.e., for R or W, in the F2 of A632 xMu195 to evaluate the effect of this marker assisted selection on grain yield and other traits. About200 random F2 plants were selfed and the resulting F3 seeds were bulked according to the cobcolour of the ears. Forty seeds were bulked from each R ear to produce the selected F3-Rpopulation; similarly, 40 seeds were bulked from each W ear to produce the selected F3-Wpopulation. Moreover, 20 seeds were bulked from each ear (regardless of the cob colour) to producethe unselected F3 population. The three populations were tested in two field trials. The F3-R meanacross the two trials was superior to the F3-W mean for number of leaves per plant (19.2 vs. 18.0),days to flowering, whole leaf area, kernel moisture, number of kernels per plant and grain yield(3.44 vs. 3.05 Mg ha-1). In contrast, the mean across the two selected populations was rather close tothe mean of the unselected F3 population for most traits, indicating a prevalence of symmetricresponses to selection. In comparison with the unselected F3 population, F3-R population showed asuperiority for grain yield of 0.22 Mg ha-1 corresponding to a gain of 7.7%, which is slightly higherthan the average gain per cycle (7.3%) achieved in the laborious recurrent selection previouslyconducted. When differences between F3-R and F3-W populations were referred to the number ofleaves per plant, either directly, as for leaf area (i.e., considering the average area per leaf), orindirectly, as for grain yield (considering the average grain yield per leaf area unit), the differencesbetween F3-R and F3-W populations were not significant any more. These findings indicate that inthe material herein investigated selection for cob colour can lead to an appreciable yieldimprovement, achieved at least partly by changes concerning the number of leaves per plant andassociated traits. These findings could be accounted for by assuming that close to the P1 gene thereis a QTL controlling the number of leaves per plant and then date of flowering, whole leaf area,number of kernels per plant, kernel moisture and grain yield, following a sequence of causallyrelated events. To gain a better insight into the genetic effects of this QTL and the frequency of

recombination with P1 gene, we are going to compare the selected F3-R and F3-W populations withthe selected F3-R and F3-W populations obtained by selfing the plants derived by one cycle ofrandom mating in the same source F2.



NEW NAKED OAT GENOTYPES FOR PRODUCING FUNCTIONALFOODS

R. REDAELLI*, D. SGRULLETTA**, G. SCALFATI**, E. DE STEFANIS**,C. M. POLLINI***

*) CRA – Experimental Institute for Cereal Research, Via Stezzano 24, 24126 Bergamo**) CRA – Experimental Institute for Cereal Research, Via Cassia 176, 00191 Rome***) Pavan Tecnologie S.p.A., Via Monte Grappa 8, 35015 Galliera Veneta (PD)

naked oats, functional compounds, pasta

In consideration of the role of diet in health and wellness preservation and the recognizedprotective effects of oats against some chronic pathologies, an innovative product was recentlyproposed (Sgrulletta et al., 2003), an oat-wheat pasta obtained through the enrichment ofcommercial durum wheat semolina with the flour of naked oat cultivars. This product could bedefined as a “whole grain food” naturally rich in functional compounds. A large variability amongoat cultivars in the response to pasta-making processes was shown, highlighting the importance ofchoosing the oat genotypes with the most suitable chemical composition for food production. Theobjectives of the present study were to examine the existing variability for functional compounds ina group of forty-one naked oat genotypes (cultivars and breeding lines) and to assess the nutritionalvalue of the pasta obtained by mixing the flours of some of them to semolina. Significant genotypicdifferences were observed in oat flours with respect to protein (range: 12.8-22.7 %d.m.), totalβ-glucan (2.2-5.4 %d.m.) and soluble β-glucan (1.2-3.6 %d.m.). The pasta making process wasfound to safeguard the content of the different components: cooked oat pasta showed an improvednutritional value in comparison with the traditional 100% durum wheat pasta, particularly inrelation to the increase of fibre (TDF and β-glucan) content. These data confirmed that: i) thesuggested oat-wheat pasta could fit well in a balanced diet, as the high soluble fibre contentcontributes to lower the level of blood cholesterol; ii) the choice of the genotype that is used forpasta production strongly affects the technological and organoleptic aspects of the process. Specificbreeding programs to develop and select the most suitable genotype have been activated.

Sgrulletta D., De Stefanis E., Conciatori A., Redaelli R., Pollini C.M. (2003). Influence ofdifferent naked-oat cultivars on the nutritional value of pasta. Tecn. Mol. Intern. 54(2/A): 125-130.



NEW EMMER AND SPELT CULTIVARS DEVELOPED BYINTROGRESSION OF SOME USEFUL DURUM AND BREAD WHEATTRAITS

P. CODIANNI, C. FARES, A. TROCCOLI, C. RIEFOLO, A. GALLO, N. DI FONZO,L. CATTIVELLI, P. DE VITA

C.R.A. Centro Ricerche per la Cerealicoltura, S.S. 16 km 675, 71100 Foggia, Italy –[email protected]

emmer, spelt, durum wheat, bread wheat, cultivar release

Emmer (Triticum turgidum L. subsp. dicoccum Schrank) and Spelt (Triticum aestivum L.subsp. spelta) hulled wheats were the main cereal crops in the Mediterranean basin during theRoman period, progressively replaced with hulless durum (T. turgidum subsp. durum Desf.) andbread (T. aestivum L. subsp. aestivum L.) wheat. Despite a number of defects as plant height, lowgrain yield and low pasta- and bread-making quality, spelt and emmer have been recovered inmodern times thanks to their adaptability to poor soils and unfavourable climatic conditions. Abreeding program based on crosses emmer x durum and spelt x bread wheat was developed by theC.R.A. Centre for Cereal Research at Foggia (Italy) to release new emmer and spelt cultivars withimproved agronomic and qualitative traits for use by grain producers in Italy. During the selectionprocess attention was given to the preservation of key morphological and functional traits of emmerand spelt species such as the tough glumes that tightly enclose the grains giving good protection ofthe stored grain against pest. The selection process was carried out in high input conditions (with Nfertilization of 90 kg ha-1) and lead to identify 3 emmer cultivars (Mosè, Padre Pio and Davide) and4 spelt cultivars (B1030, S2013, S2070 and P12). Our findings provide evidence that the breedingstrategies exploiting hulless wheat as donor parent in selection programs aiming to develop hulledwheat cultivars adapted to low-input/organic cropping conditions could be successfully adoptedoverturning the roles, often adopted in the past, to improve hulless wheats.



CITOGENETIC AND HISTOLOGICAL CHARACTERIZATION OFCITRUS REGENERATION SYSTEM

L. GIORGETTI*, V. MICHELOTTI**, M. SALVINI**,***, M. FAMBRINI**, C. PUGLIESI**,G. LUCCARINI*, C.E. GERI*

*) Istituto di Biologia e Biotecnologia Agraria, CNR Area della Ricerca di Pisa, Via Moruzzi 1,56124 Pisa, Italy**) Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica, Via Matteotti 1B,Università di Pisa, 56124 Pisa, Italy***) Scuola Normale Superiore, Piazza dei Cavalieri 7, 56124 Pisa, Italy

Citrus, somatic embryogenesis, polyembriony, gene expression

Different tissue culture systems were considered in order to establish the best experimentalapproach for the isolation and characterization of biotic and abiotic stress resistant regenerants inCitrus plants. A new protocol of in vitro culture and somatic embryogenesis, starting fromgerminated seeds (zygotic and apomittic plantlets) of different Citrus genotypes was established andcompared with the mainly used in vitro regeneration system from excised immature ovules.

Cytological and histological analyses were performed during the very early stages of callusinduction and during somatic embryogenesis in the two different regeneration systems. Whenyoung plantlets were used as source of explants every tissue can de-differentiate but the roots.Hypocotyls and cotyledons were a good source of embryos/regenerants directly, but with little/noembryogenic callus production.

An unexpected result was the stability of the callus in culture: cells are strictly diploid, therewere no anomalies, both in calluses derived from seedlings as well from ovules.

Histological analysis of cultured ovules of four different Citrus genotypes (Citrus lemon var.Eureka, Citrus lemon var Volkameriana, Citrus lemon var Macrophilla, Citrus sinensis varSanguinello) showed that embryogenic callus came from the outer integuments of the ovules andnot from nucellar tissue as supposed.

A further extensive cyto-histological analysis was performed in vivo during Citrus lemonembryogenesis on the process of apomittic embryos formation from nucellar tissues.

Moreover in situ hybridization experiments were done with the embryogenic specific cDNAprobe HaL1L recently isolated in Helianthus (HaL1L homologue of LEC1 of Arabidopsis) to studythe pattern of expression during embryogenesis in Citrus.

Preliminary results revealed a clear hybridization signal that had the same pattern of expressionas in Helianthus, and it was located in the nucellar tissues, in zygotic and apomittic embryos atglobular - torpedo stage.

Citrus embryogenesis could offer a very good system not yet exploited: Citrus polyembrionymeans the possibility to study at the same time in the same embryo-sac zygotic and somaticembryos developmental patterns.

Research programme INTERREG III A ITALIA; FRANCIA; ISOLE



GENETIC DIFFERENTIATION BETWEEN POPULATIONS OF SCOTSPINE (PINUS SYLVESTRIS L.) FROM ITALY AND OTHER EUROPEANCOUNTRIES

S. PUGLISI*, A. LOPS**, G. RAINALDI**

*) Institute of Plant Genetics - CNR, Via Amendola 165/A, 70126 Bari, Italy**) Department of Biochemistry and Molecular Biology, University of Bari, Via Orabona 4,70126 Bari, Italy

population genetics, Pinus sylvestris, genetic diversity, genetic differentiation, natural range

The aim of this research is to study the differentiation between eight Italian populations ofScots pine (Pinus sylvestris L.) - already surveyed in a previous investigation, and which arerepresentative of the Italian natural range - and some populations representative of other countriesof the boundless natural range of this species. The used technique is isozyme analysis carried out bymeans of horizontal starch gel electrophoresis. Some preliminary results are shown here, regardinga group of European countries. The obtained values of genetic distance show that the previouslyobserved strong differentiation of a relict and isolated Italian population (a remnant from glacialmigrations), located in the Emilian Apennine, has been confirmed: as a matter of fact, it is even lesssimilar to the other studied Italian populations (from the Alps) than some foreign populations whichtend to group together with them. These results confirm that this small and autochthonous stand isan important genetic resource: its differentiation reveals a different evolutionary history or adifferent origin (glacial refugium), and its values of genetic diversity parameters are similar to thosefound in the other Italian populations, in spite of its geographic isolation from the main range of thisspecies. As far as they are concerned, the seven Italian populations from the Alps appear ratherdifferentiated from the remnant, suggesting that the Alpine barrier caused a genetic isolation strongenough to condition their evolutionary pathway. These results cast new light over the availableknowledge on this species, since until now Italian populations have been never compared to otherEuropean ones, and make it possible the drafting of more accurate programmes of genetic resourceconservation. On the basis of the obtained results, some hypotheses on the postglacialrecolonization routes followed by this species are discussed.



AN IMPROVED CHARACTERIZATION OF DONKEY (EQUUS ASINUS,2N=62) CHROMOSOMES BY USING C-, G- AND R-BANDING PATTERNS

G.P. DI MEO*, A. PERUCATTI*, V. PERETTI**, F. CIOTOLA***, L. LOTTA****,D. DI BERARDINO**, L. IANNUZZI*

*) National Research Council (CNR), ISPAAM, Laboratory of Animal Cytogenetics and GeneMapping, Naples, Italy**) Department of Animal Sciences and Food Inspection, University of Naples “Federico II”,Naples, Italy***) University of Catanzaro "Magna Grecia", Catanzaro, Italy****) Department “MOBIFIPA.”, Veterinary Medicine Faculty, University of Messina, Messina,Italy

donkey, banding, karyotype, constitutive heterochromatin, breed

Few cytogenetic studies have been undertaken in the donkey (Equus asinus, 2n=62; EAS), sofar. Indeed, the main interest on the chromosomes of this species is due to the close relationshipswith the horse, especially for a better understanding of the reasons for the sterility of their hybrids.A donkey chromosome nomenclature based on GTG-banding patterns at low-medium bandingresolution was earlier presented by Raudsepp et al. (2000) and used in our study to constructimproved G and R-banded karyotypes at about 450 band level and to study the distribution ofconstitutive heterochromatin (HC). Peripheral blood cultures from 15 animals of “Ragusana” andAmiata” breeds were treated for early- and late-BrdU incorporation to obtain G- and R-bandingpatterns, respectively. Cell cultures were synchronised with MTX+BrdU (G-banding) or withthymidine (R-banding). Cell blocks were removed washing cells twice and recovering cells in freshmedium containing thymidine (G-banding) or BrdU+H33258 (R-banding). Improved bandingpatterns were obtained in all chromosomes enhancing our knowledge on the chromosomes of thisspecies. In addition, CBA-banding (C-banding by acridine orange staining) revealed large blocksof HC in many chromosomes, in particular on EAS1q-prox (the largest HC-block). EAS1 showsalso the p-arms C-band positive. HC varies among chromosomes. In particular, some animal ofRagusana breed showed a very small HC-block at the homozygous or heterozygous level, in EAS1qwhen compared with the that achieved in normal karyotypes. Further studies are necessary to betterunderstand this phenomenon. This study is also our contribution for the standardization of donkeykaryotype.



SCE-TEST REVEALS GENOMIC INSTABILITY IN CATTLE AFFECTEDBY CHRONIC ENZOOTIC HAEMATURIA

V.PERETTI*, F. CIOTOLA**, S. ALBARELLA*, C. DARIO***, G.P. DI MEO****,A. PERUCATTI****, S. ROPERTO*****, V. RUSSO*****, V. BARBIERI*, L. IANNUZZI****

*) Department of Animal Science and Food Inspection, Faculty of Veterinary Medicine, UniversityFederico II, Naples**) University of Catanzaro "Magna Graecia", Catanzaro, Italy***) Department of Animal Production, University of Bari, Bari****) CNR-ISPAAM, Laboratory of Animal Cytogenetics and Gene Mapping, Naples*****) Department of Pathology and Animal Health, University Federico II, Naples

cattle, chromosome fragility, SCE, cancer, breed

Chronic enzootic haematuria (CEH) is a pathology specifically found in cattle grazing naturallyor fed with plants rich in bracken fern (Pteridium aquilinum) which is widely spread all over theworld and particularly in southern Italy. This plant is know to contain toxic compounds such asptaquiloside and quercitin, which have mutagenic and cancerogenic effects, mainly in the bladder.

Previous cytogenetic studies on cows affected by CHE using normal staining technique andanalyses of chromosome abnormalities (gap, chromatid and chromosome breaks, fragments)revealed a significant increasing of chromosome abnormalities in cows affected by CEH, comparedto the control (Lioi et al., 2004). In the present study we applied the sister chromatid exchange(SCE) test on 30 cows with CEH (all these cows were slaughtered to check for the presence ofCEH) and 10 control cows fed without bracken fern. All animals aged from five to twelve years. Atleast 35 metaphase plates per animal were examined.

Higher levels of SCE (SCE/cell = 7.51 ± 3.58) were found in the cows affected by CEH whencompared with the control (SCE/cell = 5.82 ± 3.05) and the differences were highly significant(P<0.001). The present study confirms genomic instability in cells of animal affected by CEH, asrevealed earlier by using other cytogenetic test (Lioi et al. 2004). To our knowledge, this is the firstinvestigation in a representative sample of cattle with CEH using the SCE test.

Acknowledgements. Supported by PRIN-05



THE ITALIAN PLANT DNA BANK: TOWARDS AN INTEGRATEDSYSTEM FOR CONSERVATION AND UTILIZATION OF GENETICRESOURCES

G. SONNANTE, L. MONTI

Institute of Plant Genetics, CNR, Via Amendola, 165/A, 70126 Bari, Italy –[email protected]

DNA bank, conservation, genetic resources, landraces, model species

Human life is highly dependent on the use of plants, since they represent not only an importantcomponent of the diet, but also a source of various by-products, e.g. fibres and medicines. Toreduce the risk of a loss of genetic material, particularly wild species and old local varieties, severalInstitutes are acting all over the world for the in situ and ex situ conservation of plant germplasm.Conservation possibilities were widened by the recent advances in plant genomics which allow theretrieval of large amounts of information from DNA, in particular on genes, their function andorganization, and on possible markers. A DNA bank may represent therefore a reservoir of geneswhich is useful, both for comparison purposes, in order to monitor the changes in the geneticstructure of cultivated material, and for the development of new molecular markers and search forgene variants. In this framework, a DNA bank is an extension of the concept of gene-bank initiallyimplemented in seed genebanks and is not meant as a substitute.

In 2005 the first core of a DNA bank was set up at the Institute of Plant Genetics, CNR, in Bari,where a seed bank exists since 1970. The purposes of this DNA bank are to collect, extract andstore genomic DNA from local varieties of Mediterranean crops, and from their wild relatives. Thismaterial, in fact, can be of particular interest for the presence of useful genes (e.g. resistance tovarious stresses), which can be easily retrieved from the DNA available in the bank. For this reason,DNA was extracted from single individuals from several populations of Eruca sativa, Boragoofficinalis, Cynara cardunculus var. scolymus and var. sylvestris, Lens culinaris, Capsicumannuum, etc. and stored at -80°C. A further aim of our DNA bank is to store DNA from modelspecies, such as Arabidopsis thaliana and its relatives, collected in the Mediterranean Basin.

At present we are also implementing a database including the most relevant informationrelative to the stored samples and are developing a web interface to allow external users access theinformation and request DNA samples on the basis of a material transfer agreement.



COLLECTION, CHARACTERIZATION AND DNA STORAGE OFARABIDOPSIS THALIANA (L.) HEYNH. (BRASSICACEAE) ECOTYPESCOLLECTED IN ITALY

G. MARUCA, G. LAGHETTI, G. SONNANTE, D. PIGNONE, F. LOSAVIO, L. MONTI

Institute of Plant Genetics (IGV) – CNR, Via Amendola, 165/A, Bari

Arabidopsis thaliana, model plant, phenotypic characterization

Arabidopsis thaliana (L.) Heynh., a small annual white flowered species of the Brassicaceaefamily, has become a very important model system in plant genetic research, thanks to its smallgenome, short generation time and simplicity to be mutagenized. In contrast with plants possessinglarger genomes, it is suitable to current studies of molecular biology for the establishment of genefunctions. The importance of isolation and functional analyses of genes in A.thaliana, likewise theidentification of fundamental mechanisms is that it can be used to find their homologues in cropplants. For this reason there is a great scientific interest in this widely studied plant species, andmany stock centers store samples of A. thaliana collected in different locations in Europe, CentralAsia and Africa.

Considering that Italy and the Mediterranean Basin have been poorly explored for thecollection of this plant species, the Institute of Plant Genetics (IGV) of CNR (National ResearchCouncil) of Bari ( Italy) has started a research activity on collecting, conservation andcharacterization of A. thaliana ecotypes from Italy. Samples from several populations were sampledand collected mainly from Southern Italy. Ten plants per each populations were grown understandard conditions in a greenhouse and phenotypic traits were scored during the variousphysiological phases. Variation analysis of these characters has allowed the identification of asubset useful to assemble a short descriptor list for rapid field evaluation of the collected samples.Addditional 10 plants per population were grown and DNA was extracted from rosette leaves. DNAquality and concentration were checked and DNA was stored at –80° C in order to start a DNAcollection implemented in the recently constituted DNA bank at the IGV, Bari.



ASSESSMENT OF GENETIC DIVERSITY IN ETHIOPIAN DURUMWHEAT

P. DONATI*, M. A. PAGNOTTA*, A. FARINA*, M. ATALLAH**, E. PORCEDDU*, **

*) Department of Agrobiology and Agrochemistry. University of Tuscia, Via S. De Lellis,01100 Viterbo, Italy - pagnotta@unitus.**) Sant’Anna Higher School, Pisa. International Ph.D Program in AgroBiodiversity,00057 Maccarese -Rome, Italy

SSR, durum wheat, germplasm, diversity

Durum wheat [Triticum turgidum ssp. durum (2n = 4x = 28)] had an important centre ofdiversification in Ethiopia, whose germplasm could provide useful breeding traits, including diseaseresistance, environmental stability, drought and low temperature stress tolerance.

The analysis of a collection of 234 durum genotypes from nine populations of three Ethiopianregions (Tigray, Gonder and Shewa) by 28 SSRs markers, randomly chosen in each chromosomearm, was run in order to define: a) population structure, b) the assessment of genetic variation, c)the relationships between and within populations, d) the presence of rare or unique genotypes. Theresults indicated the presence of great variation among regions and both among and withinpopulations, the presence of specific haplotypes and rare alleles. The number of alleles per locusranged from 1 to 10. Genetic distance between population indicated that the material from Tigray isfar apart, whereas populations from Gonder and Shewa were mixed up. The expected heterozigosityover all populations was, for most of the loci, around 0.5 indicating an equivalent distribution of thealleles in the populations; whereas the observed heterozygosity was on average 10%, indicating asignificant level of outcrossing.



BARLEY (HORDEUM VULGARE L.) FROM THE CENTRAL HIGHLANDSOF ETHIOPIA

T. TANTO*, D. RAU*, L. NANNI*, T. HODGKIN**, D. JARVIS**, R. PAPA*

*) Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, Via BrecceBianche, 60131 Ancona, Italy**) International Plant Genetic Resources Institute, Via dei Tre Denari 472/a, 00157 Maccarese(Fiumicino) Roma, Italy

barley, Belg, collection, Ethiopia, diversity, landraces, Meher

Ethiopia is a secondary centre of diversity for barley (Hordeum vulgare L.), where farmersmainly grow local landraces over a wide range of environments. Barley is the third most importantcereal crop after teff (Eragrostis teff) and maize (Zea mais). It is often grown in two differentplanting seasons (Meher and Belg) per year: during the long rainy season (June-September, the‘Meher’ season) and the short rainy season (February-April, the ‘Belg’ season).

The level and structure of seed flow between seasons can have important implications fordiversity. Two extreme scenarios can be considered: (1) the continuous use of seeds from oneseason for planting for the next season, doubling the number of generations per year, and (2) theseeds flow independently across years within the two seasons. The impact of evolutionary forces(drift, mutation migration and selection) will be different on the level and structure of diversity ofthe barley landraces in the different scenarios.

In 2005 we collected barley landraces in the central highlands of Ethiopia over the two maingrowing seasons. Thus, two collection expeditions were conducted, visiting the same sites in bothseasons. We documented the occurrence of barley landraces, seed selection and seed exchangeduring both the Belg and Meher seasons. A total of 101 farms were visited in the Shewa province(central highlands), 48 during the Belg growing season and 56 during the Meher season. In eachseason, three distinct districts were visited (Ankober, Mojanawadera, and Tarmaber) so that 16-18farms per district were considered. Collection site positions and the information from each farmwere recorded. Within each district, sampling was conducted along an altitude cline (from 1500 to3500 m a.s.l.). A total of 112 barley landrace populations were sampled on a single plant basis. Foreach cultivated plot, 100 spikes were randomly sampled all along the diagonal of the field, with theplants 5-10 m apart. Each collected spike was separately threshed and stored for further analysis.Before threshing, 30 spikes per population (for a total of 3450 spikes) were characterised for thefollowing eight morphological traits: kernel row number, spike density, lemma awn barbs, glumecolour, lemma type, length of rachilla hair, kernel cover, and lemma colour. The informationcollected during the expeditions and the results of the morphological characterization will bepresented and discussed.



GENETIC DIVERSITY AND INTROGRESSION IN MAIZE LANDRACESFROM CENTRAL-ITALY

E. BITOCCHI*, L. NANNI*, A. GIARDINI*, A. BUONAMICI**, G. G. VENDRAMIN**,R. PAPA*

*) Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, Via BrecceBianche, 60131 Ancona, Italy – [email protected]**) Plant Genetics Institute – CNR -Via Madonna del Piano 10, 50019 Sesto Fiorentino (Firenze)Italy

Zea mays L., landraces, assignment methods, population structure, introgression

Maize landraces are still cultivated in many countries in Europe and they are often linked tospecific adaptations to particular farming systems and environments. Some landraces are alsoassociated with the production of traditional local food. In Italy, the flint corn landraces are stillcultivated in most regions, particularly in the mountain areas where traditional farming is stillpractised. Landraces represent a valuable source of genetic variation and an important culturalheritage. In this study, we have investigated the population structure of maize landraces from theMarche region and have estimated the impacts of introgression from modern hybrid varieties. Weused assignment-based approaches that ascertain population membership of individuals or groups ofindividuals using genetic information. Two samples of maize landraces collected in two differentperiods, one in 1950 before the introduction of hybrids and the other in 2000, were compared with asample of the main temperate maize inbred lines and modern hybrid varieties. We have considered43 accessions of maize landraces from Marche collected in 1950, and 20 accessions collected in2000, with 11 accessions of landraces from North Italy that have been used for flint corn breeding,along with 8 flint and dent modern hybrid varieties, 22 main temperate corn inbred lines 19 dent, 2flint and 1 pop corn; for a total of 218 individuals. We have analysed all the material using 20microsatellite loci (2 per chromosome) and 170 AFLP markers. The data were analysed usingdifferent approaches. SSR data were analysed using the STRUCTURE software to infer the numberof populations comparing the posterior probability for the five different populations that wespecified a priori.

Most of the modern maize landraces appear to have maintained their genetic identity, despite50 years of wide cultivation of hybrid varieties; however, in several cases we also detected asignificant introgression from hybrid varieties.



GENETIC AND REPRODUCTIVE DIVERSITY WITHIN AND AMONGKENTUCKY BLUEGRASS (POA PRATENSIS L.) WORLDWIDEACCESSIONS

L. RAGGI*, E. ALBERTINI*, G. MARCONI*, T. SHARBEL**, M. FALCINELLI*

*) Department of Plant Biology and Agro-environmental and Animal Biotechnology,University of Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy**) Apomixis Research Group, Dept. of Cytogenetics and Genome Analysis, Leibniz Institut fürPflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben, Germany

Kentucky bluegrass, apomixis, SSR, genome size, flow cytometry

Kentucky bluegrass (Poa pratensis) is a hardy, persistent, and attractive forage and turf grassadapted to a wide range of environmental conditions in continental Europe and America. The greatadaptive capacity of this species is likely associated with its variable ploidy level and versatilemode of reproduction, ranging from obligate apomixis to complete sexuality. Understanding thebiogeographic distribution of ploidy variation and apomixis in P. pratensis will lead to insights onhow apomixis has evolved and spread in this species.

We report the characterization of 33 accessions of Kentucky bluegrass, including 24 wildpopulations from North America, Eurasia and Africa, and 8 cultivated varieties. For eachpopulation genome size was investigated using flow cytometry on leaf tissue, and ploidy level wasdetermined using karyology. Various DNA markers, including nuclear and chloroplast SSRs,chloroplast SNPs, and a candidate gene-derived SCAR marker, were analyzed to infer gene flowbetween reproductive forms, and the phylogeographic relationship between different populations.

These data are being used to understand the effects of genome size and ploidy on theexpression of apomixis, and to examine the evolutionary origin(s) of this trait in differentpopulations.



MARKER-TRAIT ASSOCIATIONS IN A COLLECTION OF DIVERSIFIEDTOMATO (SOLANUM LYCOPERSICUM L.) LANDRACES ANDCULTIVARS

A. MAZZUCATO*, E. BITOCCHI**, P. MOSCONI*, L. NANNI**, V. NEGRI***, R. PAPA**,M.E. PICARELLA*, F. SILIGATO*, G.P. SORESSI*, B. TIRANTI***, F. VERONESI***

*) Department of Agrobiology and Agrochemistry, Università degli Studi della Tuscia, Via S.C. deLellis s.n.c., 01100 Viterbo, Italy – [email protected]**) Department of Food Science, Università Politecnica delle Marche, Via Brecce Bianche s.n.c.,60100 Ancona, Italy - [email protected]***) Department of Plant Biology and Agro-environmental Biotechnology, Università degli Studidi Perugia, Borgo XX Giugno 74, 06100 Perugia, Italy - [email protected]

association analysis, genetic diversity, germplasm, landraces, tomato

The tomato (Solanum lycopersicum L.) has long served as a model species in the study of fruitgrowth and development. Conventional quantitative trait loci (QTL) analysis led to theidentification of many of the regions controlling complex traits such as fruit size, shape and quality.In addition to genotyping suitable segregating populations, the identification of QTLs for importanttraits may be addressed through association genetics, after the phenotypic and genotypiccharacterization of germplasm collections, provided that a suitable amount of phenotypic andmolecular variation is available.

In this research, we exploited the genetic diversity maintained in Italian landraces and inselected cultivars to implement association mapping in tomato addressing plant, flower and fruitcharacters. Fifty-nine accessions highly diversified as for fruit size, shape and destination.werephenotyped for 15 morpho-physiological traits and genotyped at 20 microsatellite (SSR)polymorphic loci. SSR markers were selected in order to obtain a rough coverage of the tomatogenome; in addition, nine SSRs were linked to regions harboring reported QTLs affecting fruit sizeand/or shape (Q-SSRs), whereas 11 had no a priori known linkage with genes affecting fruit traits(NQ-SSRs).

Significant marker-trait associations were first detected by a non-parametric test. The matrixrevealed 29 significant combinations, among which 22 (15.6%) were due to Q-SSRs and seven(4.2%) to NQ-SSRs. When only traits directly describing fruit dimensions and shape wereconsidered, the proportion of significant associations revealed by Q-SSRs raised to 28.6%, whereasthe other groups of combinations ranged between 3.4 and 5.6%. Results obtained in parallel byGLM analysis widely overlapped those revealed by non-parametric test. In some cases associationswere obvious if population structure among accession was factored into the analysis, in others theassociations were structure-independent pointing to previously reported QTLs or to newassociations between markers and phenotypes.

Although based on a relatively small number of accessions and marker loci, the studydemonstrates that the level of diversity encountered in this material made it very attractive toimplement association analysis in tomato and that such analysis may represent a useful tool for both

validating the function of known QTLs and for highlighting genetic effects not previously reported.Such power was evident because the proportion of significant associations was much higher whenmarkers chosen as localized near QTLs controlling the trait were compared with variables related tothe trait itself. It can therefore be reasonably predicted that a suitable choice of markers, accessions,and traits may provide an analytical platform useful to get insights into the function of specificQTLs over the phenotypic germplasm variation. To translate this information into tools for markerassisted breeding would then be straightforward.



TOWARDS GENETIC ANALYSIS FOR CHLOROGENIC ACID CONTENTON AFRICAN EGGPLANT BY USING A LARGE BIODIVERSITY

F. SUNSERI*, V. ALBA**, C. LOTTI***, A. D’ALESSANDRO****, G. MENNELLA****,G.B. POLIGNANO*****, E. ALBA*, L. RICCIARDI**

*) Dip. Biologia, Difesa e Biotecnologie AgroForestali – Università degli Studi della Basilicata**) Dip. Biologia e Chimica Agro-Forestale ed Ambientale – Università degli Studi di Bari***) Dip. Scienze Agroambientali, Chimica e Difesa Vegetale – Università degli Studi di Foggia****) CRA - Istituto Sperimentale Orticoltura – Pontecagnano (SA)*****) Istituto Genetica Vegetale – CNR (BA)

Solanum aethiopicum L., molecular markers, cluster analysis, chlorogenic acid, HPLC

The scarlet eggplant (Solanum aethiopicum group gilo L.) and the gboma eggplant (Solanummacrocarpon L.) are usually named as african eggplant in that Continent (Lester et al. 1990).Scarlet eggplant is rarely spread and cultivated in Europe, despite its berries are edible such as thecultivated eggplant (Solanum melongena L.). Scarlet eggplant is instead very spread in manyAfrican areas where it can be consumed as source of edible leaves and fruits, both for nutritionaland wealthy purposes. Unfortunately, the statistics on spreading and productivity of this specie arenot easily available such as the cultivated eggplant (Solanum melongena L.). Scarlet or red eggplantis considered of great interest for its typical productions in marginal areas in Europe or in otherContinent. This species is also of interest for genetic improvement of related cultivated species(Solanum melongena L.), considering the presence in its germplasm of several traits of agronomicinterest. Important research results have been reported in order to transfer in cultivated eggplant thetolerance to Fusarium spp., by using somatic hybridization techniques considering the interspecificsexual incompatibility between S. melongena and S. aethiopicum. The level of antioxidant insubstances are now in progress, in order to transfer and identify the responsible genes of these traitsinto cultivated eggplant. Eggplant is known as a vegetable with high level of phenolic constituents.Several potential health promoting effects have been ascribed to plant phenolic phytochemicals. Afirst report on phenolic acid constituents in eggplant fruit from accessions in the USDA germplasmcollections were reported (Stommel and Whitaker, 2003), and differences in phenolic acid contentwere evident between the species and among genotypes within species.

Starting from the collection of scarlet eggplant of different origin eighth accessions wereselected and analysed by means of morphological traits, molecular markers and for the level ofchlorogenic acid in their berry. Genetic characterization of the collection confirms the geneticorigin of the accessions and the wide diversity among gilo genotypes, already revealed onmorphological traits basis.

Phenolic acid compounds separated by HPLC were tentatively identified as hydroxycinnamicacid (HCA) derivatives based on HPLC elution times, UV absorbance spectra. These phenolicswere grouped into chlorogenic acid isomers and isochlorogenic acid isomers. The total HCAcontent in scarlet eggplant was low relative to cultivated eggplant. Important differences in totalHCA content were detected among the scarlet eggplant accessions. A segregant intraspecificpopulation was obtained starting from low and high chlorogenic acid content genotypes in order to

map microsatellites and AFLP markers associate to the trait. The results will represent aninteresting tool in order to utilize interspecific hybrids obtained by somatic hybridization(considering the sexual incompatibility between S. melongena and S. aethiopicum) to characterizethis interesting trait also in cultivated eggplant.

ReferencesLester RN, Jaeger PML, Bleijendaal-Spieringe BHJ, Bleijendaal HPO, Holloway HLO

(1990). African eggplants - a review of collecting in West Africa. Plant Genetic ResourcesNewsletter 81/82: 17-26.

Stommel JR, Whitaker BD (2003). Phenolic Acid Content and Composition of Eggplant Fruitin a Germplasm Core Subset. J Amer Soc Hort Sci 128: 704-710.



TUSCANY BEANS LANDRACES, ON-LINE IDENTIFICATION FROMSEEDS INSPECTIONS BY IMAGE ANALYSIS AND LINEARDISCRIMINANT ANALYSIS

G. VENORA*, O. GRILLO*, C. RAVALLI*, R. CREMONINI**

*) Stazione Sperimentale di Granicoltura per la Sicilia, Via Rossini 1, 95041 Caltagirone (CT)**) Dipartimento di Biologia, Università di Pisa, Via L. Ghini 5, 56126 Pisa

biodiversity, image analysis, landraces, Phaseolus vulgaris

Biodiversity has contributed in many ways to the development of human culture and theprotection of biodiversity is one of the most important targets for the humanity. Therefore it isimportant to safeguard and to exploit agricultural biodiversity in order to produce and sellpeculiar products for high and characteristic quality which are property of specific regions of acountry. The long tradition of Phaseolus coltivation in Italy has permitted the evolution ofmany landraces which are adapted to microclimates in restricted growing areas and theselandraces warrant a good production. Therefore a matter of primary importance his to evaluate,characterize and protect the native germplasm and in this connection it is necessary to finddifferent parameters as morphological and molecular in order to get a “market card” forlandraces bean.

The first step must be seed identification and it is important to implement repeatable andquick automated method to identify and classify seeds. The determination, by an image analysissystem, of parameters as size, shape, colour and texture of seeds represent a non-destructivemethod to differentiate landraces. In this case, the mechanic vision field, for instance, imageanalysis algorithms implemented by classification statistical methods, is useful for automaticseed identification since artificial vision system is more accurate and efficient in measuring theseed parameters than inspectors with high experience.

We have realised a macro Bean-mcr to determine parameters referring both colour and sizeof entire seeds and peculiar spots in beans using an image analysis library KS-400 V 3.0 (C.Zeiss, Germany).

The parameters measured in this way, 26 quantitative variables, allowed to realize astatistic classifier, able to discriminate 100% samples of training set and 98.92% of test set thatis eleven different Tuscany bean landraces.



GENETIC DIVERSITY OF LOTUS CORNICULATUS BASED ON SSRsMARKERS

M. L. SAVO SARDARO*, E. TAVAKOL*, M. ATALLAH*, E. PORCEDDU*,**

*) Sant’Anna Higher School, Pisa, International Doctoral Program in Abrobiodiversity, Maccarese,Italy**) University of Tuscia, DABAC, Viterbo, Italy

Lotus corniculatus, SSRs, genetic diversity

Lotus is a large polymorphic and widely distributed genus, that comprises approximately 200annual and perennial species. Birdsfoot trefoil (Lotus corniculatus L.) is a strongly cross-pollinatedspecies. It is the most agriculturally important species of the genus, since it has a high nutritivevalue and is non-bloating when grazed directly by livestock. Birdsfoot trefoil is native to Europeand western Asia, with a probable regional centre in the Mediterranean basin, where the greatestdiversity of species occurs.. The range of phenotypes found in birdsfoot trefoil is believed to be theresult of adaptation to the environments in which it is grows and through continual intraspecifichybridization. It is also emerging species in several Mediterranean environment, since it appears tohave potentiality to develop new perennial legume cultivars for phase farming to reduce drylandsalinity. Moreover, birdsfoot trefoil is poorly studied and little information is available on thegenetic variation in the species. SSR markers, previously identified in Trifolium pratense and Lotusjaponicum, were used to assess the genetic diversity among 11 Lotus corniculatus naturalpopulations collected in Central and Southern Italy. High polymorphism was found, both within andbetween accessions, with populations from Southern Italy clearly different and identifiable.



EVALUATION OF SNOW CLOVER (TRIFOLIUM PRATENSE SUBSP.NIVALE) GERMPLASM NATIVE TO THE RETIC ALPS, FOR THERESTORATION OF DEGRADED LANDSCAPES AT HIGH ALTITUDE

M. ROMANI, L. PECETTI, R. PAOLETTI, L. DE ROSA, E. FRANZINI, E. PIANO

CRA-Istituto Sperimentale per le Colture Foraggere, Lodi, Italy

genetic resources, restoration, site-specific species, ski runs, snow clover

In the last 50 years, the Alps have experienced an extensive development of ski runs and otherinfrastructures for winter tourism. The need of an ecological restoration of the disturbed areas hasarisen, to limit or prevent environmental problems due to soil erosion, increased water runoff,depletion of the native flora, etc. The revegetation of degraded landscapes at high altitude requiresplant material able to meet the technical purposes of the restoration, adapted to the peculiar pedo-climatic conditions and compatible with the existing ecosystems (particularly in wilderness areas).Some key species, including snow clover, have been identified that may act as ‘starters’ in theprocess of renaturation, favoring the natural reentry of a wider range of species. Information isneeded on these key species to define the existing level of variation in the native germplasm, thepresence of superior variants for a possible selection, and the optimal environment for seedmultiplication in view of their commercial distribution. A collection of snow clover was carried outin the Retic section of Alps, in the framework of the Project SEMENSCI funded by the RegionLombardy, which aims at implementing the production of seed of local germplasm suitable for therestoration of degraded areas at high altitude. Three valleys were explored above 1800 m a.s.l., and12 populations were collected altogether, five each from Upper Valtellina and Valchiavenna andtwo from Valmalenco. Two evaluation fields were established for this material, in a mountain (1300m a.s.l.) and in a lowland location (81 m a.s.l.), respectively, to assess any interaction effectbetween the alpine germplasm and the site of growth, with a special emphasis on seedmultiplication in areas different from those of origin. So far, data from the lowland location only areavailable. Variation among and within valleys was examined for traits of interest in landscaperestoration, such as plant morphology, phenology and seed yield. Significant differences amongvalleys were found for flowering time, number of seeds per plant and susceptibility to mildew.Upper Valtellina and Valmalenco had contrasting behavior, with the latter showing earlier blossom,lower disease susceptibility and higher seed yield. The estimate of the variance component ‘amongpopulations within valleys’ was greater than the component ‘among valleys’ for all traits.According to this finding, exploring several sites within valleys may be more effective thanexploring several valleys to gather variable material for breeding exploitation. The greatest within-valley variation was observed in Valchiavenna. The frequency of plant flower color and growthhabit also displayed great variation among valleys, with Upper Valtellina showing 95% of plantswith white flowers, versus 74% in Valmalenco and 45% in Valchiavenna, and Valmalenco showing51% of plants with prostrate or semi-prostrate habit, versus 37% in Valchiavenna and 21% in UpperValtellina. Information on the species’ mating system was also obtained in this study, highlightingits dependence from pollinators to set seed, and suggesting a lack of self-tripping or an almost

complete self-incompatibilty. These results are just preliminary and need to be confirmed in thehigh-altitude evaluation site. Nonetheless, there appears to be an interesting variation in the geneticresources of snow clover from the Retic Alps, which is a pre-requisite for the selection of the mostinteresting germplasm. Promising populations were noted that were able to combine positive traits,such as early flowering, high vigor, great seed yield and good tolerance to mildew.



GENETIC DIVERSITY IN A COMMON BEAN (PHASEOLUS VULGARIS L.)EX SITU COLLECTION OF ITALIAN LANDRACES

B. TIRANTI*, L. MACALUSO**, P.L. SPAGNOLETTI ZEULI**, V. NEGRI*

*) Department of Plant Biology and Agro-Enviromental and Animal Biotechnology (DBVBAZ),University of Perugia, Borgo XX Giugno 74, 060121 Perugia, Italy - [email protected]**) Dipartimento di Biologia, Difesa e Biotcnologie Agro-Forestali, Università degli Studi dellaBasilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy - spagnoletti @unibas.it

genetic diversity, landraces, Phaseolus vulgaris, phaseolin, SSR marker

Landraces (LRs) are vital genetic resources for breeding purposes, diversification ofproduction, developing new farming systems and new quality products. The extent and distributionof the genetic diversity in a crop depends on its breeding system, geographical, ecological andhuman factors. Conservation of genetic variability is essential for present and future human well-being.

To date, the in situ or ex situ conservation strategies have been applied with little informationon the genetic diversity that was being conserved. In order to improve conserved germplasmmanagement, it is necessary to understand the genetic diversity that is present in collections.

Common bean LRs have been obtained from Italian farms and local markets, mostly located inCentral Italy, and their seeds samples were used to establish an ex situ collection in DBVBAZ. Inthis study the amount of genetic diversity and its distribution in 159 Italian LRs were assessed usingdifferent approaches that included morphological (international descriptors), biochemical (phaseolinseed proteins) and molecular analysis (Simple Sequence Repeats markers). Results obtained showeda wide variation overall morphological traits, especially seed characters. The three major phaseolintypes were found, ‘C’ (38.9%), ‘S’ (33.1%) and ‘T’ (28.0%) types. Nine of ten loci analysed werepolymorphic and 82 different alleles were detected overall SSR loci.

Our findings on the extent and distribution of different aspects of genetic diversity in thisItalian common bean LRs collection is an essential prerequisite to determinate what to conserve andhow to conserve it. In addiction all the information collected will offer the opportunity to rationalizethe collection, to develop a core collection and to exploit these resources for valuable traits.



ANALYSIS OF THE CONTRIBUTION OF MESOAMERICAN ANDANDEAN GENE POOLS TO EUROPEAN COMMON BEAN (PHASEOLUSVULGARIS L.) GERMPLASM

G. LOGOZZO*, R. DONNOLI**, M. DILUCA**, R. PAPA***, P. SPAGNOLETTI ZEULI**

*) Centro Interdipartimentale per la Salvaguardia delle Risorse Genetiche Vegetali, Universitàdegli Studi della Basilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy [email protected]**) Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Università degli Studi dellaBasilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy - [email protected]***) Dipartimento di Scienze degli Alimenti, Facoltà di Agraria, Università Politecnica delleMarche, Via Brecce Bianche, I-60131 Ancona, Italy - [email protected]

Phaseolus vulgaris, phaseolin, European gene pool, genetic resources, SDS-PAGE

The genus Phaseolus has contributed crop plants to agriculture in both the New and the OldWorlds. Phaseolus beans are valued grain legumes or pulse crops in many tropical countries usuallyconsumed as dry beans whereas, in temperate countries, varieties for fresh pod consumption and forprocessing as frozen vegetables have also been developed. Archaeo-morphological, biochemicaland molecular evidence suggest that common bean (Phaseolus vulgaris L.) was independentlydomesticated in Andean and Mesoamerican centres of origin As showed by a consisted set ofobservations based on morphology, hybrid inviability, biochemical and molecular markersdomestication occurred independently in Mesoamerica and the Andes leading to two differentdomesticated gene pools. Compared to the Mesoamerican gene pool the Andean gene pool ischaracterized by larger seeds and by two major phaseolin types (‘T’ and ‘C’) while the ‘S’ type isthe prevalent one in Mesoamerica. Due to adaptation to new ecological and man-made conditions, alarge diversity evolved in European germplasm that is a particular interest for plant breeding.Because of the distinct evolutionary histories, genetic differences are expected to be larger betweenaccessions that originated from the two different American gene pools.

In this study, the distribution of Andean and Mesoamerican gene pools within Europeangermplasm collection (n=544) has been studied, including for the first time accessions from almostall European countries (n=24), on the base of phaseolin electrophoretic (SDS-PAGE) patterns andseven seed characters (three quantitative and four qualitative).

Results showed that the Andean phaseolin types ‘T’ (45.6%) and ‘C’ (30.7%) prevailed on theMesoamerican ‘S’ (23.7%), and accessions with cuboid seed shape (34.9%), maroon seed coatdarker colour (44.3%), uniform seed colour (69.6%) were the most frequent. European accessionswith phaseoline ‘S’ showed on average significantly larger seed size compared to those fromAmerica in the same phaseolin class while those presenting ‘T’ and ‘C’ phaseoline did not. Thissuggests that, during crop expansion in Europe, sampling or selection favoured the large seeds raceswithin the Mesoamerican ‘S’ gene pool or, possibly, introgression from Andean germplasm didoccur.



GENETIC DIVERSITY AND HYBRIDS IDENTIFICATION WITHIN THESERAPIAS GENUS USING SSR MARKERS

M. ATALLAH*, T. KARTHIKEYAN*, M. L. SAVO SARDARO*, B. ARACRI**,E. PORCEDDU*,***

*) Sant’Anna Higher School, Pisa, International Doctoral Program in Abrobiodiversity, Maccarese,Italy**) Floramiata S.p.A. Località casa del corto, Piancastagnaio, Siena***) University of Tuscia, DABAC, Viterbo

Serapias, SSR, genetic diversity

The Serapias genus is terrestrial and some of its species are distributed in the Mediterraneanregion, including Italy. Its taxonomy is rather controversial. Some authors distinguished seven -nine species, whereas others recognized 17 species and three subspecies. In addition, most ofSerapias species are endangered because of the rapid disappearance of the scanty meadows wherethey live. SSR markers, previously identified in S. vomeracea, were used in assessing the diversityamong Italian Serapias species S. lingua, S. parviflora, S. vomeracea, S. apulica, S. nurrica, S.neglecta, S. cordigera, S. politisii, and in identifying markers for the identification of inter-specifichybrids between them. The six SSR markers used showed a significant variation among species andthe possibility of identifying hybrids and their parents.



VALORIZATION OF A VENETO GRAPEVINE GERMPLASMCOLLECTION BY MOLECULAR CHARACTERIZATION ANDBIOCHEMICAL ANALYSIS OF RESVERATROL COMPOUNDS

M. SALMASO*, C. FORESTAN*, F. MATTIVI**, M. LUCCHIN*

*) Department of Environmental Agronomy and Crop Production. Agripolis, University of Padova(PD)**) Istituto Agrario di San Michele all'Adige (IASMA), Centro Sperimentale, Via E. Mach 2,38010 San Michele all'Adige (TN)

grapevine, SSRs, Resveratrol, local varieties

Grapevine is the most important perennial crop worldwide. The world’s collections of grapeplant material are estimated to contain about 12000–15.000 cultivars. Many of these are ancient andautochthonous cultivars still not registered in the ampelographic national catalogues and thereforerisking the extinction. In order to safeguard this germplasm a correct classification solving theproblems of synonymous and homonymous in the cultivar designations is requested.

The identification and comparison of plant material by ampelographic methods is liable tomisinterpretations, while DNA-based markers provide a more reliable alternative for cultivaridentification. To preserve the genetic variability of local germplasm we initiate the characterizationof 30 North-Eastern cultivars at 23 microsatellite loci. The markers were proved to be informativein the grapevine cultivars. The genetic profiles of all 30 cultivars were searched for possible parent-offspring groups and several cases of suspected synonyms have been investigated.

The valorization of this germplasm was implemented by biochemical analysis of resveratrol(trans-3, 4 , 5-trihydroxystilbene) content. This stilbenic compound is a phytoalexin protectinggrapevine against fungal infections. The recent years have also witnessed intense research aimed toclarify the role of stilbenes (that are also present in wines) and, among them, resveratrol, in humanhealth because of their protective effects against cardiac decompensation and cancer. The potentialtherapeutic value of resveratrol has stimulated research activities on the occurrence of thismolecules in grapes and wines.



MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OFCELERY LANDRACE FROM CENTRAL ITALY, APIUM GRAVEOLENS L.VAR. DULCE (MILLER) PERS.*

G. CASTELLINI, R. TORRICELLI, E. ALBERTINI, M. FALCINELLI

Department of Plant Biology and Agro-environmental and Animal Biotechnology, University ofPerugia, Borgo XX Giugno 74, 06121 (Italy) - [email protected]

biodiversity, landrace, Apium graveolens, AFLP, cluster analysis

Reducing biodiversity between and within species is one of the most worrying environmentalproblems. Landraces are a precious repository of valuable traits and genetic diversity. A newstudied landrace of Apium graveolens L. var. dulce (Miller) Pers. is well-known like “black celeryfrom Trevi”, registered in the list of typical local products from the Umbria region (Italy). The nameblack of this celery comes from the wild physiological trait to maintain green rib colour (not self-blanching) if not subjected to agronomic whitening treatment. The objective of this study was togenetically characterize both morphologically and molecularly this celery landrace from the severalélite celery varieties cultivated within the Trevi valley. This was done by using the UPOV (UnionInternationale pour la Protection des Obtentions Végétales) guidelines for morphologicalcharacterization and Amplified Fragment Length Polymorphism (AFLP) markers. Geneticrelationships were estimated in six accessions of black celery and four élite celery varieties. Celerywild relative, Apium nodiflorum (L.) Lag., and the related species Petroselinum sativum Hoffm.(parsley) were also analyzed. Morphological characterization of celery accessions was carried out inTrevi during summer 2004. The on farm field trial was made using a randomized blocks strategywith four replicates (sixteen plants for each plot). Morphological distances among populations wereestimated employing nine quantitative and four qualitative traits. For each accession, 5 DNA bulkswere created and used for AFLP analyses using 9 EcoRI/MseI primer combinations. A total of 568bands were detected, of which 305 (53.7%) were polymorphic. Polymorphic AFLP fragments usedto calculate the Jaccard’s coefficient of genetic similarity. Morphological and molecular datasetswere also used to perform Univariate (ANOVA, ANalysis Of VAriance) and multivariate (PCA,Principal Component Analysis and PCOORDA, Principal COORDinates Analysis; UPGMA,Unweighted Pair Group Method with arithmetic Average and AMOVA, Analysis of MOlecularVAriance) statistical analysis. The six landraces populations were separated from the cultivars andshowed to belong to the same cluster and each of them was distinguished from the other landracespopulations. The germplasm of A. nodiflorum and P. sativum is distinct from the cultivated celeryforms. All celery germplasm has been collected and preserved (ex situ conservation). Broadening ofcelery collections in genebanks and detection of new genetic resources are vital for improvementsin celery breeding. On farm conservation of celery landraces is essential to protect biodiversity.

* Research project funded by Regione dell’Umbria, Programma Interregionale “Svilupporurale” – Sottoprogramma “Innovazione e ricerca - Azioni di Innovazione e ricerca a supporto delPiano sementiero”



ANALYSIS OF SPATIAL GENETIC STRUCTURE IN AN EXPANDINGPINUS HALEPENSIS POPULATION REVEALS DEVELOPMENT OF FINE-SCALE GENETIC CLUSTERING OVER TIME

D. TROUPIN*, R. NATHAN*, G.G. VENDRAMIN**

*) Movement Ecology Laboratory, Department of Evolution, Systematics and Ecology, TheHebrew University of Jerusalem, Edmond J. Safra Campus at Givat Ram, 91904 Jerusalem, Israel**) Plant Genetics Institute, Florence division, National Research Council, 50019 Sesto Fiorentino,Firenze, Italy

spatial genetic structure, spatial autocorrelation, Pinus halepensis, population expansion,microsatellites

We analyzed the change of spatial genetic structure (SGS) of reproductive individuals overtime in an expanding Pinus halepensis population. To our knowledge, this is the first empiricalstudy to analyze the temporal component of SGS by following the dynamics of successive cohortsof the same population over time, rather than analyzing different age cohorts at a single time. SGSis influenced by various factors including restricted gene dispersal, microenvironmental selection,mating patterns and the spatial pattern of reproductive individuals. Several factors that affect SGSare expected to vary over time and as adult density increases. Using air photo analysis, tree-ringdating and molecular marker analysis we reconstructed the spread of reproductive individuals over30 years beginning from five initial individuals. In the early stages, genotypes were distributedrandomly in space. Over time and with increasing density, fine-scale (<20m) SGS developed andthe magnitude of genetic clustering increased. The SGS was strongly affected by the initial spatialdistribution and genetic variation of the founding individuals. The development of SGS may beexplained by fine-scale environmental heterogeneity and possibly microenvironmental selection.Inbreeding and variation in reproductive success may have enhanced SGS magnitude over time.



GENETIC UNIFORMITY OF A WIDESPREAD MEDITERRANEAN TREE,PINUS PINEA L.

F. SEBASTIANI*, A. BUONAMICI*, F. PINAZUATI*, B. FADY**, R.J. PETIT***,I. SCOTTI****, M.L. RACCHI*, G.G. VENDRAMIN*****

*) DiBA,Genexpress, Università degli Studi di Firenze, Italy - [email protected]**) INRA, Avignon, France ***) INRA, Cestas Cedex, France****) INRA, Kourou, French Guyana*****) Istituto di Genetica Vegetale, CNR, Firenze, Italy - [email protected]

phylogeography, microsatellites, EST, diversity, human impact

Phylogeographic studies have used current geographical patterns of genetic diversity to inferthe post-glacial history of many tree species. For domesticated species, this inference may beincorrect as a result of human practices in a more recent past. We used 12 chloroplastmicrosatellites (cpSSRs) to estimate the among and within population genetic variation of 50 Pinuspinea L. populations representing the species’ current Mediterranean distribution area. A singlehaplotype was detected in all populations studied except in Lebanon and in two populations fromSpain, where 3 and 1 additional haplotypes at very low frequencies were found, respectively. Theresults found in P. pinea are therefore unique and may reflect the recent diffusion of the specieswestward across the Mediterranean, along with the beginning of offshore sailing and of long-distance trade in the Basin.. Confined to a narrow distribution range during successive glacial-interglacial cycles, P. pinea may (or not) have experienced further reduction of its genetic diversitywith the emergence of traditional agriculture and long distance trading. In order to deeplyinvestigate the genetic uniformity of P. pinea, new nuclear markers were developed. Sinceexpressed sequence tags (ESTs) offer an interesting source for marker discovery, a non-normalizedcDNA library was constructed from mRNA of young P. pinea needles. 1000 ESTs were sequencedto characterize the gene content of the library and a set of EST-SSR were developed and optimisedand then used to screen diversity in the same populations. These results are presented and discussed.



PRELIMINARY STUDIES ON GENETIC VARIABILITY OF QUERCUSPUBESCENS WILD POPULATIONS IN SICILY

A. DE CARLO*, C. VETTORI*, L. SAPORITO**, R. GIANNINI*

*) Istitute of Plant Genetics – CNR, Research Division Florence, Via Madonna del Piano 16,50019 Sesto Fiorentino (FI), Italy – [email protected]**) Regione Siciliana, Azienda Regionale Foreste Demaniale, Via della Libertà 97, 90143 Palermo,Italy

Quercus pubescens, nuclear microsatellites, conservation

In Sicily the distribution of Quercus pubescens Willd. is scattered and covers about 15.000hectares. The most important populations are located in Madonie (PA), Nebrodi (ME), Iblei (SR),and in Ficuzza Forest (PA). These forests are exsposed to the risk of genetic erosion in consequenceof small population size.

Because the presence and maintenance of genetic variation is a prerequisite for their ability tosurvive in heterogeneous temporal and spatial conditions, and to preserve their adaptability forfuture generations, in the present work, we have examined the ability of nuclear microsatellites in:i) genotyping the populations; ii) exploring the genetic variability among and within thesepopulations.

Several SSR primer sets, previously developed for Quercus spp., are tested for amplification onQ. pubescens and 4 SSRs have been selected.

Individuals to be sampled from these populations have been chosen following themorphologycal characters previuosly described for this species by Tutin et al. (1993) and Bruschi etal. (2000).

Preliminary results indicate that the genetic diversity was found within rather than amongpopulations like observed for others forest populations.

Morever, all populations have been identified by the presence of unique allele; for example, thepopulation of Madonie was characterized by allele 120 (locus 1), by alleles 226, 230 and 222 (locus2), by allele 228 (locus 3) and by allele 206 (locus 4). The population of Nebrodi was identified byallele 115 (locus 1), allele 218 (locus 2), alleles 208 and 220 (locus 3) and by alleles 228 and 210(locus 4).



LEADING SWEET CHERRY APULIAN CV. FERROVIA (PRUNUS AVIUML.) APPEARS GENETICALLY AKIN TO SEVERAL CENTRAL-EASTERNEUROPEAN CULTIVARS

M.A. PALASCIANO*, A. GODINI**, G. FANIZZA*, M. PALASCIANO**, P. RESTA*

*) Department of Agro–Forest and Environmental Biology and Chemistry (DiBCA),University of Studies of Bari, Via Amendola 165/A, Bari, Italy – [email protected]**) Department of Crop Sciences, University of Studies of Bari, Via Amendola 165/A, Bari, Italy

cultivar identification, similarity relationships, UPGMA, genetic diversity, AFLP

Genetic diversity studies in fruit trees can provide useful information for parental selection inbreeding programs, optimization of collections, and identification of synonymous denominations.DNA comparisons alone (i.e. without morpho-physiological descriptors) might not display the fullextent of genetic diversity present in reference collections or in a germplasm. Yet, DNA analysesare very powerful for the breeder in confirming or undermining expected links or associationsand/or pinpointing unforeseen relationships. Furthermore, DNA fingerprints can be a rapid andprecise tool in the field of genetic correspondence analyses, if the error associated with spuriousbands, subjective interpretation and casual association is taken into account.

The Province of Bari (in the Apulia region) stands out nationwide for sweet cherry productionand export, by growing a few key cultivars of diverse origins and a large mix of locally diffuseddenominations. Thus, a representative sample of local germplasm was compared by AFLPs withselected cultivars from France, Germany, Hungary, and Canada, chosen either because they arelocally grown or because of testing purposes (34 cultivar denominations).

Total leaf DNAs from the cherry tree collection gathered and maintained by the Department ofCrop Sciences of Bari were isolated and amplified with 13 primer combinations labeled with 33P,followed by gel-electrophoresis fragment separation and visual band scoring on autoradiographs. Aconservative scoring yielded 168 AFLP polymorphisms (a hundred without P. mahaleb), and their0/1 matrixes were analyzed with NTSYS-pc by UPGMA dendrograms of simple matching (SM)similarities. Internal controls were: i) an outgroup species (Prunus mahaleb, widely used in Apuliaas rootstock); ii) cultivars with a known family tree (Sunburst, plus its female and male parents Vanand Stella); iii) replicated samples (DNAs isolated from the same tree at different times).

As expected, inclusion or exclusion of P. mahaleb, distantly related to P. avium, had a drasticeffect on the absolute SM values only, and not the relative (within-cherry) SM values, and thus thedendrogram topologies were unchanged. The exclusion reduced the cophenetic correlation from r=0.98 to r= 0.89. Cultivars grouped at all the levels of similarity, up to 100%, and were thusclassified as ‘alike’, ‘related’, and ‘unrelated or undetermined’. Overall, more agreements withindependent observations were found than disagreements. In particular, four clusters were found atvery high similarity values, close to being identical. One of them matched Ferrovia, a well-knownlocal cultivar, with six other denominations, Schneiders, Germersdorfer and Badacsony, which arediffused in central and eastern Europe, thus, providing strong evidence that the Ferrovia genotype is

not autoctonous from Apulia. Scoring approach, experimental error, and independent evidences arediscussed.



SPATIAL DISTRIBUTION OF GENETIC VARIATION IN NATURALSILVER FIR (ABIES ALBA MILL.) STANDS BASED ONMICROSATELLITE MARKERS

M.-T. SCARANO*,**, A. BUONAMICI***, F. SEBASTIANI***, F. PILLA*, S. REALE*,R. TOGNETTI****, G.G. VENDRAMIN*****

*) Dipartimento di Scienze Animali, Vegetali e dell'Ambiente, Università degli Studi del Molise,Via De Sanctis, 86100 Campobasso**) Istituto di Genetica Vegetale, CNR, Corso Calatafimi 414, 90129 Palermo***) Dipartimento di Biotecnologie Agrarie, Genxpress, Università degli Studi, Via della Lastruccia13, 50019 Sesto Fiorentino (FI)****) Dipartimento di Scienza e Tecnologie per l’Ambiente e il Territorio, Università degli Studidel Molise, Località Fonte Lappone, 86090 Pesche (IS)*****) Istituto di Genetica Vegetale, CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI)- [email protected]

Abies alba Mill., in situ conservation, microsatellites, spatial autocorrelation

The spatial distribution of alleles is described in four natural silver fir (Abies alba Mill.) standslocated in Molise, within a large wilderness area of south-central Appennine mountains. Samplingconsisted of a total of about 150 adult trees. Each tree was genotyped at six well-scorable, highlypolymorphic, nuclear microsatellite loci. For the characterization of spatial genetic structures, twodifferent statistics were used. One method is based on the multivariate autocorrelation procedure,which strengthens the spatial signal and reduces the allele to allele stochasticity and locus to locusnoise, and the other one based on the 'Sp' statistic which is less sensitive to the sampling designused and allows the comparison of spatial genetic structure (SGS) magnitude among differentpopulations. The results show the same tendency of a family structure in the distance classes up to20 meters in comparison with that expected for a spatially random distribution of genotypes. Spatialgenetic structures are influenced by unpredictable factors such as restricted gene dispersal,microenvironmental selection, mating patterns and wind direction. Recommendations about seedcollections that should cover large areas in order to prevent a preponderance of few families and areduction of the adaptive potential of the next generation are provided.



CORK OAK DISPLAYS A STRONG GENETIC STRUCTURE:A PHYLOGEOGRAPHIC STUDY USING CHLOROPLAST MARKERS

S. FINESCHI*, D. MAGRI**, M.C. SIMEONE***, R. BELLAROSA***, B. SCHIRONE***,F. SEBASTIANI****, A. BUONAMICI****, G.G. VENDRAMIN*****

*) Consiglio Nazionale delle Ricerche, Istituto per la Protezione delle Piante, Sesto Fiorentino**) Dipartimento di Biologia Vegetale, Università di Roma “La Sapienza”, Roma***) Dipartimento di Tecnologie, Ingegneria e Scienze dell’Ambiente e delle Foreste, Universitàdella Tuscia, Viterbo****) Dipartimento di Biotecnologie Agrarie, Università degli Studi di Firenze, Sesto Fiorentino*****) Consiglio Nazionale delle Ricerche, Istituto di Genetica Vegetale, Sesto Fiorentino

Quercus suber, chloroplast microsatellites

Cork oak (Quercus suber L.) is an emblematic Mediterranean sclerophyllous tree, with amodern discontinuous distribution extending from the Atlantic coasts of North Africa and IberianPeninsula to the southeastern regions of Italy, and including the main west-Mediterranean islands aswell as the coastal belts of Maghreb (Algeria and Tunisia), Provence (France) and Catalonia(Spain). We have analysed by chloroplast DNA markers (microsatellites) of 110 populationsthroughout the distribution range. Eight microsatellite primers out of fourteen turned out to bepolymorphic and led to the identification of five different haplotypes. The distribution of thesehaplotypes reveals a strong geographic structure, as demonstrated by the high value of geneticdifferentiation between populations for unordered alleles: GST= 0.965 (s.e. 0.0155) as well as forordered alleles NST = 0.962 (s.e. 0.0180). Most populations are fixed for one haplotype. Only fourpopulations (one per region in Spain, Morocco, Italy, and Corsica) are characterised by twodifferent haplotypes. In the Italian peninsula and Sicily two closely related haplotypes (H1 and H2)are present. Haplotype 3 is found along the Mediterranean coast of Provence and Liguria, in theislands of Corsica, Sardinia, and Elba, as well as in the northern sector of Tunisia and Algeria.Haplotype 4 is distributed in the westernmost part of the range (southwest France, Portugal,southwest Spain and western Morocco). Haplotype 5 shows a discontinuous distribution inCatalonia, the Balearic islands and the Rif range in Morocco. Although the recent history of Q.suber is closely related to human activity for cork production, the geographical distribution of thecork oak haplotypes does not appear imputable to cultivation. Fossil pollen of Q. suber is notalways reported separately from other oaks in pollen diagrams. Even with these limitations, fossilpollen and wood records suggest that in pre-Neolithic times cork oak was distributed inapproximately the same areas as today. Other cultivated tree species in the Mediterranean (e.g. Oleaeuropaea and Castanea sativa) display low geographic structure in genetic variation, arguing for amultidirectional diffusion of the cultivated taxa due to human activity. Contrary to this pattern,distinct cork oak haplotypes are found even in neighbour geographic areas such as Corsica andItaly, Tunisia and Sicily, and western and eastern Morocco, indicating that human activity did notblur the original genetic structure.



SSCP POLYMORPHISMS OF GENE INTRONS AND MORPHOLOGICALTRAITS: NOVEL PROCEDURES FOR CLONAL IDENTIFICATION INPOPLAR

A. TURCHI*, S. CAPARRINI*, A. GIORCELLI**, F. PICCO**, M.L. RACCHI*, A. CAMUSSI*

*) Department of Agricultural Biotechnology, Genetics Unit, University of Florence,Via Maragliano 77 50144 Florence, Italy – [email protected]**) Istituto di sperimentazione per la Pioppicoltura- CRA, Casale Monferrato, Italy

molecular markers, SSCP, clonal identification, Poplar, Modified Location Model

The definition of genetic relationships and identity is important for germplasm resourcesmanagement, variety identification and registration to protect breeders, growers and industry.

Molecular markers may play a major role to achieve these objectives.Most of the commercially relevant cultivars in Poplar are clones and many identification

procedures based on molecular markers are in progress. The use of AFLP and SSR markers isrecently applied by Fossati et al. (2005) to classify a collection of 66 clones. In our laboratory SSCP(single-stand conformational polymorphism) markers were developed by designing specific primersflanking introns of two specific genes of the Catalase family. These SSCP marker have been testedon a sample of more than 100 poplar clones representative of the germplasm collection includingsome experimental clones of known genetic relationships.

The results show that SSCP intron markers have a very high efficiency, allowing todifferentiate most of the clones defined as identical in previous reports.

Though molecular marker are considered a powerful tool for clonal identification, the UPOVconvention still requires a detailed description of 64 morphological and phenological traits to allowto a novel clone to access to the DUS (Distinction-Uniformity-Stability) tests and, consequently, theinscription to National and International Registers of forest clones. In fact, neutral molecularmarkers cannot predict from the level of genetic identity the effective amount of diversity whenmany complex morphological traits are considered.

We developed a novel approach for the joint analysis of molecular diversity and morphologicalvariability by means of Modified Location Models (MLM). Without inferring any causalrelationship between SSCP polymorphism and the morphological characteristics of the clones, theprocedure allows to evidence the degree of morphological differentiation existing within clusters ofclones with a reduced level of marker based diversity.

The results are discussed in the view of possible integrate strategies to simplify registrationprocedures in poplar breeding.

Fossati et.al. (2005) Tree Genetics & Genomes 1: 11-19

Acknowledgements: the work is supported by the National Project “RI.SELV.ITALIA”



MOLECULAR CHARACTERIZATION OF ITALIAN HAZELNUTCULTIVARS AND SELECTION OF NEW GENOTYPES

R. DI BONITO, M. ARAMINI, L. BACCHETTA

ENEA Biotec-Gen, Via Anguillarese 301, 0060 S. Maria di Galeria, Roma [email protected]

Corylus avellana, AFLP, intraspecific variability

The cultivation of hazelnut (Corylus avellana) is relevant in the regions Pimonte, Lazio,Campania and Sicilia and the main production is addressed to the food industry, with a local marketfor the nut consumption. In the typical regions of cultivation, several varieties are present with alarge number of local genotypes and consequent heterogeneity of morphological traits and problemsrelated to the presence of synonyms or homonyms. In the last few years some of the major Italiancultivars (Tonda Romana from Lazio, Tonda di Giffoni from Campania and Tonda delle Langhefrom Piemonte) have obtained the quality certification from the European Community. Traditionalmethods for cultivar identification are usually based on phenotypic observations; however, this is aslow process for the long juvenile period of the trees and the presence of environmental factors.The aim of this work is the molecular characterization of Italian genotypes using AFLP techniqueand the development of markers able to distinguish each cultivar. The study included the mainItalian varieties (Tonda delle Langhe, Tonda Gentile Romana, Tonda Ghiffoni, Tonda Bianca,Tonda Rossa), local genotypes and Mediterranean cultivars. The second objective of the study wasthe evaluation of the clonal variability among genotypes of TGR, collected in different locations ofthe typical areas of cultivation.

The results show a distinct fingerprinting for all the main cultivars. A phylogenetic analysispoints out the relationships among genotypes and the presence of cultivars of different origin in theregions Campania and Sicily. The AFLP markers were also used to explore the genetic variabilitypresent in clones of the cultivar Tonda Gentile Romana.



MOLECULAR CHARACTERIZATION OF ITALIAN OLIVE CULTIVARSBY MICROSATELLITE MARKERS

I. MUZZALUPO, N. LOMBARDO, A. SALIMONTI, M. PELLEGRINO, E. PERRI

C.R.A. - Istituto Sperimentale per l’Olivicoltura, C.da Li Rocchi, I-87036 Rende (CS), Italy

Olea europaea, DNA, molecular markers, SSR, genetic diversity

Olive (Olea europaea L.) is a very important oil-producing crop in the Mediterranean area.Despite the long cultivation history and the great social-economic interest of the olive tree, itsgermplasm is today poorly characterized on the whole. Indeed, elaiographical and biometricalstudies are surprisingly insufficient to well address its management and preservation. In addition,reliable molecular standardized methods in order to elucidate the potential occurrence of homonyms(one denomination for several genotypes) or synonyms (one genotype with several denominations)are needed to eliminate ambiguities in variety identification.

Genetic diversity studies using microsatellite analysis were carried out in a set of 100 Italianolive (Olea europaea L.) cultivars (14 cvs from Abruzzo region, 9 cvs from Basilicata, 7 cvs fromCalabria, 2 cvs from Campania, 4 cvs from Lazio, 1 cv from Liguria, 12 cvs from Molise, 12 cvsfrom Apulia, 29 cvs from Sicilia and 10 cvs from Toscana region). Samples of olive leaves wereharvested from plants growing in the olive germplasm collection of the Consiglio per la Ricerca eSperimentazione in Agricoltura (C.R.A.) – Istituto Sperimentale per l’Olivicoltura (ISOl) of Rende(CS), Italy.

All microsatellites were polymorphic. A total of 52 alleles over nine loci were observed,ranging from 2.0 at UDO01 to 11.0 alleles at UDO39, with a mean of 5.8 alleles per locus. Theexpected heterozygosity ranged from 0.39 at UDO01 to 0.79 at UDO39 and the discriminationpower varied from 0.40 at UDO01 to 0.89 GAPU103A.

The results of alleles identification were then used to create a qualitative data matrix ofpresence (1) and absence (0) that was processed using NTSYS-PC software. Pairwise similaritiesbetween cultivars were calculated using Dice coefficient for qualitative data. The resultingsimilarity matrix was used to construct a dendrogram by means of the UPGMA (unweighted pair-group method with arithmetical averages) algorithm.

The following three presumable synonyms were detected: 1- ‘Filare’ and ‘Grossa di Venafro’,2- ‘Giarraffa’ and ‘Pizzo di Corvo’ 3- ‘Frantoio’ and ‘Ogliarola barese’. Therefore, synonymscharacterisation is very important to avoid genotype redundancy in order to maximise geneticdiversity in Italian olive germplasm collection.

This study showed that the use of molecular markers like SSRs is very useful to build a database available for variety analysis and for olive germplasm collection management.

Acknowledgments. This research was supported by Forestry and Agricultural Policy Ministry ofItaly, C.R.A. as Ordinary research and Project RIOM ‘Ricerca ed Innovazione per l’OlivicolturaMeridionale’.



ASSESSING GENETIC DIVERSITY OF NERO D’AVOLA GRAPEVINECULTIVAR BY USING SSR MARKERS

P. RICCARDI*,**, G. LAURIA**, R. GRILLO**, M.C. FIORE**, R. CIFARELLI**,F. SUNSERI*

*) Dipartimento di Biologia, Difesa e Biotecnologie AgroForestali, Università degli Studi dellaBasilicata, Via Ateneo Lucano 10, 85100 Potenza**) Metapontum Agrobios – SS Jonica 106 km 448,2, 75012 Metaponto (MT)

genetic characterization, diversity, PCR, microsatellites, multiplex

Grapevine (Vitis vinifera L.) is highly polymorphic since more than 7000 cultivars are believedto exist in the world. This variability can be observed at the morphological level as well as at theagronomical and technological level (Alleweldt and Possingham., 1988).

The methods based on morphologic and ampelographic features commonly used for Vitisvinifera cultivar identification not always allow the most accurate information because of theinteraction of the genotype with the environment (Dettweiler, 1993); on the contrary it is wellreported (Imazio et al. 2002) that genetic methods (molecular marker characterization) overcomethis problematics. With the aim to characterize and clarify the diversity of the Nero d’Avolagrapevine cultivar it has been developing a DNA fingerprinting method. For this purpose it has beenused different accessions belong the cultivar studied from different Sicilian areas, in particular 15vineyards from 3 different areas (Caltanisetta, Trapani and Ragusa) were sampled.

Within a wide range of molecular markers we used a PCR-based method named SimpleSequence Repeats (SSRs), also called microsatellites. This work included all steps ofcharacterization from sampling through fingerprinting.

Genomic diversity and differentiation in accessions was analyzed using polymorphism attwenty-four microsatellite loci (VVS2; VVMD5, 7, 27,14,17, 21, 26, 31, 24, 25, 28, 32, 34, 36;VrZAG62, 79, 7, 12, 14, 15, 21, 25, 67). Forward primers of the twenty-four SSR loci were labelledwith one of the three unique ABI PRISM fluorescent dyes: 6-FAM, TET, HEX, and the PCRreactions were run on ABI PRISM.

The automated sequence system ABI PRISM combined with fluorescent labelling of expectedfragments has been applied as an alternative to radioactivity detection using [32P] or [33P]-labelling.This technology provides an automatic and rapid sizing of the fragments through the use of specificinternal size standard (GS500-Liz) and allows analysis of fragments obtained by simultaneouslymultiplex amplification up to three different labeled PCR products in the same reaction. The datacollected from each sample were automatically analysed by GeneMapper Analysis Software.

The genetic diversity between accessions were estimated and multivariate analysis methodswere applied for the analysis of genetic relationships among the vineyards and the Nero d’Avolaareas.

ReferencesAlleweldt G, Possingham JV (1988). Progress in grapevine breeding. Theor Appl Genet 75 :

669-673

Dettweiler E (1993). Evaluation of breeding characteristics in Vitis, influence of climate onmorphologic characteristics of grapevines. Vitis 32: 249–253.

Imatio S, Labra M, Grassi F, Winfield M, Bardini M, Scienza A (2002). Molecular tools forclone identification: the case of the grapevine cultivar 'Traminer'. Plant Breeding 121:6, 531-535



CHARACTERIZATION OF NEWLY ISOLATED MICROSATELLITEMARKERS FROM ARTICHOKE

G. SONNANTE*, A. DE PAOLIS**, A. V. CARLUCCIO*, D. PIGNONE*

*) Institute of Plant Genetics (IGV), CNR, Via Amendola 165/A, 70126 Bari, Italy –[email protected]**) Institute of Food Science Production, CNR, Str. Prov. Lecce-Monteroni, 73100 Lecce, Italy

Cynara cardunculus, microsatellites, characterization, variety identification

Artichoke, Cynara cardunculus L. var. scolymus (L.) Fiori is a diploid outcrossing species,originated in the Mediterranean basin, whose value is much recognised since ancient times both forits tasty heads and for pharmaceutical properties.

Only a few varieties are widely cultivated in Italy; nevertheless, a number of landraces arediffused on a small scale which have been poorly characterized to date. Some problems arise forvariety identification, due to the fact that landraces are normally topologically named irrespectivelyof their genetic constitution or morphological characters, thus resulting in synonymy and/orduplications. To overcome this problem, molecular markers able to fingerprint the varieties can bedeveloped. One class of such markers are microsatellites (SSRs). They consist of tandem repeats ofdi-, tri- or tetra-nucleotide patterns, are frequent and usually well distributed in plant genomes, andcan be exploited to develop locus-specific codominant markers, useful for studies on geneticdiversity and mapping.

In artichoke, only few SSRs have been isolated to date. For this reason, a genomic libraryproduced from the variety "Locale di Mola", was used in order to identify microsatellite regions.Hundreds of recombinant phages were screened by using oligonucleotides labelled withdigoxygenin and containing SSR sequences. Positive clones were obtained with (CAT)8 and(GATA)5 probes. The nucleotide sequence of the insert was determined and the repetitive stretchesof di- and tri- nucleotides were identified. Primers specific to SSR flanking regions were designedand DNA fragments in the range 150-350 bp were amplified, cloned and sequenced, in order toascertain the repetitive nature of the sequences. A set of artichoke accessions belonging to the mainmorpho-agronomic groups and from various geographical origins was selected, together with somewild C. cardunculus and other Cynara species. A total of 20 microsatellite regions were amplifiedusing one of the primers fluorescently labelled; the amplification product was then analysed usingan automated sequencer. Most of the SSRs produced more than two alleles and for some of themallele distribution was related to morpho-agronomic traits.



GENETIC MAPPING IN CYNARA CARDUNCULUS L.

E. PORTIS*, A. ACQUADRO*, R. MAURO**, G. MAUROMICALE **, S. LANTERI*

*) DiVaPRA, Plant Genetics and Breeding, University of Turin, Via L. da Vinci 44,10095 Grugliasco (TO), Italy - [email protected]**) Dipartimento di Scienze Agronomiche, Agrochimiche e delle Produzioni Animali –sez. Scienze Agronomiche, University of Catania, Via Valdisavoia 5, I-95123 Catania, Italy

globe artichoke, cardoon, genetic map, QTLs

The complex Cynara cardunculus, includes the globe artichoke (var. scolymus L.), thecultivated cardoon (var. altilis L.) and the wild cardoon (var. sylvestris (Lamk) Fiori).

Globe artichoke (C. cardunculus var. scolymus L.) contributes significantly to theMediterranean agricultural economy, with an annual production of about 750Mt (more than 60% ofglobal production) from over 80kha of cultivated land. Italy is the leading world producer (about470Mt) followed by Spain (188Mt), France (52.5Mt) and Greece (35Mt). Although to a lesserextent globe artichoke is also cultivated in the Near East, North Africa, South America, UnitedStates (mainly in California), and its cultivation is spreading in China (FAO data 2005:http://faostat.fao.org/).

The C. cardunculus genome is as yet poorly investigated. In order to move to a crossingstrategy for breeding, a greater knowledge of artichoke and cardoon genetics will be essential. Inparticular, it will be advantageous to establish a framework of linkage relationships to allow theidentification and localization of genes controlling important yield traits or resistance againstpathogens.

Recently we generated the first genetic maps of globe artichoke, based on a two-way pseudo-testcross strategy. An F1 population was created by crossing a clone of ‘Romanesco C3’ (a late-maturing, non-spiny type) with ‘Spinoso di Palermo’ (an early-maturing spiny type), and theprogeny were genotyped using AFLP, M-AFLP, SSR and retrotrasposon based SSAP markers. Thefemale map comprised 204 loci, spread over 18 linkage groups and spanned 1330.5cM with a meanmarker density of 6.5cM. The equivalent figures for the male parent map were 180 loci, 17 linkagegroups, 1239.4cM and 6.9cM. The presence of 78 loci in common to both maps allowed for thealignment of 16 of the linkage groups.

At present we are applying the same strategy to developing further genetic maps on F1

progenies obtained by crossing the same clone of ‘Romanesco C3’, previously used as femaleparent, with either a cultivated cardoon (‘Altilis 41’) and a wild cardoon (‘Creta 4’) accessions usedas pollen sources. Wide cross populations of this type are suited for the investigation of the geneticcontrol of quantitative characters in exotic genetic backgrounds. Furthermore, both wild andcultivated cardoon represents the most straightforward resource to exploit for globe artichokeimprovement, since they are full cross-compatible to it.

A very high molecular variation was detected and a high phenotypic variation was observed forimportant commercial traits like: size, shape, weight, tightness, presence/absence and length ofspines, peduncle length of the capitula, size and branching of the plants, etc. Since C. cardunculusis easily vegetatively propagated, the mapping populations are immortalised, and thus will be grown

in contrasting environments to investigate genotype x environment interaction for importantcommercial traits.



PRODUCTION AND FINE CHARACTERIZATION OF NEAR ISOGENICLINES FOR HETEROTIC QTL IN MAIZE

P. PAULINE SANDRA*, G. PEA**, M.-L. SAVO SARDARO*, M. A. CANÈ***,E. FRASCAROLI***, P. LANDI***, M. MORGANTE****, E. PORCEDDU*,*****, M. E. PÈ**

*) Sant’Anna Higher School, Pisa. International Ph.D Program in AgroBiodiversity,00057 Maccarese -Rome, Italy - [email protected]**) Department of Biomolecular Science and Biotechnology – Università degli Studi di Milano,Via Celoria 26, 20133 Milano, Italy***) Department of Agroenvironmental Sciences and Technologies “DiSTA” – Università diBologna, Viale Fanin 44, 40100 Bologna, Italy****) Dip. di Scienze Agrarie e Ambientali – Università degli Studi di Udine, Via delle Scienze208, 33100 Udine, Italy*****) Dip. Agrobiologia e Agrochimica – Università degli Studi della Tuscia, Via S. C. de Lellis,01100 Viterbo, Italy

heterosis, QTL, maize, Near Isogenic Lines, quantitative genetics

Although heterosis is largely exploited for crop improvement and breeding, its genetic basis isstill poorly understood. Integrating biometrical and molecular marker approaches can produceuseful information to elucidate the genetic basis of complex traits and, therefore, be particularlyadvantageous for studying heterosis. This approach was originally applied on materials developedfrom the maize single cross B73 x H99 in order to: (i) study the level of heterosis for traits ofagronomic importance; (ii) detect the genetic effects involved (i.e., allelic and non-allelicinteractions) by following procedures of both classical and neoclassical (i.e. QTL) genetic analyses;(iii) investigate the relationships between the level of molecular marker heterozygosity and thephenotypic performance; (iv) identify the genomic regions most involved in heterosis (i.e. showingoverlaps among QTLs for the most heterotic traits). The mapping population is represented by 142Recombinant Inbred Lines (RILs) genotyped for almost 200 molecular markers. Several QTLs forheterosis were identified, and both biometrical and QTL analyses indicated that heterosis wasmainly due to overdominance and/or pseudo-overdominance, whereas epistasis was negligible.

Here we present the advancements of the subsequent introgression program aimed at theproduction of sets of Near Isogenic Lines (NIL) for a subset of selected QTLs for heterosis, carriedout with the purpose of obtaining a more accurate estimate of their effects on hybrid vigor andeventually isolating the implicated genetic factors. Where possible, two sets of NIL were producedstarting from different RILs. Our “short-cut” strategy adopted for NILs production and preliminaryresults of fine mapping for the selected QTL intervals are here described.



A NEW MUTANT ALLELE OF BRACHYTIC 2 MAIZE GENE

E. CASSANI*, D. VILLA*, S. CURIALE*, M. LANDONI**, F. CERINO BADONE*,D. PANZERI*, D. REGINELLI***, R. PILU*

*) Dipartimento di Produzione Vegetale - University of Milan, Via Celoria 2, 20133 Milano, Italy**) Dipartimento di Scienze Biomolecolari e Biotecnologie - University of Milan, Via Celoria 26,20133 Milano, Italy***) Azienda Agraria “Angelo Menozzi” - University of Milan, Landriano (PV), Italy

maize, mutant, green revolution, brachytic, P-glycoprotein

Plants with short stature have had a big impact on agriculture. World rice and wheat grainyields increased dramatically in the l960s and 1970s (green revolution) because farmers adoptednew varieties that were shorter and more resistant to storm damage.

Brachytic/dwarf varieties have not been exploited commercially in maize, partly because ofthe excessively severe nature of the original mutant alleles. However similar mutations have beenused extensively in sorghum production since the 1950s.

To our knowledge, there are three brachytic mutants isolated so far in maize: brachytic1 (br1),brachytic2 (br2 ) and brachytic3 (br3) that show a short stature and gibberellin insensitivephenotype

A maize brachytic mutant of agronomic potential is the recessive br2 mutation, which results inthe shortening of lower stalk internodes. br2 was cloned by transposon tagging with Mu element byMultani et al., in 2003 and it encodes a putative protein similar to adenosine triphosphate (ATP)-binding cassette transporters of the multidrug resistant (MDR) class of P-glycoproteins (PGPs)involved in polar movement of auxins.

The spontaneous maize mutant, named brachytic-23* (br*-23), described in this work has ashort stature and compact lower stalk internodes compared to wild type control. The geneticanalysis indicated that br*-23 was inherited as a monogenic recessive trait.

In allelism tests our mutant failed to complement the br2 mutant, thus it became obviousthat br*-23 represents a mutation in the br2 gene. Following the guidelines provided by the Maize-GDB, we renamed the new mutation br2-23.

In order to investigate the molecular lesion in the br2-23 allele, we designed specific primerson the basis of the sequence of the br2 gene cloned by transposon tagging by Multani et. al., in2003. The genomic PCR fragments obtained from a homozygous mutant and a wild type plant(B73 inbred line) were subcloned and sequence analyses were performed. Preliminary alignmentof Br2 (B73 Allele) and br2-23 sequences, performed using the CLUSTALW program, revealedthat the mutant carries a eight nucleotide deletion in the coding region. The presence of thisdeletion in the coding region was also confirmed by using allele-specific primers.

Details of further genetic, molecular, and histological characterization of this mutant will bepresented.



STUDIES ON EPIGENETIC ASPECTS OF lpa1-241 TRAIT IN MAIZE

D. PANZERI*, F. CERINO BADONE*, E. CASSANI*, M. LANDONI**, R. PILU*

*) Dipartimento di Produzione Vegetale - University of Milan, Via Celoria 2, Milano, Italy**) Dipartimento di Scienze Biomolecolari e Biotecnologie - University of Milan, Via Celoria 6,Milano, Italy

maize, mutant, low phytic acid, epigenetic, DNA methylation

Phytic acid is a nearly ubiquitous component of plant seeds, supplying both phosphate (P) andcations during germination. However, during digestion, the phytic acid form of P is not bio-available for monogastric animals. A possible solution to this problem is the isolation of cerealmutants accumulating less phytic P and more free P and cations in the seed (low phytic acid, lpa).

Several mutants have been isolated in recent years in maize, barley (Hordeum vulgare L.), rice(Oryza sativa L.) and soybean (Glycine max L.). These low phytic acid (lpa) mutants produce seedsin which the chemistry of seed P, but not the total amount of P, was greatly altered.

All lpa1 mutations affect the first committed step in inositol biosynthesis, i.e. the production ofmyo-inositol-3-phosphate (Ins3P) from glucose-6-phosphate (G6P), catalysed by the enzyme myo-inositol-3-phosphate synthase (Ins3P synthase, MIPS).

In previously published studies, we described a single, recessive lpa mutation (originallynamed lpa241) in maize (Zea mays L.) mapping on chromosome 1S, which resulted allelic to thelpa1 mutant, showing a decrease in the expression of the myo-inositol-3-phosphate synthase locatedon the short arm of the chromosome 1 (MIPS1S).

In this work, we present some genetic and molecular analyses of the lpa1-241 trait that mayindicate an epigenetic origin of the mutation.

Some families show a Mendelian segregation of the lpa phenotype, while others seem toproduce more mutant individuals than expected. We measured this phenomenon on both backcrossand BC1F1 families.

Also, a high rate of spontaneous occurrence of this mutation was found and its value has beenestimated for different families belonging two different inbred lines.

The analysis of the coding region of the MIPS1S gene using MS-PCR (methylation-sensitivePCR) showed many methylated sites at its 3’ portion. So far, we could not find noticeabledifferences between the lpa1-241 mutant and the wild type.

Further details on MIPS1S gene methylation pattern, as well as the genetic segregation of thetrait, will be presented.



IDENTIFICATION OF NATURAL OCCURING EPIALLELES IN MAIZEINBRED LINES USING A WIDE-GENOME METHYLATION ANALYSIS

M. LAURIA, M. MOTTO

C.R.A., Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo, Via Stezzano 24,24126 Bergamo (BG), Italy

DNA methylation, epialleles, MSAP

In the past, genetic variation among individuals was considered only as a result of alterationsin the primary nucleotide sequences due to mutation or gene recombination. However, it is nowknown that epigenetic factors such as DNA methylation or histone modification can play afundamental role in generating alternative states (epialleles) of gene expression among biologicalstrains or individuals in a population. In maize plants, spontaneous epialleles have been identified atsome loci as those which encode transcription factors that activate the biosynthesis of flavonoidpigments. For example, the b1 locus can shift from a high expressed form (B-I epiallele) to a low-expressed form (B’ epiallele). Importantly this transition correlates with both altered level of DNAmethylation and differential chromatin structure within the regulatory region 100 kb upstream of thelocus.

In order to gain further insight into rules governing both the establishment and thepropagation of epialleles in maize, the maize genome for target regions showing methylationdifferences among individuals of the Mo17 inbred line was screened. The methylation state ofapproximately 1000 DNA sequences was investigated during different maize plant developmentalstages using a methylation sensitive amplified polymorphism (MSAP) approach. While the majorityof methylation profiles analyzed were faithfully maintained during plant development, individual toindividual methylation differences were observed representing 2% of total DNA sequencesanalyzed. Newly formed epialleles were successfully transmitted meiotically, thereby mimickingtraditional genetic mutations.

Evidence providing a possible explanation of alternative phenotypes observed, for example, inthe progeny of long-term inbred lines, through the production at each plant life generation of novelepigenetic states, has been shown by our study.



SCREENING OF MAIZE GENOTYPES FOR RESISTANCE TOASPERGILLUS FLAVUS*

C. BALCONI, N. BERARDO, V. PISACANE, M. FERRARESE, A. FERRARI, F. FUMAGALLI,G. DELLA PORTA, A. VERDERIO, M. MOTTO

CRA-Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo, Via Stezzano 24,24126 Bergamo, Italy - [email protected]

Aspergillus flavus, Zea mays L., artificial inoculation, aflatoxin

The fungus Aspergillus flavus is responsible for both pre- and post-harvest accumulation ofaflatoxin in maize (Zea mays L). Concern about aflatoxin contamination is due to its potentpotential carcinogenicity.

In maize, resistance against A.flavus infection and aflatoxin accumulation is a complex trait thatis influenced by genotype, agronomic practics and environamental conditions. Beneficial secondarytraits such as husk covering and tightness, physical properties of the pericarp, and drought or heatstress tolerance are factors contributing to aflatoxin resistance.

The availability of reliable methods for the screening and evaluation of maize genotypes forimproving tolerance to Aspergillus attacks is an unvaluable tool in breeding programmes to increasecrop protections against fungal infection. The aim of our research is to evaluate and compare 34maize hybrids for A. flavus resistance and for aflatoxin accumulation under field condition. The testincluded: i)self pollinated non–inoculated ears, ii)self-pollinated inoculated (A.flavus) ears. iii)open-pollinated non inoculated ears (in 10-20 different locations)- The inoculation experiment wasreplicated at two different planting dates. At pollination, silk channel (region within the huskbetween the tip of the cob and tip of the husk where the silks emerge) length was recorded for eachgenotype.

Ten per plot hand pollinated plants were inoculated with a fresh spore suspension (mixture offive A.flavus isolates from Northern Italy), 7 days after pollination by the non-wounding silkchannel inoculation technique applied to each primary ear. Controls were non-inoculated and sterilewater-inoculated plants. At maturity, ears were manually harvested. For husk cover visual ratingranging from 1 (good tight long husks extending beyond the tip of the ear) to 5 (poor:loose shorthusks with exposed ear tips) has been recorded. After hand de-husking; the severity of ear A. flavusattack was evaluated using rating scales based on the percentage of kernels with visible symptomsof infection, such as rot and mycelium growth(Disease Severity Rating-DSR-:from1=0%-noinfection to 7=76-100% of visibly infected kernels/ear). Inoculated materials showed a higher DSRin comparison to control or open-pollinated non-inoculated plants, that did not show any or verylow disease symptoms. This result indicates that the non-wounding silk channel inoculationtechnique applied was effective in inducing A.flavus attack. In addition, variability in the responseof the maize genotypes tested to Aspergillus attack has been shown. After visual inspection earswere dried and shelled. The kernels were bulked within plots. To evaluate internal kernel infectionfifty kernels were randomly chosen from each sample, were surface-disinfected and plated onpotato PCNB agar.

Content of Aflatoxin B1 in the control, inoculated and open-pollinated non inoculatedmaterials, has been evaluated using enzyme-immunoassay-ELISA kit. The aflatoxin content in theinoculated ears resulted higher than in the controls and in the open-pollinated non inoculatedmaterials;.this indication confirm that the A.flavus isolates used for the inoculum procedure weresuccessful in accumulating mycotoxin in grains. Also in this case variability has been foundbetween the genotypes under tested.

Correlations between visual ear rot ratings, internal kernel infection evaluation, aflatoxincontent, silk channel length at pollination, husk cover ratings, are in progress.

*Research developed in the Program AFLARID



FINE QTLs MAPPING FOR SALINITY TOLERANCE IN RICE

Y. Z. EL-REFAEE*, M. ATALLAH*, M. L. SAVO SARDARO*, E. PORCEDDU*,**

*) Sant’Anna Higher School, Pisa, International Doctoral Program in Abrobiodiversity, Maccarese,Italy**) University of Tuscia, DABAC, Viterbo, Italy

Oryza sativa, salinity tolerance, QTLs, SSR

Rice (Oryza sativa) is one of the agronomically and nutritionally important cereal crops. It is amajor source of food for more than 2.7 billion people on a daily basis and is planted on about one-tenth of the earth’s arable land. Soil salinity is considered one of the major and widespread abioticstresses, limiting rice production worldwide. A project aiming to identify chromosomal regionsassociated salinity tolerance is being carried out. A total of nine rice genotypes, including threeEgyptian and six introduced varieties having three different degrees of salinity tolerance (tolerant,moderate and sensitive) are being used. DNA polymorphism (SSR) among genotypes, differing insalt tolerance, was assessed. A total of 272 SSR primer pairs covering the whole rice genome wereused. Results indicated that 80% of the all tested primers showed polymorphic pattern among thesegenotypes. The number of alleles ranged from two with RM 428 to six with RM 206 primers. F2plants are being grown to perform the phenotypic and genotypic characterisation.



DEVELOPMENT OF MOLECULAR MARKERS FOR THEINTROGRESSION OF BROAD SPECTRUM BLAST RESISTANCE GENESINTO RICE GERMPLASM CULTIVATED IN ITALY

C. LANZANOVA*, D. BULGARELLI**, V. BALDASSARRE**, G. VALÈ**, E. LUPOTTO*

*) Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo, Via Stezzano 24,24126 Bergamo, Italy**) Istituto Sperimentale per la Cerealicoltura, Sezione di Fiorenzuola d’Arda, Via S. Protaso 302,I-29017 Fiorenzuola d’Arda (PC), Italy

rice, blast, resistance gene, molecular markers

Rice blast, which is caused by the fungus Magnaporthe grisea, is the most economicallyimportant fungal disease in the world’s rice-growing areas. Development of resistant cultivars isconsidered to be the most effective method against blast. Currently, more than 40 rice blastresistance genes have been identified and over 25 of them have been mapped on the rice genome(Chao et al., 1999, Euphytica 109: 183-190). The resistance in newly released rice cultivars to riceblast can be lost quickly due to the high level of instability in the genome of this fungus (Bonman etal., 1992, Annu Rev Phytopathol 30: 507-528); this problem can be overcome by pyramidingmultiple resistance genes each recognizing different sets of Magnaporthe grisea isolates into asingle cultivar, or deploying rice cultivars with broad spectrum resistance or utilizing both theapproaches at the same time.

To our knowledge, no known blast resistance genes have been introgressed into elite Italianrice cultivars and this result in a generalized susceptibility of the cultivated Italian rice germplasmwith respect to the rice blast disease. Genetic improvement of blast resistance therefore represents apriority of the rice breeding in Italy.

With this purpose in mind, we have collected rice germplasm bearing several known broadrange effective blast resistance genes. PCR-based molecular markers linked to these genes havebeen developed from published primers or by designing primers in genomic regions tightlyassociated to the genomic map position of the selected blast resistance genes. Allelic variation ofthe molecular markers obtained (SSR, CAPS, STS) was evaluated into the donors of the blastresistance genes and within a representative collection of rice germplasm that include cultivated,traditional and of general interest rice genotypes. Polymorphic combinations that could allow boththe introgression of the broad spectrum resistances into susceptible genetic background and thepyramiding of resistance genes have been identified, suggesting that the markers identified couldrepresent suitable tools for the breeding of rice blast resistance of Italian rice germplasm.



EVOLUTION OF MICROSATELLITES WITHIN LTRRETROTRANSPOSONS IN THE RICE GENOME

F. MAGNI*, A. ZUCCOLO**, M. MORGANTE*

*) Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi di Udine, Via delle Scienze208, 33100 Udine, Italy – [email protected], [email protected]**) Arizona Genomics Institute, Department of Plant Sciences, University of Arizona, Tucson, AZ85721, USA – [email protected]

Gypsy and Copia like LTR-retrotransposon, microsatellite evolution, plant genomics, Oryza sativa.

The aim of this study was to investigate microsatellite evolution in gypsy-like and copia-likeLTR retroelements in Oryza sativa L. var. japonica. We screened a collection of 2807 members ofthe Ty-3-gypsy group and 776 members of the Ty-1-copia group, isolated from the draft genomesequence of rice, for a total amount of almost 35 Mb, searching for microsatellites in their LTR andinternal domains. We characterised the microsatellites present in the gypsy-like and copia-likeretroelement populations, in terms of motif class distributions and length class distributions; weestimated the abundance and distribution of the identified microsatellites in LTR retroelementsgrouped according to their time of insertion; finally, we investigated the mutability ofmicrosatellites residing within the LTR regions.

We found similar microsatellite frequencies in copia and gypsy elements, according with thecoeval major waves of propagation inferred for the two groups on the basis of sequence divergencebetween LTRs of every retroelement. Moreover our estimates confirmed the relative paucity ofmicrosatellites in the repetitive fraction of the rice genome, in comparison to more ancientcompartments, such as genes. We observed a sharp difference in the microsatellite frequenciesbetween different structural retroelement domains, with LTRs displaying more than doubledconcentration of microsatellites compared to the internal domains. Moreover, microsatellites appearmore frequent in internal regions of ancient retroelements than recent ones, while reach very highfrequencies in LTRs of the youngest elements. We investigated the kind of mutations responsiblefor length differences between pairs of microsatellites belonging to the two LTRs of the sameretroelement and we observed a predominance of stepwise events followed by point mutations andother rearrangements.

We discuss these features under the hypothesis that in grasses microsatellites residing inrecently amplified LTR-retrotransposon DNA are still reaching an equilibrium state.



MAPPING OF NEW EST-DERIVED SSR ON 6A AND 6B CHROMOSOMESOF WHEAT

A. GADALETA, A. GIANCASPRO, S. ZACHEO, G. MANGINI, R. SIMEONE, A. BLANCO

Department of Environmental and Agro-Forestry Biology and Chemistry, University of Bari sect.of Genetics and Plant Breeding, Via Amendola 165/A, 70126 Bari, Italy

molecular markers, EST-derived SSR, wheat, linkage maps

The level of molecular polymorphism in cultivated hexaploid and tetraploid wheats was foundto be low as compared to many other species. Use of molecular markers in genome analysis, thesystematic mapping of agricultural important traits, and marker-assisted selection have been greatlyadvanced by the development of reliable PCR-based markers, such as amplified fragment lengthpolymorphism (AFLP) and microsatellite o simple sequence repeat (SSR). Over the past decademicrosatellites have attracted considerable attention of researchers. The overall frequency ofmicrosatellites among species was found inversely related to genome size and to the proportion ofrepetitive DNA but remained constant in the transcribed portion of the genomes. Bioinformaticanalysis indicated that the frequency of microsatellites was significantly higher in ESTs than ingenomic DNA across all species. At present over three million sequences from approximately 200plant species have been deposited in the publicly available plant expressed sequence tag databases.Many of the ESTs have been sequenced as an alternative to complete genome sequencing and thiscreates a formidable resource for microsatellite marker development. The objectives of this studywere to construct a high density EST chromosome map of wheat chromosome group 6 to determinethe distribution of ESTs, examine patterns of duplication and investigate the putative function. Atotal of 23 new EST-derived SSRs were genetically mapped on chromosome 6A and 6B in a RILmapping population developed by crossing two durum wheat cultivars (Ciccio and Svevo). Twogene rich islands flanked by relatively gene-poor regions on both the short arms of chromosome 6Aand 6B were discovered. Three times more loci were mapped on the short arms than on the longarms. A higher number of EST-SSRs, detecting multiple loci mapped on the same chromosomewas found on chromosome 6B. The co-localization of separate PCR fragments detected by onecouple of primer to the same chromosome region could be due to duplication of genetic material.Good colinearity was observed among the two homoeologous chromosomes.



USE OF GENOMIC AND EST-DERIVED SSR MARKERS IN WHEATPHILOGENETIC ANALYSIS

A. M. DIGESÙ, A. GADALETA, R. SIMEONE, G. MANGINI, A. V. CARLUCCIO,A. BLANCO

Department of Environmental and Agro-Forestry Biology and Chemistry, University of Bari,Via Amendola 165/A, 70126 Bari, Italy

gSSR, EST-SSR, wheat, transferability, phylogenetic analysis

Molecular markers are used for a wide range of purposes in crop genetics and breedingincluding genetic linkage and comparative mapping, positional cloning, genetic diversityassessment, phylogenetic analysis, genotypic profiling, detection of quantitative trait loci (QTLs)and marker-assisted selection (MAS). Genomic microsatellites (or Simple Sequence Repeats,gSSR) have attracted relatively more attention because of their abundance in plants genome,reproducibility, high level of polymorphism, and codominant inheritance. Recently, due to theavailability of enormous data for expressed sequence tags (ESTs), more emphasis has been given toEST-derived SSRs, which belong to the transcribed regions of DNA and are expected to be moreconserved and have a higher rate of transferability across species than genomic SSR markers. In thepresent study, 79 gSSR and 61 EST-SSR markers were used to investigate their transferability andlevel of DNA polymorphism in different ancestral tetraploid and diploid Triticum and Aegilopsspecies more or less closely related to common wheat, and their applicability for phylogeneticanalysis of wheat. The transfer rate was correlated with the genetic telatedness of the examinedspecies. The average transfer rate of gSSR was different from the average transfer rate of EST-SSRmarkers. The range and the number of alleles per locus indicated that gSSRs are more polymorphicthan EST-SSRs. Both phylogenetic trees based on gSSR and EST-SSR markers were in agreementwith the phylogenetic relations based on cytogenetic and molecular analysis. The use of transferredpolymorphic SSR markers for the characterization and evaluation of germplasm and forphylogenetic analysis of wheat are discussed.



QTL ANALYSIS FOR YIELD COMPONENTS IN A SET OF RECOMBINATINBRED LINES OF DURUM WHEAT GROWN IN DROUGHTENVIRONMENTS

G. MANGINI*, L. MATTEU**, A. SIGNORILE*, A. GADALETA*, A. MASTRANGELO**,L. CATTIVELLI**, A. BLANCO*

*) Department of Environmental and Agro-Forestry Biology and Chemistry, University of Bari,Via Amendola 165/A, 70126 Bari, Italy**) CRA-Experimental Institute for Cereal Research Section of Foggia, S.S. 16 km, 675,71100 Foggia, Italy

QTLs, yield components, stress conditions, genetic map, durum wheat

Grain yield of durum wheat (Triticum turgidum L. var. durum) under Mediterranean conditionsis frequently limited by both high temperature and low-erratic distribution of rainfall during graingrowth. The plant’s response in this stress conditions depends on its metabolic activity, morphologyand stage of growth. Identifying QTLs that show consistent in expression across environments,even in different environments, would be desirable for marker assisted selection.

A set of 120 recombinant inbred lines (RIL) derived by single seed descent from the cvs Svevoand Ciccio were grown during the seasons 2004, 2005 and 2006 at Valenzano (BA, Italy), Gaudiano(PZ, Italy) and Foggia (Italy). Yield components (grain yield, kernel number, 1000 kernel weight,grain yield per spike and test weight) and plant adaptive traits (plant height and heading time) wereevaluated. QTLs analysis was carried out using a genetic linkage map of the RIL populationobtained by genomic microsatellite and EST-derived SSR markers. QTL detection was based onKruskal-Wallis analysis (van Ooijen, 2004) related to each RIL phenotypic mean values in singleand across environments and data of polymorphic markers in the segregant population. Thepresence of a putative QTL was judged if a significant effect were observed in two or more trialswith P≤0.01. A number of QTLs for yield were identified on group-2 chromosomes. In fact, QTLsfor yield per spike, kernel weight and number kernels per spike were detected on 2BS chromosomearm. Moreover QTLs for several yield components were localized on 2AS chromosome arm thatsuggested that chromosome regions on 2AS and 2BS are involved in the control of grain. For yieldper spike, one QTL on 2BS chromosome was detected in all environments, two QTLs, localized on2A and 6B chromosomes, were identified in four environments and one QTL in three environments,indicating that individual QTLs seem to be sensitive to the environment. In situation with largegenotype x environment interactions, such as in this study, it would be common to find a QTLsignificant in some trial but not in other. Three QTLs on 3BS, 3AL and 4BL chromosome armswere detected for the plant height in several experiments. Kernel weight and number of kernels perspike showed QTLs in the same regions: chromosomes 2BS, 2AS, 4BL and 6B. This resultsupposed that the same QTLs were involved in phenotypic expression of the two traits. Markersassociated with variation in heading time were identified on 1AS and 2AS. Finally three QTLs forthe test weight were detected on 2BS 7AS and 6AS.



GENETIC LINKAGE DISEQUILIBRIUM FOR DROUGHT TOLERANCE INETHIOPIAN DURUM WHEAT

A. FARINA, P. DONATI, L. MONDINI, M. A. PAGNOTTA, E. PORCEDDU

Department of Agrobiology and Agrochemistry. Tuscia University, Via S. De Lellis 01100 Viterbo,Italy - pagnotta@unitus

SSR, durum wheat, germplasm, drought tolerance, linkage disequilibrium, QTL

Ethiopian durum wheat [Triticum turgidum ssp. durum (2n = 4x = 28)] germ plasm showspeculiar characteristics and is a source of many traits, including disease resistance, environmentalstability, tolerance to environmental stresses, such as drought and low temperature.

A durum wheat collection of 234 genotypes from nine populations of three Ethiopian regions:(Tigray, Gonder and Shewa) were used to define the linkage disequilibrium in drought tolerances’QTL regions by 60 SSRs markers located closely to these QTL on chromosomes 2, 4 and 6.

The assessment of genetic variation and its components (i.e. among regions,populations/region, and within populations) was variable when different QTL regions wereconsidered. The markers located on chromosome 4 showed statistically significant differences onlybetween regions, whereas those located on chromosome 2 and 6 were significantly different also forthe components between and within populations. Some loci were in linkage disequilibrium at all theanalysed regions whereas others were in disequilibrium only in a single region, indicating differentselection pressures in the analysed regions.



ASSOCIATION MAPPING USING CANDIDATE GENES FOR DROUGHTAND SALINITY RESISTANCE IN DURUM WHEAT

F. ARZENTON*, I. JURMAN*, A.M. MASTRANGELO**, L. CATTIVELLI**,M. MORGANTE*

*) Dipartimento di Scienze Agrarie ed Ambientali, Università degli Studi di Udine, Via delleScienze 208, 33100 Udine, Italy**) Istituto Sperimentale per la Cerealicoltura, sezione di Foggia, S.S. 16 Km. 675, 71100 Foggia,Italy

association mapping, candidate genes, stress resistance

Linkage disequilibrium mapping is a method to determine whether candidate genes areassociated with variation in a trait of interest using natural populations and to localizepolymorphisms that contribute to this variation. In this project we used the candidate gene approachto identify genes that contribute to the drought and salinity resistance in durum wheat (Triticumturgidum ssp. durum). A set of candidate genes that includes stress-inducible transcription factorsbelonging to many different classes, were preliminarily sequenced on 12 inbred lines, that greatlydiffer in terms of genetic diversity and stress resistance. Hence, each polymorphic site found in thepreliminary analysis has been genotyped in 95 samples (87 inbred lines and 8 individuals fromdifferent Ethiopian populations) that were characterized phenotypically during the project. In LDmapping studies the population structure has to be taken into account because it may lead tononfunctional, spurious associations. In order to infer the population structure with random andunlinked genetic markers, we selected, on the basis of a simple sequence repeat (SSR) map of T.turgidum previously constructed in our lab, the two most distal SSR markers of each major linkagegroup. Therefore, all the samples have been genotyped with 28 SSR markers. Here we present anddiscuss results on nucleotide diversity in a large set of genic regions, on the extent of LD in durumwheat genome and on the genetic variability and the phylogenetic relationships among the linesused in the present study. Association tests, that incorporate estimates of population structure, willenable to detect statistically significant associations between sequence variants in the candidategenes and the phenotypic variation and may lead to the identification of the alleles responsible forthis variation in the trait of interest.



PCR ANALYSIS OF X- AND Y-TYPE GENES PRESENT AT GLU-1 LOCI INOLD DURUM WHEAT CULTIVARS

M. URBANO, G. COLAPRICO, B. MARGIOTTA

Institute of Plant Genetics – CNR, Bari sede, Via Amendola 165/A, 70126 Bari, Italy –[email protected]

HMW-GS, durum wheat, SDS-PAGE, PCR

High molecular weight glutenin subunits (HMW-GS) have been extensively investigatedbecause of their impact on quality of wheat gluten and dough elasticity. In durum wheat they areencoded by genes at two complex loci (Glu-A1 and Glu-B1) present on the long arms of thechromosomes of the homeologous group 1. Each locus consists of two tightly linked genes oneencoding a subunit of higher, and the other of lower molecular weight, designated as x- a y-typerespectively. Despite the fact that durum wheat possess four HMW-GS genes, the number ofexpressed subunits ranges from one to three because of gene silencing processes. The y-type gene atthe Glu-A1 locus is always silent in cultivated wheat, while the x-type at the same locus isexpressed only in some cultivars. The identification of “quality-associated” glutenin subunits thatcan be incorporated into durum wheat, has led to wider studies on allelic variation at each of theencoding loci which has been shown to have different effects on technological performance ofdurum semolina.

In order to identify sources of novel genes and to study changes occurred in encoded proteins,the HMW-GS present in durum wheat cultivars grown in Italy seventy years ago, have beenanalysed by biochemical and molecular techniques. HMW-GS alleles encoded at two glutenin locihave been identify, from a set of old durum wheat cultivars and commercial varieties recentlyreleased, by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). At theGlu-A1 and Glu-B1 loci, one and six alleles have been observed respectively. Comparison ofglutenin alleles among old cultivars and modern commercial varieties confirmed the presence ofallelic pairs associated to poor quality score. Genes corresponding to these subunits were amplifiedby polymerase chain reaction (PCR) using specific primers. DNA fragments corresponding to x-and y-type genes associated to the A and B genomes were detected in all accessions including thosewithout expressed x- and y-type subunits. The presence in the y-type genes associated to A genomeof the particular 8-kb transposon-like insertion, usually present in unexpressed y-type genes at theGlu-A1 locus in Chinese Spring, has been also shown confirming previous results obtained in wildand cultivated tetraploid wheat.



IDENTIFICATION AND MOLECULAR TAGGING OF A POWDERYMILDEW RESISTANCE GENE IN TETRAPLOID WHEAT

A. M. M. ABD-ELBACKI, R. SIMEONE, A. V. CARLUCCIO, A. GADALETA, A. BLANCO

Department of Environmental and Agro-Forestry Biology and Chemistry, Section of Genetics andPlant Breeding, University of Bari, Via G. Amendola 165/A, 70126 Bari, Italy

Triticum turgidum, powdery mildew, resistance gene, molecular markers

Powdery mildew caused by the pathogen Blumeria graminis f.sp. tritici is a destructive foliardisease in regions with a maritime climate. Breeding for varieties with resistant alleles is the mosteconomical and effective way for controlling the disease. Triticum turgidum L. var. dicoccoides(2n=4x,AABB) the progenitor of cultivated wheats, is crossable with both durum and commonwheat and simply breeding procedures enable an efficient transfer of desirable alleles from eachwild chromosome into its cultivated homoeologue. In a previous screening of emmer wheatcollection, the accession MG29896 resulted particularly interesting for high seed protein contentand resistance to powdery mildew. It was crossed to susceptible durum wheat cultivar Latino and aset of backcross inbred lines (BILs) was produced. These lines were essayed for powdery mildewresistance in field condition: one BC5F5 line (5BIL-42) resulted highly resistant and was crossed toLatino. Segregation analysis of F2 plants showed that the resistance was inherited as a single anddominant locus. One hundred and twenty F3 progenies were also essayed in field condition toconfirm F2 phenotype and to distinguish resistant and susceptible homozygous from segregantprogenies.

Ten homozygous resistant and ten homozygous susceptible F3 progenies were used in thebulked segregant analysis (BSA). Molecular markers (SSR and EST-SSR) were used to identifygenetic markers associated to the resistance gene. Fifty hundred and twenty eight SSRs and 409EST-SSRs have been tested, 194 and 76 were polymorphic between Latino and MG29896respectively. Among them 9 SSRs and 8 EST-SSRs were polymorphic between Latino and 5BIL-42and one marker, EST-SSR BJ261635(AC), was polymorphic also between the resistant andsusceptible bulks. Such marker was screened on the complete F3 progeny and from the regressionanalysis resulted completely linked to the resistance gene. The marker was mapped on chromosomearm 5BL using nulli-tetrasomic and ditelosomic lines of Chinese Spring.

In order to saturate the chromosome region with the resistance gene, analysis by new PCR-based markers (Target Region Amplification Polymorphism) is in progress.

The molecular marker identified in this study has potential use in marker-assisted selection andpyramiding of genes for resistance to powdery mildew in wheat.



GENETIC STRUCTURE AND DIVERSITY IN PHASEOLUS COCCINEUS L.BY USING SSR (SIMPLE SEQUENCE REPEAT) MOLECULAR MARKERS

P. ARCALENI*, B. TIRANTI*, G. SPATARO*, G. ATTENE**, R. PAPA***,P. SPAGNOLETTI ZEULI****, V. NEGRI*

*) Department of Plant Biology and Agro-environmental and Animal Biotechnology, University ofPerugia , Borgo XX Giugno**) University di Sassari***) Università Politecnica delle Marche, Ancona, Faculty of Agriculture****) Università della Basilicata

P. coccineus, genetic diversity, molecular markers, SSR, European germplasm

P. coccineus L. (2n = 2x = 22) originated in Mexico and Central America. It was introduced inEurope after the discovery of America and, nowadays, is widely cultivated because of its suitabilityto cool environments.

A first taxonomic treatment recognized five subspecies: coccineus, formosus, glabellus, griseusand darwinianus. Two of these subspecies are cultivated, coccineus and darwinianus, but only theformer was present in Europe. A recent classification redefined the subspecies darwinianus as P.dumosus .

At the present, few information about the level of genetic diversity of the whole Europeangermplasm of P. coccineus are available, even thought genetic variation and structure in somelandraces and populations of this crop from Spain, Central Italy and Poland were carried out. Allthese studies detected a high level of polymorphism.

The aim of this study was to compare the level of genetic diversity in landraces of P. coccineusfrom European and central America by using SSR (Simple Sequence Repeat) molecular markers.The comparison between the European and the Mesoamerican germplasm “core collections”allowed to:

I. investigate the genetic variation and structure in P. coccineus landraces from a worldwidecollection.

II. improve the knowledge of the evolutive process of the European landraces, evidencing thatgenetic diversity was reduced and that a distinct genetic pool originated;

III. obtain an overall picture for future plant breeding activities.

Data show that two distinct runner bean gene pools exist. The diversity present inMesoamerican gene pool is higher in respect to the European gene pool. Introduction in Europe hasoriginated a particular gene pool which should be the primary source of genetic variability forbreading activities in Europe.



DEVELOPMENT AND USE OF CHLOROPLAST MICROSATELLITES(CPSSR) IN PHASEOLUS SPP. AND OTHER LEGUMES

S.A. ANGIOI*, F. DESIDERIO**, D. RAU**, E. BITOCCHI**, L. NANNI**, G. ATTENE*,R. PAPA**

*) Dipartimento di Scienze Agronomiche e Genetica Vegetale Agraria, Università degli Studi diSassari, Via de Nicola, 07100 Sassari, Italy**) Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, Via BrecceBianche, 60131 Ancona, Italy

chloroplast microsatellites, leguminosae, Phaseolus spp.

Due to their relatively high levels of polymorphism and because they are generally inheriteduniparentally, chloroplast microsatellites represent a useful tool for the study of genetic variationand evolution of plants. The flanking regions of chloroplast microsatellite loci are conserved amongdifferent related species, and so this allows the testing of primer pairs that were developed for onespecies also in species belonging to the same genus, to the same family, and even to differentfamilies.

We screened 35 primer pairs from those published in the literature, and 27 of them gave asingle and scorable fragment in a small set of Phaseolus accessions. For one locus, we redesignedthe primer pair using the published Phaseolus sequence to optimise the quality of the PCR product.Finally, a further two markers were designed using published Phaseolus sequences, for a total of 29markers that were selected for further analysis. All 29 primer pairs were used to analyse a core setcomposed of 8 different genera of Leguminosae (Phaseolus, Lupinus, Pisum, Arachis, Vigna, Cicer,Glycine and Medicago) and 5 different species of Phaseolus (P. vulgaris, P. coccineus, P. dumosus,P. lunatus and P. acutifolius). Among all the 29 primer pairs tested, 27 were polymorphic amongthe genera, of which 18 were polymorphic within the Phaseolus genus and 14 were polymorphic inP. vulgaris or P. coccineus. Our results suggest that these sets of markers could be very useful toolsto study the diversity and the evolution of several legumes, and particularly of the Phaseolusspecies like P. vulgaris and P. coccineus.



NUCLEAR AND CHLOROPLAST MICROSATELLITE DIVERSITY INPHASEOLUS VULGARIS L. FROM SARDINIA (ITALY)

S.A. ANGIOI *, M. RODRIGUEZ*, D. RAU **, F. DESIDERIO **, R. PAPA**, G. ATTENE*

*) Dipartimento di Scienze Agronomiche e Genetica Vegetale Agraria, Università degli Studi diSassari, Via de Nicola, 07100 Sassari, Italy**) Dipartimento di Scienze degli Alimenti, Università Politecnica delle Marche, Via BrecceBianche, 60131 Ancona, Italy

genetic resources, Phaseolus vulgaris L., chloroplast and nuclear microsatellites

Domesticated Phaseolus vulgaris L. originated in America and was introduced into Europeafter the travels of Columbus in 1492. The European common bean comes from two centres ofdomestication of the species, Mesoamerica and the Andes. Over the last few decades, the commonbean agro-ecotypes have been gradually replaced by modern cultivars. However, as with otherEuropean countries, in Italy it has been reported that several local populations still survive on-farmin marginal areas. When possible, these should be first conserved and then characterized, to maketheir conservation, evaluation and use for breeding purposes possible and easier. Moreover, ananalysis of local landraces could also help to reconstruct the dissemination pathway and theevolution of the crop into the Old World.

In collaboration with the ‘Centro per la Conservazione e Valorizzazione della BiodiversitàVegetale’ (Centre for Conservation and Evaluation of Vegetal Biodiversity), we collected 73 localvarieties in Sardinia, Italy (i.e cultivated by the same farmer for at least 30 years) in areas wheretraditional farming is still practised. The genetic diversity was investigated with molecular andmorphological traits with three main aims: 1. to determine the relative contribution of the Andeanand the Mesoamerican gene pools to the Sardinian collection; 2. to compare local landraces withcommercial varieties that have been introduced over the last few decades in Sardinia and to assessthe degree of distinctiveness of the local accessions; 3. to estimate and compare the relative levelsof genetic diversity in the common bean species in a single region of Europe (Sardinia, Italy).Molecular analyses were carried out using simple sequence repeats (SSRs) developed from gene (5loci) and genome (5 loci) sequences, and with chloroplast SSRs (cpSSRs, 14 loci) as cytoplasmicmarkers, including all of the Sardinian landraces, 22 American genotypes (13 Andean and 9Mesoamerican) and 15 commercial varieties. A morphological characterisation of the seeds basedon the IPGRI descriptor list for the common bean was also performed.

The results suggest that: 1. the majority of the local P. vulgaris varieties is of Andean origin; 2.the Sardinian accessions share the same multilocus genotypes with the commercial varieties morefrequently using cpSSRs compared to nuclear SSRs; 3. a relevant but not high overall level ofdiversity is present within the P. vulgaris genetic resource collection from the Sardinia region.



THE EFFECT OF SELECTION ON LOCI WITHIN CLOSE PROXIMITY OFDOMESTICATION LOCI IN COMMON BEAN (PHASEOLUS VULGARIS L.)

M. ROSSI*,**, S. MAMIDI**,***, E. BELLUCCI*, M.D. MCCONNELL**,***, R.K. LEE**,***,R. PAPA*, P.E. MCCLEAN**,***

*) Dipartimento di Scienze degli Alimenti, Facoltà di Agraria, Università Politecnica delle Marche,Via Brecce Bianche, I-60131 Ancona, Italy - [email protected]**) Department of Plant Sciences, North Dakota State University, Fargo, ND, 58105 [email protected]***) Genomics and Bioinformatics Program, North Dakota State University, Fargo, ND, 58105USA

crop improvement, domestication, Phaseolus vulgaris, selection

Identifying regions of the genome that have been the targets of selection will provide importantinsights into the evolutionary history and facilitate the identification of important agronomic genes.The structure of genetic diversity of modern crops is deeply influenced by the processes ofdomestication and plant breeding. Both domestication and plant breeding reduced genetic diversitydue to random genetic drift (bottlenecks) and because of selection for target genes. World-widecommon bean (Phaseolus vulgaris L.) is the most important source of proteins for direct humanconsumption, and the identification of genes of agronomic importance may facilitate improvedproductivity and quality of this important crop. We sequenced gene fragments spanning 6 cM of agenomic region of the linkage group B8 where several major QTLs related to the domesticationsyndrome have been previously located. We compared three sets of accessions representing thevarious stage of common bean improvement (wild, landraces and improved cultivars). Genefragments representing other genomic regions were sequenced for comparison. Overall, weanalysed 48 genotypes chosen on the basis of SSR data in order to represent the largest diversitywithin each set of accessions. We performed several statistical tests to identify the signature ofselection due to domestication and crop improvement. This research is very promising because wemay be able to identify genes of potential agronomic importance and to determine the effect ofdomestication and breeding on the structure of genetic diversity in the common bean genome.



GENETIC RELATIONSHIPS AMONG LENTIL (LENS CULINARISMEDIK.) LOCAL POPULATIONS ASSESSED BY AFLP AND I-SSR

F. FIOCCHETTI*, B. LADDOMADA**, M. ROSELLI*, P. CRINÒ*, S. LUCRETTI*

*) ENEA C.R. Casaccia, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, Sez.Genetica e Genomica Vegetale, Via Anguillarese 301, 00060 Roma**) CNR ISPA, Via Prov.le Lecce-Monteroni, 73100 Lecce

biodiversity, ecotype, Lens culinaris, molecular markers

Lentil (Lens culinaris Medik.) is an important seed legume crop cultivated worldwide as humanfood and dating back to pre-historic times. In the last century, lentil was widely cultivated in Italy,providing a cheap source of dietary proteins and allowing spreading of a lot of local biotypes.Socio-economic changes which occurred in the last 50 years in Italy had conducted to a drasticreduction of lentil cultivation (by 93%) resulting in the disappearance of several local populationsand exposing the ones still in cultivation to a high risk of genetic erosion. In order to preventdisappearance of genetic variation inside landraces it is important the improvement and theconservation of the already existing biodiversity. To estimate the level and distribution of geneticvariation in endangered local populations using molecular markers is a primary objective ofconservation genetics. The aim of the current research was to investigate the level of geneticvariation within and among three of the most appreciated Italian common lentil ecotypes: Onano,Altamura and Villalba.

For Altamura, Onano and Villalba, 29, 30 and 25 plants respectively were analysed atindividual level by fluorescent AFLP (Amplified Fragment Lenght Polymorphism) markers usingsix primer combinations and by means of bulks using I-SSR (Inter-Simple Sequence Repeats)markers utilizing 14 primer. Out of the 643 discernible DNA fragments generated by AFLP, 565(87.87%) were polymorphic. The percentage of polymorphic bands within populations ranged from49.92 to 62.99. Genetic diversity (H) within populations ranged from 0.147 to 0.167. The fixationindex (Gst = 0.219) showed that about 78% of the total genetic variability observed can be attributedto within population differences and approximately 22% is due to differences among the threelandraces. The estimated gene flow ( Nm = 1,779) confirmed the condivision of a common genepool.

Genetic similarity between individuals estimated by Dice coefficient was calculated in allpossible pair-wise comparisons and the correspondent matrix was used for the construction of anUPGMA dendrogram, showing that genotypes belonging to the same landrace clustered in the samegroup. The genetic similarity estimates within and between populations ranged from 0,82 to 0,86and from 0,71 to 0,75, respectively. The low level of genetic diversity exhibited by Altamura,Onano and Villalba suggests a common origin for them. Our results also show the ability of AFLPmarkers to distinguish the genotypes analyzed into different clusters according to their geographicorigins.

The comparison between the AFLP dendrogram obtained from markers performed at individuallevel and the one provided by I-SSR markers performed by bulked DNA showed a high correlation

(96%) suggesting a good efficiency of both methods with a greater discriminative ability of AFLPmarkers in comparison with I-SSR.



MORPHOLOGICAL, BIOCHEMICAL, AND MOLECULARCHARACTERIZATION OF GPI “SENISE” PEPPER LANDRACE

G. SARLI, A. DE LISI, D. PIGNONE, G. SONNANTE

Institute of Plant Genetics, National Research Council, Bari, Italy

Capsicum annuum, landrace, evaluation, promotion

The landrace “Peperone di Senise” is a long standing typical crop, specifically produced in alimited area along the Serrapotamo river, in the Basilicata region, adjacent to the “Pollino” NationalPark, and represents a characteristic element of the traditional gastronomy. It is consumed bothfresh and dried and is also used to produce a spice (fruit powder) largely used in food preparation.Due to its specific traits, the “Peperone di Senise” landrace has been attributed the PGI mark (EUregulation 163/96). Nowadays it is cultivated over some 50 ha (ca 125 acres) with a yearlyproduction around 900.000 kg (ca 142.000 stones).

In order to support the economic promotion of this landrace, in collaboration with theBasilicata Region extension service (ALSIA), 18 different populations of this landraces werecollected from the local farmers production fields. From these populations, after three years ofagronomic evaluation, 8 biotypes were selected for superior fruit and plant characters.

These biotypes were cultivated in the experimental field of Policoro, in metapontino’s area,under pollination isolation, and morphological data collected over two years (2004-2005).Biometric characters, derived from the descriptors for Capsicum outlined by IPGRI and agreed atthe international level, were used to describe the plants and regarded mostly plant habit, flowerscharacters and fruit morphology, position and colour; also qualitative characters of the fruits wereconsidered, such as low water content or pericarp width.

Biochemical characterization was conducted on mature fruits and regarded soluble solids,capsicin, ascorbic acid, capsanthin, cryptoxanthin (the red components of pericarp colour) and β-carotene (the yellow component) content.

Molecular characterization made use of AFLP markers and was performed on 10 individualsper sample using an automated sequencer. Five primer combinations produced useful dominantalleles to be used in similarity and population analyses. In order to compare the subpopulations withthe original landrace samples collected, the molecular analyses were carried out on both sets ofsamples.

The selected subpopulations are now being cultivated under strict isolation in order to increaseseed availability.

Aknowledgements: the authors wish to thank Dr. Vincenzo Montesano and Ms LorenzaSannino for their assistance to this research.



CAPSICUM EST-DERIVED MICROSATELLITES AND THEIR POTENTIALUSE FOR MAPPING STUDIES

E. PORTIS*, I. NAGY**, Z. SASVÁRI**, A. STÁGEL**, L. BARCHI*, S. LANTERI*

*) DiVaPRA , Plant Genetics and Breeding, University of Turin, Via L. da Vinci 44,10095 Grugliasco (TO), Italy - [email protected]**) Agricultural Biotechnology Center, H-2100 Gödöllö Szent-Györgyi Albert u. 4, Hungary

Capsicum spp., EST, linkage mapping, SSR

The genus Capsicum belongs to Solanaceae family and includes five cultivated species: Cannuum, C. frutescens, C. chinense, C. pubescens, C. baccatum. C. annuum, known as pepper,sweet pepper or paprika is the most cultivated worldwide.

Several genetic maps have been constructed for Capsicum spp. in the past twenty years byusing F2, BC1, RILs or doubled haploid (DH) populations of interspecific crosses C. annuum x C.chinense or C. annuum x C. frutescens and from intra-specific crosses of C. annuum.

To date there is no map of Capsicum that achieve the goal of completely delineating andsaturating pepper chromosomes, however DNA markers linked to some agriculturally importantcharacters have been identified and marker assisted selection (MAS) has become feasible for sometraits.

Among the different classes of molecular markers available, microsatellites are particularlyuseful for mapping studies because of their high variability and ease of detection by PCR. Here wereport on the development of EST-derived microsatellites which show the advantage of detectingvariation in the expressed portion of the genome, allowing greater transferability across species, andin some cases having additional utility as anchor markers for comparative mapping.

A computer search of 8.094 sequence from Capsicum species in the EMBL NucleotideSequence Database (http://www.ebi.ac.uk/embl), corresponding to approximately 4.1 Mb, yielded1325 microsatellite in 899 sequences. This corresponds to an average distance between SSRs ofapproximately 3.1 kb or one SSR-containing sequences every 9.0 sequences.

In order to obtain non-redundant sequences containing SSRs, a cluster analysis wasperformed; the contigs obtained were carefully evaluated and redundancies were removed. In total,783 microsatellites in 576 non–redundant sequences were identified; among them 531 weremononucleotide, 103 dinucleotide and 137 trinucleotide motifs. Trimeric SSR repeats appeared tobe more abundant than the dinucleotide ones.

Primer pairs could be designed for 348 out of the 576 SSR-containing sequences (60.4%).The remaining sequences contained either too little DNA sequence flanking the microsatellite or thesequences were inappropriate for primer modelling. A subset of 204 primer pairs flanking SSR lociwas used for screening polymorphisms among four C. annuum inbred lines (‘P4’, ‘GD1’, ‘YoloWonder’ and ‘Criollo de Morellos 334’) and one C. frutescence accession (cv. Tabasco) beingparents of three mapping populations. As a result, 49 EST-derived SSR-markers were foundvaluable for genetic mapping; 47 of them were polymorphic in the inter-specific population‘Tabasco’ x ‘P4’, while 21 might be mapped in both the intra-specific populations ’GD1’ x‘CM334’ and ‘Yolo Wonder’ x ‘CM 334’. In order to assess marker diversity and judge the utility

of these markers for fingerprinting, PIC (polymorphic information content) values were assessedusing 16 Capsicum accessions, including 6 accessions of C. annuum and 10 genotypes belonging toeither the species C. frutescens, C. chinense, C. pubescens, C. chacoense, C. eximium, C. baccatumvar. baccatum, C. baccatum var. pendulum, and C. praetermissum. A total of 219 PCR fragmentswere amplified and the estimated PIC values ranged from 0.24 to 0.86, with an average of 0.62.



THE USE OF A NEW MOLECULAR MARKER FOR ASSISTEDSELECTION OF SAN MARZANO, THE MOST FAMOUS TOMATO IN THEWORLD

E. SABATINI***, S. VOLTATTORNI*, G. L. ROTINO**, N. ACCIARRI*

*) C.R.A. Istituto Sperimentale per l’Orticoltura, Via Salaria 1, 63030 Monsampolo del Tronto(AP)**) C.R.A. Istituto Sperimentale per l’Orticoltura, Via Paullese 28, 26836 Montanaso Lombardo(LO)***) C.R.A. Istituto di Sperimentazione per la Pioppicoltura, Strada Frassineto 35, 15033 CasaleMonferrato (AL)

ovate, CAPS marker, Campania, virus, pear shape

Tomato is one of the most important vegetable crop in the world and particularly in thecountries of the Mediterranean basin. Approximately 65% of the tomatoes grown in theMediterranean countries are for processing, while the rest are marketed fresh. The Campania regionin Italy produces 37.400 tons of tomatoes for processing, which represents 6% of Italy’s totalproduction.

The typical and famous peeled tomato grown for processing in Italy and particularly in theCampania region is the San Marzano variety. This traditional variety, grown for fresh market too,has been awarded the protected origin label “Pomodoro San Marzano Dell’Agro SarneseNocerino”.

The San Marzano fruit is thin, elongated and pointed. The conjecture is that this tomatoresulted from a spontaneous hybridization of two other varieties and was distinct from both of themand from the Roma plum tomato. The pulp has a distinctive taste: it has less sugar than other tomatovarieties. According to Neapolitan tradition, pizza was invented as a vehicle for the consumption ofthe San Marzano.

S. Marzano production in the Campania region has declined significantly in the last decadesdue to high sensibility to vascular diseases and to epidemics of cucumber mosaic virus. In the1980s, this region was the number one for peeled tomatoes production in Italy. Now, Campania isthe 4th or 5th produced with 35% of the peeled tomatoes grown in Italy. Production of typicalS.Marzano variety in Campania is declining at a rate of about 12-16% a year. The concern is thatthe San Marzano tomato will disappear from the Campania region.

Therefore, for processing and for fresh market consumption, new F1 hybrids, with similarshape, have been preferred to the original San Marzano; these hybrids have a different taste and areoften genetically far from San Marzano, but possess genetic resistance to the most threateningtomato pathogens.

Some genetic tools have already been developed to characterize the original accessions. Thesemethod are mainly based on the characterisation of hyper variable regions of the genome, like SSR,but are less used in marker assisted selection for the introgression of genetic resistances, where afast and high-throughput method is required.

We developed a CAPS marker for the “ovate” gene in tomato (Sabatini et al, 2005). It wasdeveloped to assist selection when genetic resistances or other features are introgressed fromelongated or round-fruited cultivar to neck-constricted ones.

Here we report the finding that the original accessions of San Marzano carry the ovate gene inits mutated form, which confer neck constriction, while the most modern F1 hybrids, resemblingSan Marzano, do not. This CAPS marker can be usefully exploited for genetic improvement of theoriginal San Marzano.

Sabatini E., G.L. Rotino, S. Voltattorni and N. Acciarri, 2005.A novel CAPS marker derivedfrom the ovate gene in tomato ( L. esculentum M.) is useful to distinguish two Italian ecotypes andto recover “pear” shape in marker assisted selection. European Journal of Horticoltural Science, inpress.



CHARACTERIZATION OF 3X X 2X ANEUPLOID HYBRIDS IN POTATO

M. IORIZZO*, A. ZOINA**, L. FRUSCIANTE*, A. BARONE*, D. CARPUTO*

*) Department of Soil, Plant and Environmental Sciences, University of Naples “Federico II”**) Department of Arboriculture, Botany and plant Pathology, University of Naples “Federico II”

potato, ploidy bridge, aneuploidy, resistance to abiotic and biotic stresses, cDNA AFLP

A ploidy bridge strategy was used to overcome isolation barriers due to endosperm balancenumber (EBN) differences between wild Solanum commersonii (cmm 2n=2x=24, 1EBN) and S.tuberosum haploids (tbr 2n=2x=24, 2EBN). Following 3x x 2x crosses between triploid cmm x tbrhybrids and 2x genotypes, progenies with 29-36 somatic chromosomes were obtained. Clearly, onlyeggs with 17-24 chromosomes of the triploid parents were able to develop mature embryos. Theseresults may be explained on the basis of a post-zigotic barrier operated by the EBN. To estimate theintrogression of interesting traits from the wild parent, 3x x 2x genotypes were screened forresistance to abiotic and biotic stresses. Most hybrids showed tolerance to low temperatures underacclimated conditions. The screening for resistance to Ralstonia solanacearum allowed resistanthybrids to be identified. In order to further study the resistance to R. solanacearum, cDNA AFLPtechnology was applied. Differential banding patterns between susceptible tbr and resistant cmmgenotypes were detected. Polymorphic fragments were sequenced and compared to the GenBankdatabase. Preliminary results confirmed the switched on of genes involved in resistance response.The specific fragments from the resistant genotypes up till now analyzed did not find similarity inthe database.



MICROSATELLITE MARKERS ARE POWERFUL TOOL FORDISCRIMINATING AMONG OLIVE CULTIVARS AND ASSIGNING THEMTO GEOGRAPHICALLY DEFINED POPULATIONS

V. SARRI*, L. BALDONI**, A. PORCEDDU**, A. CONTENTO*, N. CULTRERA**,M. FREDIANI***, P.G. CIONINI*

*) Department of Cellular and Environmental Biology, University of Perugia, Via Elce di Sotto,06123 Perugia, Italy – [email protected]**) Institute of Plant Genetics, Perugia, CNR, Via Madonna Alta 130, 06128 Perugia, Italy***) Department of Agrobiology and Agrochemistry, University of Tuscia, Via S. Camillo deLellis, 01100 Viterbo, Italy

Olea europaea, cultivar discrimination, olive domestication, olive germplasm, SSR markers

More than 1,275 olive cultivars have been described. Their uncertain origin, the simultaneouspresence, in a given area, of local, cosmopolitan and patchy distributed varieties, the clonalvariation found in some of them, their denominations related to fruit shape or color, oil taste andtree structure, or associated to the toponym of the cultivation site, seriously affect the identificationof the cultivars, making the management of olive as a resource extremely complex. Furthermore,the spread of olive from few centers of origin and a further multilocal selection, joined with thecontinuous interchange of plant material within different regions, have contributed to the confusedpattern of cultivar geographical distribution. However, the possibility of discriminating withcertainty and relative ease between olive cultivars would be of great interest, e.g., in order tocertificate the origin and quality of oils. Moreover, it would be of interest to be able to distinguishbetween local and allochthonous germplasm, in order to recognize the regional resources, evaluatethe adaptation capacity of each variety and support the initiatives to identify and promote typicalolive oils.

Simple sequence repeats (SSRs) in the nuclear DNA have been suggested to be reliable andeasy-to-use markers for genotype identification. In an attempt to establish their effectiveness indiscriminating among olive cultivars and assessing their ability in assigning individuals to varietalpopulations according to geographic criteria, twelve SSR loci were selected and used to analyze thegenetic structure of olive cultivar gene pools and differentiate among one hundred and eighteencultivars sampled in several countries of the Mediterranean Basin. The markers were found to havea high discrimination power. On average, with a single assay was possible to discriminate 96% ofthe pairwise comparisons and, with a combination of three loci, virtually all cultivars weredistinguished. The markers were also tested for their utility to assign cultivars to their geographicpopulation of origin. A selection of six markers was found to maximize assignment accuracy,correctly reallocating up to 75.4% of cultivars to their aboriginal population.



DEVELOPMENT AND APPLICATION OF MOLECULAR MARKERS FORGENETIC VARIATION ASSESSMENT IN A PIEDMONTESE LANDRACEOF CELERY

F. MAGURNO, A. ACQUADRO, E. PORTIS, S. LANTERI

DiVaPRA, Plant Genetics and Breeding, University of Turin, Via L. da Vinci 44, 10095 Grugliasco(TO), Italy - [email protected]

Apium graveolens, landrace, AFLP, SSR, genetic variation

In Piedmont, as well as in other Italian regions, a large number of vegetable landraces are stilllocally cultivated. These landraces have a certain genetic identity, are morphologically recognisableand their local demand is growing because of the added value consumers attributes to the taste,appearance, nutritive value of their production and their link with the culture and tradition of thearea. Currently, organic farmers are also interested in introducing crop landraces in cultivation,because of their higher adaptation to local pedo-climatic conditions and/or supposed resistance tobiotic stresses.

During the past years efforts have been made by the Piedmont Region, section AgriculturalDevelopment , to ca ta logue autochthonous loca l vegetable ecotypes(www.regione.piemonte.it/agri/biodiversita/orticolo/schede.htm). At present activities involvingfarmer organizations as well as research institutions are funded, with the main goal to obtain, for atleast some of them, the registration as variety and fit the present seed trade regulations. However, asregistered varieties are required to meet distinctiveness, uniformity and stability (D.U.S.)parameters, it is at first necessary to quantify the amount of genetic variation which is present in thegermplasm in cultivation.

Here we report our preliminary results on the molecular assessment of genetic variation in alocal ecotype of celery (Apium graveolens var. dulce): i.e. ‘Dorato d’Asti’, which is grown insouth–east Piedmont and is well appreciated due to its flavour and the production of highlyvigorous fleshy stalks with golden ribs.

We quantified genetic variation in tree populations by applying two complementary classes ofmolecular markers: amplified fragment length polymorphism (AFLP) and simple sequence repeats(SSR = microsatellites), the latter not available at the time of starting the work. Our first goal wasthus the in silico mining of SSRs. We screened 2277 DNA accessions, downloaded from GenBank,and we found a total of 73 microsatellites containing sequences; the number of potential markerswas reduced by means of a cluster analysis and using the sequence editor of MEGA software:redundant accessions and sequences presenting the repetitive motif at one end were discarded. Afterthat, 39 sequences, all derived from ESTs, were selected for PCR amplification. On the whole wedeveloped 11 dbEST-derived microsatellite markers, whose polymorphism was at first explored insixteen celery commercial varieties, and marker transferability was tested on three accessions ofceleriac (A. graveolens var. rapaceum).

The analysis of AFLP and SSR molecular variance in the three populations of celery ‘Doratod’Asti’ provided useful information for future application of appropriate mass selection and

breeding strategies aimed at restricting the genetic basis of the material in cultivation an promotethe registration of the landrace as a variety.



SPECIES RELATIONSHIP AND HYBRID IDENTIFICATION IN LILIUMUSING RAPD MARKERS

M. CAMMARERI*, F. CAMPANILE**, C. CONICELLA*, A. ERRICO***

*) Institute of Plant Genetics – CNR, Research Division Portici, Italy**) Research Institute for Industrial Crops, MIPAF - SS.18 n° 156, 84091 Battipaglia, Italy***) Department of Soil, Plant and Environmental Sciences, University of Naples “Federico II”,Via Università 100, 80055 Portici, Italy

Lilium, chromosome number, genetic diversity

The genus Lilium includes about a hundred species classified into seven Sectionsdistributed through the cold and temperate region of the Northern hemisphere. Thisgermplasm represents an important source of useful genes as well as a "reservoir" of allelicdiversity for breeding purposes. However, gene introgression through the wide inter-specifichybridization is hampered by incompatibility barriers at pre- and post-fertilization levelsincluding F1 hybrid sterility.

In the present investigation, putative inter-specific hybrids of Lilium obtained byconventional crosses were characterized. The hybrids and their parents were analyzed byRAPD markers at an early seedling stage as well as by morphology at differentdevelopmental stages. In addition, RAPDs were used to estimate the genetic diversity in asample of Lilium species that was also characterized for chromosome number and pollenstainability. The chromosome number of the material used in the experiments includingdifferent Lilium species (L.bulbiferum, L.candidum, L. martagon ,L. .formosanum Lmiryophyllum,.L. pyrenaicum, L.pumilum, L.willmottiae, L.regale) as well as some cultivars(Snow Queen, Cascade, Pollyanna) resulted diploid (2n=2x=24). The exception wasrepresented by Elite cultivar that is triploid.

Inter-specific crosses were made between three species, L. formosanum, L. miryophillum,L. regale, and four cultivars: Cascade, Elite, Pollyanna, Snow Queen. Capsules were obtainedfrom all cross combinations, except from Elite x L. miryophillum, Elite x L. regale andPollyanna x L. regale. Average seed number per capsule was generally lower in the crossesthan in selfings. Average germination time was also estimated: in particular, L. formosanumx Elite germinated significantly later as compared to parents.

Firstly, RAPD analysis was performed on the parental lines and on the other Liliumspecies. 35 random primers revealed polymorphic amplified DNA fragments. The percentageof polymorphic bands for each primer ranged from 0.1 to 9 %. Genetic similarities amongLilium species, estimated through simple matching coefficient, based on both shared andunique amplification products, ranged from 0.53 to 0.69 across nine species. The highestsimilarity was detected between L. regale and L. myriophyllum. The dendrogram obtainedusing UPGMA cluster analysis evidenced the Lilium species subdivided into two majorgroups, one including L. formosanum and L. willmottiae. To identify the hybrids, nine randomprimers were used since they were informative to discriminate between parents. So far, L.formosanum x Elite hybrids have evidenced the paternal band.

DNA markers generated by RAPDs allowed the identification of genotypes during a veryearly stage of plant development giving an helpful support to the breeding programmes ofLilium.



INHERITANCE OF A TASSEL SEED CHARACTERISTIC IN MAIZERESULTING FROM IN VITRO CULTURE

S. SALVI, F. ROTONDO, M. A. CANÈ, E. FRASCAROLI, P. LANDI

Department of Agro-environmental Science and Technology (DiSTA), University of Bologna,Viale Fanin 44, I-40128, Bologna, Italy

somaclonal variation, tassel seed, tissue culture, Zea mays

Regeneration from in vitro tissue culture can give rise to plants showing genetic variation,which can be of classical as well as of novel types. In a previous study in maize (Zea mays, L.), weconducted three cycles of recurrent selection aimed at improving plant regeneration capacity fromcallus cultures of immature embryos, using as source population the double cross (A188 x W64A) x(A634 x B79). The population resulting from the third selection cycle (C3) was subjected to oneselfing generation and in the subsequent S1 population a plant showing tassel seed phenotype (ts)was identified. An inbred line (Bo22) was then developed from this ts plant. Throughout the selfingprocess the ts phenotype proved to be stable. Objectives of this study were to: (a) gain informationon the inheritance of the trait, and (b) map the locus/loci responsible for such phenotype by meansof molecular marker analysis.

The ts inbred line Bo22 was crossed to the three wild type (wt) inbred lines W23, B73 andLo976; then corresponding F2 as well as BC1 (using Bo22 as recurrent parent) generations wereproduced. The three F1’s always showed the wt phenotype. For the cross Bo22 x W23 and Bo22 xB73, the F2 and BC1 generations showed phenotypic frequencies (wt:ts) consistent with 15:1 and3:1, respectively, in accordance with the hypothesis of duplicate gene action. On the other hand, thegenerations derived from Bo22 x Lo976 showed segregating ratios not consistent with this simpleinheritance hypothesis.

Bulk Segregant Analysis (BSA) using 114 publicly available SSR markers was carried out onbulked samples of ts and wt plants from the two BC1 populations (Bo22 x Lo976) x Bo22 and(Bo22 x B73) x Bo22. Association of the ts phenotype with one chromosome region (chrom. bin6.07, near umc2165) was confirmed after the analysis of both BC1 populations. The same 6.07markers confirmed the association of bin 6.07 with the ts phenotype in two other small F2

populations. No ts mutations have been previously mapped in this chromosome region.In conclusion, two genes with duplicate action seem to be responsible of the ts phenotype

shown by the inbred line Bo22 at least in two of the three analysed crosses, and one of these twogenes appears to map in bin 6.07. Further analyses are now in progress to identify and map the othergene involved.

Proceedings of the 50th Italian Society of Agricultural Genetics Annual Congress

Ischia, Italy – 10/14 September, 2006

ISBN 88-900622-7-4


TISSUE CULTURE RESPONSE OF SOLANUM BULBOCASTANUM (+) S.

TUBEROSUM SOMATIC HYBRIDS

S. SAVARESE, M. IOVENE, R. AVERSANO, L. MONTI, D. CARPUTO

De partment of Soil, Plant and Environmenta l Scienc es, University of Naples “Fede rico II”,

Via Università 100, 80055 Portici, Italy - sa sa vare@ unina.it

wild species, potato, anther culture, haploid, somaclonal variation

In the present study, Solanum somatic hybrids were tested for their ability to respond to tissue

culture. Plant material consisted of 4x and 6x genotypes obtained from Solanum bulbocastanum (+)

S. tuberosum haploids fusions. They carry noteworthy traits from a breeding standpoint, but they

are sterile and therefore cannot be crossed both as female and male parent.

In order to utilize them, we carried out two in vitro approaches. The first is based on

regeneration from leaf/internode explants, in order to exploit somaclonal variation. The second is

based on haploid extraction through anther culture. We may expect that due to recombination,

haploids may be fertile in crosses with potato genotypes. From four somatic hybrids internode and

leaf explants were excised from in vitro plantlets and a two-step regeneration protocol was

employed. All the clones tested regenerated, with an average regeneration frequency of 60% and 4

shoots explant. Molecular analysis through ISSR markers provided evidence that genetic

rearrangements occurred. Plant material is being tested for fertility. As for anther culture, in vitro

androgenesis through direct embryogenesis was carried out for six somatic hybrids. Two thousand

and five hundred anthers were cultured and 27 produced microspores-derived embryo structures. In

total, 10 green plants were regenerated. From two somatic hybrids we obtained embriogenetic mass.

They generated 80 embryos which evolved later in shoots. Haploids derived from interspecific

somatic hybrids are relevant research materials for inheritance studies and for cytological

determination of transmission of parental species chromosomes into microspores and haploid

plants. Crosses will be performed between haploids and S. tuberosum genotypes with various

Endosperm Balance Number. Alternatively, haploids will be fused with potato haploids.



ISBN 88-900622-7-4


TISSUE CULTURE RESPONSE OF SOLANUM BULBOCASTANUM (+) S.

TUBEROSUM SOMATIC HYBRIDS

S. SAVARESE, M. IOVENE, R. AVERSANO, L. MONTI, D. CARPUTO

Department of Soil, Plant and Environmental Sciences, University of Naples “Federico II”, Via

Università 100, 80055 Portici, Italy - [email protected]

wild species, potato, anther culture, haploid, somaclonal variation

In the present study, Solanum somatic hybrids were tested for their ability to respond to tissue

culture. Plant material consisted of 4x and 6x genotypes obtained from Solanum bulbocastanum (+)

S. tuberosum haploids fusions. They carry noteworthy traits from a breeding standpoint, but they

are sterile and therefore cannot be crossed both as female and male parent.

In order to utilize them, we carried out two in vitro approaches. The first is based on

regeneration from leaf/internode explants, in order to exploit somaclonal variation. The second is

based on haploid extraction through anther culture. We may expect that due to recombination,

haploids may be fertile in crosses with potato genotypes. From four somatic hybrids internode and

leaf explants were excised from in vitro plantlets and a two-step regeneration protocol was

employed. All the clones tested regenerated, with an average regeneration frequency of 60% and 4

shoots explant. Molecular analysis through ISSR markers provided evidence that genetic

rearrangements occurred. Plant material is being tested for fertility. As for anther culture, in vitro

androgenesis through direct embryogenesis was carried out for six somatic hybrids. Two thousand

and five hundred anthers were cultured and 27 produced microspores-derived embryo structures. In

total, 10 green plants were regenerated. From two somatic hybrids we obtained embriogenetic mass.

They generated 80 embryos which evolved later in shoots. Haploids derived from interspecific

somatic hybrids are relevant research materials for inheritance studies and for cytological

determination of transmission of parental species chromosomes into microspores and haploid

plants. Crosses will be performed between haploids and S. tuberosum genotypes with various

Endosperm Balance Number. Alternatively, haploids will be fused with potato haploids.



NUCLEAR AND CYTOPLASMIC CHARACTERIZATION OF CITRUSASYMMETRIC SOMATIC HYBRIDS BY MEANS OF ISSR-PCR AND CAPS

M.-T. SCARANO*,**, N. TUSA**, L. ABBATE**, S. LUCRETTI***, S. FERRANTE**

*) Dipartimento S.A.V.A., Università degli Studi del Molise, Via De Sanctis, 86100 Campobasso**) Istituto Genetica Vegetale sez. di Palermo - C.N.R., Corso Calatafimi 414, 90129 Palermo –[email protected]***) ENEA C.R. Casaccia, Sezione Genetica e Genomica Vegetale (026) Via Anguillarese 301,00060 S.M. di Galeria (Roma)

cybrid, ISSR-PCR, CAPs, flow cytometry

In order to obtain new somatic hybrids for genetic improvement of mandarin, several protoplastfusion experiments have been carried out. Isozyme banding pattern analysis and flow cytometrywere used for early nuclear characterization. DNA of the hybrids was then extracted and subjectedto ISSR-PCR using different primers anchored at 5’ and 3’ ends. ‘Murcott’ tangor + ‘Duncan’grapefruit genotype resulted to be diploid, with morphological traits very similar to ‘Duncan’ (leafparent) and displayed only the banding pattern of ‘Duncan‘ grapefruit. Successively, forcytoplasmic characterization of the hybrids, CAPs using universal mtDNA and cpDNA primerswere carried out; PCR products were digested with different restriction enzymes and resolved on3% Metaphore agarose gel. All the hybrids presented the mtDNA banding pattern of the ‘Murcott‘tangor (callus parent); as for cpDNA, among the 10 analized genotypes, 2 showed the same bandingpattern of ‘Duncan’ grapefruit (leaf parent) and 8 the banding pattern of ‘Murcott’ tangor. Ourresults showed that these new genotypes are nevertheless cybrids resulting asymmetric hybrids, inwhich the nucleus is inherited from the leaf parent and mitocondria from callus parent.



MUTATIONAL EFFECTS OF SYNTHETIC CYTOKININSDIPHENYLUREA-DERIVED ON PLANTS REGENERATED BY SOMATICEMBRYOGENESIS

M. SIRAGUSA, A. CARRA, C. MORGANA, L. ABBATE, F. DE PASQUALE,F. CARIMI

Institute of Plants Genetic – CNR, Research Division Palermo, Corso Calatafimi 414,I-90129 Palermo, Italy

genetic polymorphisms, molecular markers, mutations, plant regeneration, somaclonal variability

Style-stigma explants from calamondin (Citrus madurensis Lour.) were cultured in mediasupplemented with three synthetic phenylurea derivatives, N-(2-chloro-4-pyridyl)-N-phenylurea(4-CPPU), N-phenyl-N’-benzothiazol-6-ylurea (PBU) and N,N’-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU). A cytokinin adenine-derived N6-benzylaminopurine(BAP) supplemented medium and an hormone-free (HF) medium were used as negativecontrols. Somatic embryos were regenerated in all the conditions used and plantlets were obtained.Among the phenylurea analogous compounds, the 2,3-MDPU showed the highest embryogenicpotential (68.0, 49.3 and 43.3% of responsive explants in 2,3 MDPU, PBU and 4-CPPU,respectively) and all of them showed a significantly higher percentage of responsive explants thanthat obtained with BAP and HF conditions (33.7 and 10.0, respectively). In order to verify thepresence of somaclonal variability of the regenerants, twenty-seven somaclones, coming fromdifferent embryogenic events, were randomly selected from each different culture condition andanalyzed by using inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA(RAPD) analyses. We observed that plantlets regenerated in media supplemented with 2,3-MDPUand PBU gave 3.7% of somaclonal mutants, whereas plantlets from medium supplemented with 4-CPPU gave 7.2% of mutants. No somaclonal variability was observed when plantlets wereregenerated in BAP or HF medium. Our results suggest that, although diphenylurea derivativesshow an higher embryogenic potential as compared to BAP, they induce higher levels ofsomaclonal variability. This finding should be taken into consideration when new protocols forclonal propagation have to be set up. On the other side, the regeneration of somatic embryos inpresence of diphenylurea derivatives, that induce somaclonal variability, can contribute to plantgenetic improvement programs.



ISBN 88-900622-7-4


TWO NOVEL TESTS BASED ON GENETIC POLYMORPHISMS FOR

SPECIES IDENTIFICATION IN MILK AND MEAT PRODUCTS

S. REALE, A. CAMPANELLA, M. D’ANDREA, F. PILLA

Department of Animal, Vegetal and Environmental Sciences, University of Molise, Via De Sanctis

snc, 86100 Campobasso, Italy – [email protected]

SNPs, food traceability, domestic animals, k-casein gene, myostatin gene

Identification of the species of origin in animal derived-food has a remarkable importance

because of the possibility of detecting fraudulent procedures, such as the substitution of one type of

ingredient with a cheaper one or accidental adulteration. These facts are of concern particularly in

relation with authenticity and labeling regulations which require accurate declaration of product

compounds, adulterations and human adverse reactions (allergies).

The aim of this work was to develop two sensitive tests that allow to detect and quantify

specie-specific genomic DNA from different animal species in a single assay using genomic DNA

instead ofmitochondrial DNA. A set of primer was designed in conserved region on the basis of the

alignment of the genomic kappa casein sequences from the main milk-producing species (cattle,

sheep, goat and water buffalo). Likewise a primer pair was designed in conserved region of the

myostatin gene in chicken, turkey and pig. In both cases, alignment harvested single nucleotide

polymorphisms (SNPs) that were confirmed and detected via minisequencing using extension

primers designed in conserved sequences for haplotype determination that permits unambiguous

assignment to each species.

The first method was successfully applied to the detection of the different analyzed species in

raw and pasteurized milk as well as in mixed species cheese products from the retail trade,

recovering genomic DNA from somatic milk cells that persist in cheese. The second method was

successfully applied to the quantitative detection of species in meat mixtures such as sausages,

wurstel and hamburgers made up with mixed chicken, turkey and pork meat.

Limit of detection (LOD) estimation was carried out using serial dilutions of genomic DNA

first, and DNA isolated from milk of known number of somatic cells and meat in different species.

The ability of detection resulted to be as low as 0,1% of bovine milk mixed with buffalo milk as

well as with chicken and turkey meat.



ISBN 88-900622-7-4


MOLECULAR CHARACTERIZATION OF DURUM WHEAT “S. AGATA”

BY fAFLP

C. BLANCO*, C. FICHERA**, F. SCIACCA*, S. DI SILVESTRO**, M. PALUMBO*

*) C. R.A.- Experime ntal Institute for Cere al Researc h – Section of Cata nia, Via Varese 43,

95123 Catania, Italy - [email protected]

**) Science and Te chnology Pa rk of Sicily – Zona Industria le Blocc o Palma I, 95030 Catania, Ita ly

- [email protected]

durum wheat, fAFLP, genotype identification, SDS-PAGE

Genotype identification is a fundamental requirement for traceability in the cereal food chain.

Amplified fragment length polymorphism in fluorescence (fAFLP) has become a popular tool for

quickly assessing the genetic background of selected lines or populations. This technique, by

performing a reproducible fingerprinting profiles, allows to add an exact evaluation of amplicons

size to intraspecific risolution and to high polymorphism determined by AFLP markers.

In this research a simplified fAFLP method was developed for identification and molecular

characterization of a new variety of durum wheat, namely Sant'Agata, recently selected by the

Catania Section of C.R.A, by fAFLP. Sant'Agata shows good agronomic and qualitative

characteristics, like optimal growth in Mediterranean environment, good vocation to make bread

and/or make pasta, as demonstred by grain storage proteins characterization, carried out by

identifying HMW and LMW subunits. In SDS-PAGE Sant’Agata shows HMW glutenin subunits

pair “7+8” and LMW glutenin subunits type “2”, both related with good gluten quality.

A selective amplification was performed on fifteen genotypes booked in National Register of

Variety, including Sant'Agata, with sixty-four fluorescence labelled primers combinations in order

to determine polymorphic profiles. The amplified fragments were detected on capillary gel

electrophoresis using the automated DNA sequencer Beckman & Coulter CEQ 8000 with the

analysis fragment option. Four different primer combinations produced most polymorphic bands.

Selective primers generated a total of 2142 AFLP fragments, including 826 (38.6%) polymorphic

on 15 analyzed variety. These primer combinations were selected to identify fingerprinting profile

of Sant’Agata yielding a total of 154 AFLP fragments, including 41 (26.6%) polymorphic. The best

Sant'Agata profile was performed with primer combination EcoRI+AAG* MseI+CTT, obtaining a

66% of polymorphic peaks. The achieved results confirm the usefulness of the proposed

modification of the AFLP method for diversity studies and identification of durum wheat cultivars.

This technique, could be used by the breeders for protecting the property of the cultivars constituted

and by other actors of the cereal sector for the traceability of monovariety products.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION … · ... 60131 Ancona, Italy - r.papa@ ......

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