Microsatellite variability among wild andcultivated hops (Humulus lupulus L.)

Jernej Jakse, Zlatko Satovic, and Branka Javornik

Abstract: Hop (Humulus lupulus L.) is a dioecious perennial plant native to the northern hemisphere cultivated for itsuse in the brewing industry. To investigate the genetic diversity present in wild hop accessions in comparison with cul-tivated hops, microsatellite marker variation was assessed at four loci in 124 accessions of wild (from Europe, Asiaand from North America) and cultivated (varieties and breeding lines) hops. A total of 63 alleles were identified, withan average of 15.7 alleles per locus and an average PIC of 0.64 over four loci. The average number of alleles per lo-cus in groups of accessions ranged from 5.75 to 8.30, with the highest number detected in groups of wild hops eitherof European (EU) or North American (NA) origin. Accessions from NA revealed the highest number of unique allelesindicating the high diversity present in this gene pool. Cluster analysis based on the DD or Dsw distance matrix dividedaccessions into 10 different clusters, which reflect the relationship among geographically diverse wild accessions andhop cultivars. The highest genetic differences were found between NA wild accessions, forming one distant cluster, andall the other accessions. The differentiation between European wild and cultivated accessions was revealed by PCoAbased on the DD distance matrix and by AMOVA results. Cultivated hops differ significantly from wild ones, althoughmost of the variability was found within groups. The molecular variances within groups of cultivated and wild hopswere homogeneous, suggesting that a similar level of molecular variability is found in both groups of accessions. Theanalysis of allele polymorphism and of allele sequences showed that hop germplasm can be differentiated to NA andEU geographic types according to the differences of allele sizes at three loci or by the specific microsatellite repeattype at one locus. The analysis also indicates the different evolutionary dynamics and complex mutations ofmicrosatellite sequences within loci that can be followed in the two biogeographically separated germplasms.

899Key words: Humulus lupulus L., genetic diversity, germplasm, microsatellites, allele sequence variation.

Résumé : Le houblon (Humulus lupulus L.) est une plante vivace dioïque originaire de l’hémisphère nord et cultivéepour son emploi dans l’industrie brassicole. Afin d’étudier la diversité génétique au sein d’accessions du houblon sau-vage en comparaison au houblon cultivé, la variation allélique à quatre locus microsatellites a été mesurée chez 124 ac-cessions de houblon sauvage (d’Europe, d’Asie et d’Amérique du Nord) ou de houblon cultivé (variétés et lignées desélectionneur). Au total, 63 allèles ont été identifiés pour une moyenne de 15,7 allèles par locus et un indice PIC(« polymorphism information content ») moyen de 0,64 pour les quatre locus. Le nombre moyen d’allèles par locuschez les groupes d’accessions variait entre 5,75 et 8,30, le plus grand nombre d’allèles étant détecté chez les houblonssauvages d’Europe (EU) ou d’Amérique du Nord (NA). Les accessions nord-américaines montraient le plus grandnombre d’allèles uniques ce qui indique la présence d’une grande diversité au sein de ce matériel. Une analyse degroupement fondée sur la matrice des distances (DD ou Dsw) a séparé les accessions en dix groupes, lesquels reflétaientles relations parmi les accessions sauvages d’origines géographiques distinctes et les cultivars. Les plus grandes diffé-rences génétiques ont été observées entre les accessions sauvages nord-américaines, lesquelles formaient un groupe dis-tant des autres, et toutes les autres accessions. La différenciation entre les accessions européennes sauvages et cultivéesa été révélée par analyse PCoA basée sur la matrice des distances DD ainsi que par analyse AMOVA. Les houblonscultivés diffèrent significativement des houblons sauvages, bien que la plus grande part de la diversité existe au seindes groupes. Les variances moléculaires au sein des groupes de houblons cultivés et sauvages étaient uniformes, ce quisuggère qu’un degré semblable de variabilité existe au sein des deux groupes d’accessions. L’analyse du polymor-phisme et de la séquence alléliques a montré qu’on peut distinguer des types nord-américains et européens au sein desressources génétiques du houblon en examinant la taille des allèles à trois locus ou encore en déterminant le type demicrosatellite présent à un locus. Cette analyse montre également les différentes dynamiques évolutives et les mutations

Genome 47: 889–899 (2004) doi: 10.1139/G04-054 © 2004 NRC Canada

889

Received 21 November 2003. Accepted 11 May 2004. Published on the NRC Research Press Web site at http://genome.nrc.ca on12 October 2004.

Corresponding Editor: G.J. Scoles.

J. Jakse and B. Javornik.1 Centre for Plant Biotechnology and Breeding, Biotechnical Faculty, University of Ljubljana,Jamnikarjeva 101, 1000 Ljubljana, Slovenia.Z. Satovic. Department of Seed Science and Technology, Faculty of Agriculture, University of Zagreb, Svetosimunska 25,10000 Zagreb, Croatia.

1Corresponding author (e-mail: branka.javornik@uni-lj.si).

complexes au sein de microsatellites situés dans des locus qu’on peut suivre dans deux bassins biogéographiques deressources génétiques.

Mots clés : Humulus lupulus L., diversité génétique, ressources génétiques, microsatellites, variation de la séquenceallélique.

[Traduit par la Rédaction] Jakse et al.

Introduction

Microsatellite loci consist of tandem arrays of short nucle-otide repeats of 1–5 bases per unit that evolve faster thannon-repetitive DNA (Weber and Wong 1993). Slipped strandmispairing between repeats during replication or repair ofDNA synthesis is the most likely mechanism that explainsalterations in microsatellite length variation (Levinson andGutman 1987; Schlötterer and Tautz 1992) and the structureand length of microsatellite repeats are considered to be themajor factor affecting microsatellite variability (Estoup andCornuet 1999). Microsatellites are ubiquitous, abundant andhighly dispersed in eukaryotic genomes, with high variabil-ity at most loci. They are being extensively used in geneticanalysis and evolutionary studies in many organisms. Con-siderable efforts have been made to develop microsatellitemarkers for most important plant crops (Morgante andOlivieri 1993; Powell et al. 1996; Gupta and Varshney 2000).An increasing number of reports on new microsatelliteisolations in other plants, such as grape (Thomas and Scott1993), kiwifruit (Huang et al. 1998), apple (Guilford et al.1997), peach (Testolin et al. 2000), olive (Rallo et al. 2000),chickpea (Udupa et al. 1999), oats (Li et al. 2000) have alsobeen reported, showing successful application of micro-satellites in germplasm management, genetic diversity stud-ies, genetic mapping, and marker-assisted selection. Inaddition to these applications, information about the distri-bution and variability of microsatellite sequences in the ge-nome of a given species provides a means of elucidating thegenetic history of the species from the standpoint of evolu-tion and artificial selection (Alvarez et al. 2001; Matsuoka etal. 2002; Tang and Knapp 2003).

In hop, 4 microsatellites were initially developed andcharacterized (Brady et al. 1996; Jakse et al. 2001) and 14new markers are now available (Jakse et al. 2002). Hop is adioecious perennial plant native to the northern hemisphere.It is cultivated for its female inflorescences (hop cones),which are covered with lupulin glands. These glands secreteresin containing bitter acids and essential oils, componentsof the bitterness, flavour, and aroma of beer. Breeding forimproved cultivars in terms of yield, high resin content anddisease resistance has been a principle aim of hop breedingprograms. Broad-based germplasm collections (wild hops,breeding lines, diverse cultivars) in main hop breeding cen-tres provide good starting material for breeding. The geneticdiversity of these collections, mainly female accessions andcultivars, has been studied by molecular tools (Sustar-Vozlicand Javornik 1999; Seefelder et al. 2000; Jakse et al. 2001;Patzak 2001). These studies have well documented the pres-ence of two geographically distinct germplasms in hopcultivars – European (EU) germplasm, characteristic for itsaroma, and North American (NA) germplasm, with a distinctbitter taste. Wild hops, which were the source of many im-

portant genes (Neve 1991), have been studied to a lesser ex-tent. Wild hops have been classified by leaf morphology andgeographic distribution into five taxonomic varieties (Small1978), although some authors have argued against such a di-vision of the species (Neve 1991). Humulus lupulus var.lupulus E. Small was the denomination given to Europeanhops; wild American hops were divided into three varieties,H. lupulus var. lupuloides E. Small (central and easternAmerica), H. lupulus var. pubuscens E. Small (midwesternAmerica), H. lupulus var. neomexicana E. Small (westernAmerica); and Japanese hops was designated H. lupulus var.cordifolius (Miquel) Maximowicz. Such a divison ofH. lupulus L. has been supported to some extent by chemo-taxonomic (Stevens et al. 2000) and molecular studies(Pillay and Kenny 1996; Murakami 2001). Recent phylo-genetic studies (Murakami et al. 2003) using non-codingcpDNA have suggested that there are primarily two, Euro-pean and Asian – North American types, which divergedapproximately 1.12 million years ago. Within the Asian –North American hop type, further differentiation might laterhave occurred, resulting in Chinese, Japanese, and NorthAmerican geographic populations.

We report here on the use of hop microsatellites to investi-gate the genetic diversity present in wild hop accessions incomparison to cultivated hops and to determine the utility ofmicrosatellites in classifying hop germplasms. Our resultscomfirmed that two geographically diverse hops showedhigh genetic variability and that microsatellites in the twohop germplasms revealed distinctive features.

Materials and methods

Plant materialHop accessions were provided by the hop germplasm col-

lection and hop genebank held at the Institute of Hop Re-search and Brewing, Zalec, Slovenia. Fourteen NorthAmerican wild male accessions were obtained from OregonState University, Corvallis, Ore.

A total of 124 hop accessions were included in the analy-sis (Table 1): 48 cultivated females with known pedigree(Jakse et al. 2001); 3 female and 14 male breeding lines withknown pedigree (provided on request, Institute of Hop Re-search and Brewing, Zalec, Slovenia); 25 wild females and34 wild males of known geographic origin.

Microsatellite amplification and sequencingDNA was extracted from young fresh leaves or, in the

case of the 14 wild USA samples from lyophilised materialusing a standard CTAB buffer (Kump and Javornik 1996),followed by several phenol : chlorophorm : isoamyl alcohol(25:24:1, Sigma-Aldrich Chemie GmbH, Deisenhofen, Ger-many) extractions. DNA was precipitated in high salt condi-

© 2004 NRC Canada

890 Genome Vol. 47, 2004

© 2004 NRC Canada

Jakse et al. 891

No Genotype Sex Breeding status Origin

1 Pride-Of-Ringwood F C Australia2 Eurohop-T F C Belgium3 Osvald Clone 126 F C Czech Republic4 Osvald Clone 72 F C Czech Republic5 Sirem F C Czech Republic6 Zlatan F C Czech Republic7 Ging Dao Do Hua F C China8 Chang Bei No.1/2 F C China9 Nordgard-978 F C Denmark

10 Hallertauer MTF F C Germany11 Wurtemberger F C Germany12 Spalter F C Germany13 Hallertauer Gold F C Germany14 Hûller Aroma F C Germany15 Hallertauer Tradition F C Germany16 Spalter Select F C Germany17 Perle F C Germany18 Magnum F C Germany19 Taurus F C Germany20 Coobs F C Great Britain21 Early Bird Golding F C Great Britain22 Tutsham F C Great Britain23 Fuggle’s 37 F C Great Britain24 Keyworth Midseason F C Great Britain25 Brewers Gold F C Great Britain26 Northen Brewer F C Great Britain27 Wye Target F C Great Britain28 Zenith F C Great Britain29 Yeoman F C Great Britain30 Kirin 1/2 F C Japan31 Golden Star F C Japan32 First Choice F C New Zealand33 Callicross F C New Zealand34 Smooth Cone F C New Zealand35 Serebrianka F C Russia36 Vojvodina F C Serbia and Montenegro37 Savinjski Golding F C Slovenia38 Aurora F C Slovenia39 Bobek F C Slovenia40 Buket F C Slovenia41 Ahil F C Slovenia42 Apolon F C Slovenia43 Atlas F C Slovenia44 200/69 F C Slovenia45 217/169 F C Slovenia46 251/116 F C Slovenia47 Southern Brewer F C South Africa48 Galena F C USA49 Nugget F C USA50 Comet F C USA51 Cascade F C USA52 I F W Bosnia and Herzegovina53 J F W Bosnia and Herzegovina54 K F W Bosnia and Herzegovina55 L F W Croatia56 M F W Croatia57 N F W Croatia

Table 1. Hop (Humulus lupulus L.) accessions screened for microsatellite variability.

© 2004 NRC Canada

892 Genome Vol. 47, 2004

No Genotype Sex Breeding status Origin

58 O F W Croatia59 P F W Croatia60 R F W Croatia61 A F W FYR Macedonia62 B F W FYR Macedonia63 C F W FYR Macedonia64 K5 F W Georgia65 K7 F W Georgia66 K11 F W Georgia67 A12 F W Russia (Altai)68 A16 F W Russia (Altai)69 D F W Serbia and Montenegro70 E F W Serbia and Montenegro71 F F W Serbia and Montenegro72 G F W Serbia and Montenegro73 H F W Serbia and Montenegro74 S F W Slovenia75 T F W Slovenia76 U F W Slovenia77 4-2/137 M C Slovenia78 5-146/104 M C Slovenia79 6-106/32 M C Slovenia80 10-168/31 M C Slovenia81 11-31/107 M C Slovenia82 16-223/85 M C Slovenia83 28-1/63/38 M C Slovenia84 29-167/92 M C Slovenia85 33-206/8 M C Slovenia86 34-205/3 M C Slovenia87 35-257/15 M C Slovenia88 39-150/109 M C Slovenia89 42-49/116 M C Slovenia90 43-107/118 M C Slovenia91 8-13P10 M W Croatia92 27-20PO9 M W Croatia93 9-27P14 M W Croatia94 13-40P15 M W FYR Macedonia95 12-253/95 M W Georgia96 22-253/1 M W Georgia97 No3-38 M W Japan98 No2-35 M W Japan99 2-R30 M W Russia (Altai)

100 17-R16 M W Russia (Altai)101 18-R27 M W Russia (Altai)102 21-R17 M W Russia (Altai)103 23-R21 M W Russia (Altai)104 26-R22 M W Russia (Altai)105 14-35PO1 M W Serbia and Montenegro106 3/3 M W Slovenia107 1-4/4 M W Slovenia108 7-9/2 M W Slovenia109 36-O7P14 M W Slovenia110 40-2/1 M W Slovenia111 OSU1-9005 M W USA112 OSU2-9007 M W USA113 OSU3-9006 M W USA114 OSU4-9012 M W USA

Table 1 (continued).

tions using ice-cold absolute isopropanol, washed in ice cold70% v/v ethanol, and resuspended in 100 µL of TE buffer(10 mM Tris–HCl, 1 mM EDTA, pH 8.0). Concentrations ofDNA samples were determined using a Mini TKO100fluorimeter (Hoefer Scientific, San Francisco, Calif.) andcalf thymus DNA as a standard (Sigma-Aldrich ChemieGmbH, Deisenhofen, Germany).

One plant from each hop accession was analysed usingfour sequence-tagged microsatellite loci with the followingprimer pairs: 11a59, 7a82, 3a88, and 5-2 (Brady et al. 1996).The composition of 50- or 25-µL PCRs was as follows: 50 or25 ng of hop genomic DNA prepared in water-diluted stocksto a concentration of 20 ng DNA/µL, 1 U Taq DNA poly-merase (Roche Molecular Biochemicals, Mannheim, Ger-many), 1× supplied standard PCR buffer (10 mM Tris–HCl,1.5 mM MgCl2, 50 mM KCl, pH 8.3), 0.5 µM concentrationof each primer (MWG Biotech) and 0.8 mM mixture of fourdNTPs (Sigma–Aldrich Chemie GmbH, Deisenhofen, Ger-many). Reactions were subjected to an amplification proto-col consisting of a first denaturation step at 94 °C for 3 minand 26 cycles of denaturation at 94 °C for 45 s, annealing at55 °C for 30 s and extension at 72 °C for 1 min 30 s, fol-lowed by a final extension step at 72 °C for 7 min. AppliedBiosystems PE480 or PE9700 thermocyclers were used forDNA amplification, in the case of the first cycler, reactionswere overlaid with a drop of mineral oil.

The same volume of formamide loading dye (98% de-ionized formamide, 10 mM NaOH, 0.05% xylene cyanol,and 0.05% bromophenol blue was added to samples prior toa 5-min denaturation period at 95 °C. Five microlitres ofeach sample was applied to a vertical, 6% w/v denaturingpolyacrylamide gel (19:1, 1× TBE, and 8 M urea). One hun-dred nanograms of denaturated AFLP™ Ladder (GibcoBRL, Paisley, UK) was used for allele sizing. Gels werestained following the silver staining protocol (Echt et al.1996) and permanent records of gels were made using a flat-bed scanner or by exposure of APC film (Promega, Madi-son, Wis.) for 30 s.

Individual alleles were reamplified immediately afterstaining of gels. The surface of the allele band was scratchedusing a sterile pipette tip wetted in 1× PCR buffer and thetip was transferred to 50 µL of PCR solution of the samecomposition as described previously, for 2 min. Reactionswere subjected to the same cycling protocol as described, forthe most appropriate number of cycles determined by repeti-

tion screening (between 20 and 30 cycles). A 5-µL aliquot ofreaction mixture was used for 2% w/v agarose gel electro-phoresis to confirm the desired amplification of the allele.Samples were cleaned using a Qiaquick PCR purification kit(Qiagen, Hilden, Germany) and the blunt end cloned into apCR-Script vector using a PCR-Script™ Amp Cloning Kit(Stratagene, La Jolla, Calif.) according to the manufacturer’sinstructions. Presence and correct insert length were con-firmed by bacterial colony PCR. Plasmid DNA was isolatedfrom an overnight culture using Plasmid Mini Kit (Qiagen,Hilden, Germany). DNA was quantified using a spectro-photometer, precipitated by 2 volumes of absolute ethanol,and air dried. A fluorescent labelled primer sequencingmethod was performed by the MWG Biotech SequencingService, Eberberg, Germany. All sequences have been sub-mitted to GenBank (accession Nos. AF155625–AF155634and AY382838–AY382868).

Data analysis

The degree of polymorphism was quantified using thepolymorphic information content (PIC):

PIC 1 22 2 2= −⎛

⎝⎜⎜

⎞

⎠⎟⎟

−= = +=

−

∑ ∑∑p p pii

n

j i

n

i

n

i j1 11

1

where pi and pj are the frequencies of the ith and jth allelesat a locus with n alleles in a population, respectively(Botstein et al. 1980).

Individual genetic distances were calculated using the pro-portion of shared alleles distance (Dps) (Bowcock et al.1994), Dice distance coefficient (DD) (Dice 1945), and thestepwise distance (Dsw) (Shriver et al. 1995). The first twodistance measures are based on the infinite allele model(IAM) and were calculated from a binary data matrix inwhich the presence or absence of each band was coded as 1or 0, respectively. The third distance measure assumes Ohtaand Kimura’s (1973) stepwise mutation model (SMM) andwas calculated from a data matrix containing repeat countsof each microsatellite marker allele. The software used forcalculation of genetic distances include MICROSAT ver.1.5 d (http://hpgl.stanford.edu/projects/microsat/), POPULA-TIONS version 1.2.28 (distributed by Olivier Langella,http://www.pge.cnrs-gif.fr/bioinfo/populations/), and NTSYS-pc version 2.02 (Rohlf 1998). Cluster analyses based on the

© 2004 NRC Canada

Jakse et al. 893

No Genotype Sex Breeding status Origin

115 OSU6-9011 M W USA116 OSU7-9010 M W USA117 OSU8-9017 M W USA118 OSU9-9008 M W USA119 OSU10-9046 M W USA120 OSU11-9041 M W USA121 OSU13-9013 M W USA122 OSU14-9001 M W USA123 OSU16-9002 M W USA124 OSU17-9015 M W USA

Note: F, female; M, male; C, cultivated; W, wild.

Table 1 (concluded).

unweighted pair-group method with arithmetic average(UPGMA) and principal coordinates analyses (PCoA) wereperformed on individual genetic distance matrices usingNTSYS-pc. The cophenetic correlation coefficients werecalculated, and Mantel’s test (Mantel 1967) was performedto check the goodness of fit of a cluster analysis for the ma-trix on which it is based. The same procedure was employedto calculate cophenetic correlation coefficients among matri-ces based on different distance measures.

Individual pairwise genetic distance matrices were ana-lysed by the analysis of molecular variance (AMOVA) ap-proach (Excoffier et al. 1992) using the WINAMOVA 1.55program (Excoffier 1992). Total genetic variation was parti-tioned according to the breeding status (cultivated vs. wild),and sex (female vs. male) of the accessions. The significanceof φ statistics was obtained non-parametrically by 1000 per-mutations. The homogeneity of intra-group molecular vari-ances (homoscedasticity) was tested using the HOMOVAprocedure (Barlett’s test), also carried out in WINAMOVABarlett’s statistics (Barlett 1937) null distributions were ob-tained after 1000 permutations.

Multiple sequence alignments of alleles were performedusing the standard version of the Clustal X 1.81 program(Thompson et al. 1997).

Results

Characteristics of microsatellitesFour primer pairs amplified a total of 63 alleles in a panel

of 124 hop accessions (Table 2) with an average of 15.7 al-leles per locus. Two microsatellite loci amplified well in all124 accessions, whereas no amplification was detected intwo accessions at locus 3a88, and 14 accessions at locus7a88. Both accessions were wild males of North American(NA) origin. Primer pair 5-2 amplified more fragments thanthe expected number of alleles of a diploid organism. Never-theless, as has already been shown by segregation analysis(Jakse et al. 2001), the third fragment (147 bp) correspondsto an allele of a duplicated locus (5-2K) found in some Eu-ropean accessions in a heterozygous state in combinationwith a null allele (Jakse et al. 2001). In the present analysis,duplicated locus 5-2K was found in 41 accessions of Euro-pean (EU) origin (data not shown).

PIC values ranged from 0.41 to 0.83 over four loci, withan average of 0.64. These values are very similar to thosecalculated for the 55 hop cultivars analysed with the samemicrosatellites (Jakse et al. 2001). Among three loci with 21,19, and 17 alleles, the highest PIC value was at the locuswith 17 alleles, and not that with 21 alleles as would be ex-pected. The number of alleles in groups of accessions isshown in Table 3. Twenty-nine alleles were amplified in 51hop cultivars and 23 in 14 male breeding lines, of which 2and 3 alleles, respectively, were unique to a single accession.In 45 wild male and female accessions of EU origin, 33 al-leles were detected, of which 11 were unique; in Americanmale accessions, a total of 25 alleles were scored over 3 loci,of which 19 were unique.

The most frequent alleles at loci 11a59, 7a82, 3a88, and5-2 were found in 48%, 39%, 44%, and 30% of the acces-sions. The frequency distribution of microsatellite alleles(data not shown) at loci 11a59, 7a82, and 3a88 showed

some gaps in the observed range of repeat counts and someunexpectedly large alleles were detected at loci 11a59 and3a88, although outlier alleles seem to be rare. Nevertheless,the distribution patterns of repeat counts could fit the step-wise mutation model (SMM) of microsatellite evolution ana-lysing a broader sample representing the entire geneticdiversity of wild hops. Stepwise changes in the copy numberof repeats were also revealed by allele sequences, since mostmutations were within microsatellite repeats (GenBank ac-cession Nos. AY382838–AY382868).

The distribution of microsatellite alleles between Euro-pean and North American accessions was observed. Table 4shows the detailed distribution of alleles, taking into accountgeographic origin of wild accessions (Table 1) and molecu-lar clustering and pedigree data of cultivated hops (Sustar-Vozlic and Javornik 1999; Jakse et al. 2001) and breedinglines.

At locus 11a59 (Table 4), 14 alleles in the range of 253–207 bp were found in NA germplasm and 5 shorter alleles(197–181 bp) in EU germplasm, with a 205-bp allele ap-pearing in both germplasms. The two longest alleles at thislocus (269 and 253 bp) were exceptions, being found inthree accessions from the Caucasus. The genotypic structureof these three accessions is quite unique, since the longestand a very short allele (193 bp) are in heterozygotes, whichis not very commonly found. Sequences of 12 (AY382838–AY382849) out of 21 alleles of locus 11a59 showed veryfew substitutions and no deletions in the flanking region,confirming that allele size variation was due to mutations incompound microsatellite repeats. In American alleles, longerrepeats were detected in both motifs ((TG)n(AG)n) than inthe four EU alleles (Table 4).

Locus 7a88 was the only one with a perfect microsatelliterepeat, with the lowest number of alleles. No microsatellitealleles were amplified in wild American accessions, so noobvious division of alleles between germplasms was observed.

Twelve alleles (249–199 bp) were detected in Americangermplasm that were longer than the six alleles (197–187 bp)found in EU accessions at locus 3a88. Allele 3a88-201seemed to be unique to Japanese accessions. The flankingregion of nine sequenced alleles (AY382854–AY382862)showed six substitutions and two deletions of 2-bp but theallele sizes differ primarily owing to changes in the repeatnumber. American alleles expand or contract in both motifs((CA)n(TA)n) of the compound microsatellite repeat. How-ever, it appeared that the sizes of EU alleles varied only inthe repeat number of the first motif ((CA)n) of the compoundrepeat (Table 4).

Different lengths in the two repeat motifs were also foundwhen two alleles with the same size, 213 bp, from two di-verse accessions were sequenced (AY382858 andAY382859). In one allele, both microsatellite motifs showeda more similar number of repeats ((CA)15(TA)10) than wasfound in the other allele ((CA)21(TA)4), where the first motifexpanded much more than the second motif. Thesemutational events in the compound repeat of locus 3a88 area good example of allele homoplasy, which is expected to behigh for microsatellite markers (Murray 1996).

An even more complex allele structure was found at locus5-2. At this locus, eight short alleles were characteristic ofAmerican accessions (170–154 bp) and eight longer ones for

© 2004 NRC Canada

894 Genome Vol. 47, 2004

EU germplams (186–172 bp) (Table 4). An allele (150 bp)unique to Japanese accessions was also discovered at this lo-cus. At locus 5-2, alleles in two distinct germplasms differnot only in their sizes but also in the type of microsatelliterepeat motifs. Perfect (AT) repeats were characteristic ofAmerican alleles (AY382865–AY382868, AF155631–AF155634) and compound interrupted repeats ((CT)n(AT)nGT(AT)3) were found in European hops (AY382863,AY382864, AF155625–AF155630). Differences in allele se-quences between the two groups of alleles were also dis-played in the flanking region.

Genetic relationships among accessionsTo evaluate intra-specific relationships among the ana-

lysed accessions, two types of genetic distances were calcu-

lated, i.e., infinite allele model (IAM) distances (Dps andDD) and stepwise mutation model (SMM) distance (Dsw).Dps and DD coefficients have very similar statistical proper-ties and their cophenetic correlation was highly significant(r = 0.98, p < 0.001) yielding virtually the same results withthe UPGMA clustering procedure, PCoA, and AMOVAanalyses. Cophenetic correlations between Dsw and IAM dis-tances were also highly significant (r = 0.86, p < 0.001 forDsw/Dps; r = 0.89, p < 0.001 for Dsw/DD). Results are thusshown only for the analyses based on the DD and Dsw dis-tance matrices.

On the basis of DD and Dsw distance matrices, UPGMAtrees were constructed that clustered accessions in a similarmanner, so only the tree based on the DD distance matrix isshown (Fig. 1). The tree can be divided into ten different

© 2004 NRC Canada

Jakse et al. 895

Locus n No. of alleles Size range (bp) No. of sequences Repeat type PIC

11a59 124 21 181–269 12a (TG)n(AG)n 0.58647a88 110 6 192–208 4b (CA)n 0.41153a88 122 19 183–249 9c (CA)n(TA)n 0.71315-2 124 8 172–186 8d (CT)n(AT)nGT(AT)3

9 150–170 8 (AT)n 0.831Average 15.75 0.6355

aAY382838–AY382849.bAY382850–AY382853.cAY382854–AY382862.dAY382863–AY382864, AF155625–AF155630.eAY382865–AY382868, AF155631–AF155634.

Table 2. Number of accessions analysed, size range of alleles, number of scored alleles, and sequence data microsatelliterepeat type and PIC values over four loci.

LocusCultivars(51 F)

Breeding lines(14 M)

EU Wild accession(45 F+M)

NA Wild accession(14 M)

11a59 9(1) 8 8(1) 10(9)7a88 3 3 6(3) nd3a88 8(1) 5(2) 7(4) 8(5)5-2 9 7(1) 10(3) 7(5)Total 29(2) 23(3) 33(11) 25(19)Average 7.25 5.75 8.25 8.30

Note: Numbers of unique alleles are in parentheses. F, female; M, male; EU, European origin; NA, North Americanorigin; nd, not determined.

Table 3. Number of alleles in groups of accessions.

Locus No. of alleles EU accessions NA accessions

11a59 21 205a, 197, 195, 193, 191, 181,253b, 269b

253, 247, 245, 239, 231, 229, 227, 225, 223,217, 215, 211, 209, 207, 205a

Repeat changesc (TG)5–9(AG)8–16 (TG)8–15 (AG)16–37

3a88 19 197, 195, 193, 191, 189, 187,201d

249, 241, 237, 235, 219, 217, 215, 213, 211,209, 205, 199

Repeat changesc (CA)13–15 (AT)3 (CA)15–39 (TA)4–10

5-2 17 186, 184, 182, 180, 178, 176,174, 172, 150d

170, 168, 166, 164, 160, 158, 156, 154

Repeat changesc (CT)6–11 (AT)6–9 GT (AT)3 (AT)7–15

aCommon allele to both germplasms.bAlleles in Georgian accessions.cRepeat changes of sequenced alleles.dAlleles in Japanese accessions.

Table 4. Geographic distribution of alleles (bp) and their sequenece repeat changes at three microsatellite loci.

clusters, which reflect the relationship among geographicallydiverse wild accessions and hop cultivars. In clusters I, II,and III, some wild accessions from Europe and breedinglines grouped together with hop cultivars containing pre-dominantly European germplasm. Cluster I, for example,combined the majority of Slovene cultivars, breeding linesand wild accessions; cluster III contained typical Central Eu-ropean hop cultivars together with wild accessions from re-gions of former Yugoslavia. Cluster IV was an interestingmixture of three predominantly European cultivars, Japanesewild male and wild female or male accessions ranging fromAltai to Slovenia. Similarly, one of the smaller clusters(VIII) combined North American type hop cultivars and onewild accession from Japan. However, no clustering of wildaccessions with European cultivars (VI) or with cultivarswith predominately NA germplasm (VII) was found. Themajority of wild accessions of European origin formed aseparate cluster (V) showing genetic differences from culti-vated hops. This cluster grouped 14 wild male and 4 wild fe-male accessions from the region of former Yugoslavia and 5wild male accessions from the Altai region, while two wildaccessions from Georgia formed a separate cluster (IX). Thegenetic differences were even larger with American wild ac-cessions, which all clustered together in a distant group (X),thus clearly distinguishing NA germplasm from the other ac-cessions, at high genetic dissimilarity values. High variabil-ity can be also seen among accessions within the cluster ofwild NA hops, indicating high genetic diversity also withinnative NA hops.

To have a better insight into the genetic structure of Euro-pean hops, NA wild male accessions were excluded in thePCoA analysis. Accessions were classified according to gen-der (male M and female F) and breeding status (cultivated Cand wild W). PCoA based on the DD distance matrix ex-plained 44.82% of the variation along the first two axes anddivided groups of wild and cultivated accessions, but no dif-ferentiation between female and male hops was noticed. ThePCoA plot based on the DSW distance matrix explained amuch higher percentage of variation (84.37%) along the firsttwo axes, and particularly clearly differentiated two groupsof accessions from the others. One group contained three fe-male accessions from Georgia and the other group consistedof accessions with NA germplasm (9 cultivars and 3 breed-ing lines).

Furthermore, AMOVA and HOMOVA analyses were per-formed on both DD and Dsw distance matrices to test for par-titioning of the genetic diversity between and within groupsof wild and cultivated hops, as well as between and withingroups of female and male accessions. Analyses based onboth distance measures yielded similar results and only theresults based on the DD distance matrix are shown (Table 5).It thus seems that for the analysed groups of accessions (cul-tivated vs. wild and female vs. male) the SMM based repeatcount variability matches the variability expected, with theinfinite-allele model not having a significant impact ondiversity partitioning between and within groups. In bothcases, most of the molecular diversity was attributable to dif-ferences among accessions within groups. The cultivatedhops differ significantly from the wild ones, although mostof the variability (88.95%) was found within groups. Molec-ular variances within groups of cultivated and wild hops

were homogeneous, suggesting that a similar level of molec-ular variability can be found in both groups of accessions.The proportion of the total diversity found within groups offemale and male accessions was 97.39%, leaving only 2.61%

© 2004 NRC Canada

896 Genome Vol. 47, 2004

Fig. 1. UPGMA tree based on Dice’s distance matrix among124 hop accessions.

of the diversity between groups. However, the φ-statistic wasstill significant.

Discussion

In total, 63 microsatellite alleles were amplified over fourloci in the array of 124 hop accessions. There was no obvi-ous correlation between the number of alleles per locus andthe PIC value, as reported in some studies (Huang et al.2002; Matus and Hayes 2002). The number of alleles did notdiffer much between four different classes of accessions (Ta-ble 3) but diversity was apparent in the number of uniquealleles. The wild accessions of the EU and NA regions pos-sessed a high number of unique alleles in comparison to hopcultivars or breeding lines. In particular, the occurrence ofthe highest number of unique alleles in the NA wild acces-sions is an indication of the high diversity present in thisgene pool. Identified unique alleles can also be diagnostic ofparticular germplasm (NA or EU types) or genotype (uniqueallele 11a59-239 in ‘Kirin 1/2’) and they may be of impor-tance for effective cultivar identification (Cerenak et al.2003). The high diversity of NA wild accessions and theirdifferentiation from other hops was well presented by clusteranalysis. Wild NA accessions formed a distinct cluster,showing high variability among accessions and within thecluster two main groups can be identified corresponding tothe collection sites of NA accessions. In one group were ac-cessions collected in New Mexico, Arizona, and Colorado;in the other group prevailed accessions from Montana, Ne-braska, and Colorado. Such a geographic division of NA ac-cessions agrees with the two geographic and (or) chemicalgroups found by Stevens et al. (2000), but we could notspeculate on Small’s classification of American native hop(1978) because of the limitations of available samples. Nev-ertheless, our results are a valuable contribution to the dataon native American hops which seem to be highly variableand genetically highly different from European wild hops.

These differences were even more pronounced in relationto the features of the microsatellite alleles contained in eachgermplasm. First, there was a very distinct distribution of al-leles between the two (wild EU and NA) germplasms atthree loci (Table 4) and at one locus no amplification wasdetected in wild American accessions. Although the latterlocus was not very polymorphic, it seems that no primingsites were found in wild NA accessions despite the fact thatthe source hop for these microsatellites was T6, a 3n hopcultivar containing both germplasms (Brady et al. 1996).

Second, alleles of the two germplasms were different intheir sizes within loci. At two loci (11a59 and 3a88), wefound a higher number and longer alleles in NA germplasm

than in EU germplasm. Such number and size differencesimplies that variations in mutation rates within loci mighthave occurred in both germplasms. It is now well acceptedthat the mutation rate of microsatellites differs greatly be-tween loci and that the mutation rate increases with thelength of the microsatellite (Ellegren 2000; Schlötterer2000). It has also been shown that there is a tendency formicrosatellites to increase rather than decrease in size overtime (Amos et al. 2003). In maize, for example, an increasewas shown in the average allele size in two geographicalgroups relative to the ancestral one (Vigouroux et al. 2003).Features of hop microsatellite alleles at 11a59 and 3a88 locisuggest that NA alleles mutate at a higher rate than alleles inthe EU germplasm and thus reveal a variation of mutationrate also within loci. The picture was a quite different at lo-cus 5-2, where almost the same number of alleles were de-tected in both germplasms but allele sequences showed twodistinct types of microsatellite repeat motif in biogeogra-phically partitioned hops. The NA accessions displayed al-leles with perfect repeat motifs and smaller sizes and EUalleles were longer but with compound interrupted repeats. Itseems that more complex mutations occurred at this locusthan only an increase (decrease) of repeats in geographicallydiverged European and American hops.

The analysis of allele polymorphism at microsatellite lociof different hop accessions showed that hop germplasm canbe differentiated to NA and EU geographic types accordingto the allele sizes at three loci or by specific microsatelliterepeat type at locus 5-2. Such properties of alleles can be ex-ploited for the development of molecular markers applicablein hop genetic research. The development of microsatellitescan be followed in the two biogeographically separatedgermplasms, indicating different evolutionary dynamics andcomplex mutations of microsatellite sequences within loci.Allele sequences reveal features such as allele homoplasy,which is important for the correct estimation of genetic vari-ability and (or) similarities among accessions.

Genetic diversity between EU and NA germplasms hasthus been proven to be very high but the diversity present inthe European gene pool is an important question, since itprovides a major part of the germplasm in cultivated hop.Modern hop cultivars were either derived from wild EUplants by clonal selections or by hybridization between EUand NA native or wild hops. A representative sample of EUgermplasm consisted of wild accessions covering a widegeographic span from the Altai region (area bordering Rus-sia, Mongolia, China, and Kazakhstan), Caucasus (area be-tween the Black and Caspian Seas), over Russia to Europe(Eastern, Central, and Western) and cultivated hops, the ma-jority derived primarily from the EU gene pool. Forty-five

© 2004 NRC Canada

Jakse et al. 897

Genetic distance Source of variationDegrees offreedom

Variancecomponents

% Totalvariance

φstatistic p(φ)

Bartlett’sindex p(B)

Cultivated vs. Wild Between groups 1 0.032 11.02 0.110 <0.001 0.009 0.667Within groups 122 0.261 88.98

Female vs. Male Between groups 1 0.007 2.61 0.026 <0.001 0.006 0.719Within groups 108 0.274 97.39

Note: p(φ), φ-statistics probability level after 9999 permutations; p(B), Bartlett’s index probability level after 9999 permutations.

Table 5. Results of AMOVA and HOMOVA analysis for the partitioning of microsatellite variation between and within cultivated andwild hop accessions and between and within female and male hop accessions.

EU wild accessions revealed 33 alleles over four loci, ofwhich 11 were unique, a much higher number than found incultivated accessions and thus indicating a certain degree ofdiversity present in these accessions. This was also shownby the clustering analysis, where a significant number of EUwild hops formed a “pure wild” cluster (Fig. 1, V) distantfrom other groups of wild and cultivated hops.

The results of PCoA analyses based on DD and Dsw dis-tance matrices showed different aspects of microsatellite di-versity in hops. The PCoA plot based on the DD distancematrix revealed the existence of some level of differentiationbetween European wild and cultivated accessions, as shownby AMOVA results. On the other hand, the PCoA plot basedon the Dsw distance matrix indicated the differentiation of agroup of Georgian accessions, as well as a group of hopswith NA germplasm. The depicted group of Georgian acces-sions contained some very specific alleles, which classifythese accessions as highly diverse hops within European ac-cessions. A group of hops with NA germplasm (cultivars andbreeding lines) differed clearly from accessions with onlyEU germplasm, which was not apparent in the cluster analy-sis. In the cluster analysis, particularly male breeding linescontaining half or one quarter of NA germplasm were notgrouped but dispersed in different clusters (I, IV, and VII).

Our results demonstrate that the four microsatellite mark-ers were informative enough (average PIC = 0.64, manyunique alleles) to assess diversity in hop germplasm. Thegeographic classification of hop germplasm into Europeanand North American was confirmed not only by the micro-satellite allele distribution and analysis of genetic distancesbut also by notable differences in the structure of allele se-quences. The results also showed variability present withineach of the hop geographic groups, indicating very highvariability within NA wild hops. The genetic diversitywithin wild European hops and hop cultivars was smaller,indicating a relatively narrow genetic base of the existingEU gene pool for hop breeding.

Acknowledgements

This work was supported by research grant PO-0513-0481from the Slovene Ministry of Education, Science and Sport.The authors thank the Institute of Hop Research andBrewing, alec, Slovenia, and Oregon State University,Corvallis, Ore., for kindly providing hop material. We alsothank Ms. M. Kurumata for skillful technical assistance inthe laboratory.

References

Alvarez, A.E., van de Wiel, C.C.M., Smulders, M.J.M., and Vosman,B. 2001. Use of microsatellites to evaluate genetic diversity andspecies relationships in the genus Lycopersicon. Theor. Appl.Genet. 103: 1283–1292.

Amos, W., Hutter, C.M., Schug, M.D., and Aquadro, C.F. 2003. Di-rectional evolution of size coupled with ascertainment bias forvariation in Drosophila microsatellites. Mol. Biol. Evol. 20: 660–662.

Barlett, M.S. 1937. Some examples of statistical methods of re-search in agriculture and applied biology. J. Roy. Stat. Soc.Suppl. 4: 137–147.

Botstein, D., White, R.L., Skolnick, M., and David, R.W. 1980. Con-struction of a genetic linkage map in man using restriction frag-ment length polymorphisms. Am. J. Hum. Genet. 32: 314–331.

Bowcock, A.M., Ruiz-Linares, A., Tomfohrde, J., Minch, E., Kidd,J.R., and Cavalli-Sforza, L.L. 1994. High resolution of humanevolutionary trees with polymorphic microsatellites. Nature(London), 368: 455–457.

Brady, J.L., Scott, N.S., and Thomas, M.R. 1996. DNA typing ofhops (Humulus lupulus) through application of RAPD and micro-satellite marker sequences converted to sequence tagged sites(STS). Euphytica, 91: 277–284.

Cerenak, A., Jakse, J., and Javornik, B. 2003. Identification anddifferentiation of hop varieties using simple sequence repeatmarkers. J. Am. Soc. Brew. Chem. 62: 1–7.

Dice, L.R. 1945. Measures of the amount of ecologic associationbetween species. Ecology, 26: 297–302.

Echt, C.S., May-Marquardt P., Hseih, M., and Zahorchak, R. 1996.Characterization of microsatellite markers in eastern white pine.Genome, 39: 1102–1108.

Ellegren, H. 2000. Microsatellite mutations in the germline: impli-cations for evolutionary inference. Trends. Genet. 16: 551–558.

Estoup, A., and Cornuet, J.M. 1999. Microsatellite evolution: infer-ences from population data. In Microsatellites, evolution and ap-plications. Edited by D.B. Goldstein and C. Schlötterer. OxfordUniversity Press, Oxford, UK. pp. 49–65.

Excoffier, L. 1992. WinAMOVA version 1.55. Analysis of Molecu-lar Variance, Graphical Windows 3.x program for the analysis ofpopulation structure from molecular or conventional genetic data.Available from http://cmpg.unibe.ch/excoffier/

Excoffier, L., Smouse, P.E., and Quattro, J.M. 1992. Analysis ofmolecular variance inferred from metric distances among DNAhaplotypes: application to human mitochondrial restriction sites.Genetics, 131: 479–491.

Guilford, P., Prakash, S., Zhu, J.M., Rikkerink, E., Gardiner, S.,Bassett, H., and Foster, R. 1997. Microsatellites in Malus ×domestica (apple): abundance, polymorphism and cultivar iden-tification. Theor. Appl. Genet. 94: 249–254.

Gupta, P.K., and Varshney, R.K. 2000. The development and use ofmicrosatellite markers for genetic analysis and plant breedingwith emphasis on bread wheat. Euphytica, 113: 163–185.

Huang, W.G., Cipriani, G., Morgante, M., and Testolin, R. 1998.Microsatellite DNA in Actinidia chinensis: isolation, characteri-sation, and homology in related species. Theor. Appl. Genet. 97:1269–1278.

Huang, X.Q., Börner, A., Röder, M.S., and Ganal, M.W. 2002. As-sessing genetic diversity of wheat (Triticum aestivum L.)germplasm using microsatellite markers. Theor. Appl. Genet.105: 699–707.

Jakse, J., Kindlhofer, K., and Javornik, B. 2001. Assessment of ge-netic variation and differentiation of hop (Humulus lupulus L.)genotypes by microsatellite and AFLP markers. Genome, 44:773–782.

Jakse, J., Bandelj, D., and Javornik, B. 2002. Eleven new micro-satellites for hop (Humulus lupulus L.). Mol. Ecol. Notes, 2:544–546.

Kump, B., and Javornik, B. 1996. Evaluation of genetic variabilityamong common buckwheat (Fagopyrum esculentum Moench.)populations by RAPD markers. Plant Sci. 114: 149–158.

Levinson, G., and Gutman, G.A. 1987. Slipped-strand mispairing:a major mechanism for DNA sequence evolution. Mol. Biol.Evol. 4: 203–221.

Li, C.D., Rossnagel, B.G., and Scoles, G.J. 2000. The developmentof oat microsatellite markers and their use in identifying rela-

© 2004 NRC Canada

898 Genome Vol. 47, 2004

tionship among Avena species and oat cultivars. Theor. Appl.Genet. 101: 1259–1268.

Mantel, N. 1967. The detection of disease clustering and a general-ized regression approach. Cancer Res. 27: 209–220.

Matsuoka, Y., Mitchell, S.E., Kresovich, S., Goodman, M., andDoebley, J. 2002. Microsatellites in Zea — variability, patternsof mutations, and use for evolutionary studies. Theor. Appl.Genet. 104: 432–450.

Matus, I.A., and Hayes, P.M. 2002. Genetic diversity in three groupsof barleys germplasm assessed by simple sequence repeats. Ge-nome, 45: 1095–1106.

Morgante, M., and Olivieri, A.M. 1993. PCR-amplified micro-satellites as markers in plant genetics. Plant J. 3: 175–182.

Murakami, A. 2001. Structural differences in the intergenic spacerof 18S–26S rDNA and molecular phylogeny using partial exter-nal transcribed spacer sequence in hop, Humulus lupulus. Breed.Sci. 51: 163–170.

Murakami, A., Darby, P., Javornik, B., Pais, M.S.S., Seigner, E.,Lutz, A., and Svoboda, P. 2003. Molecular evolution of hops,Humulus lupulus. In Proceedings of the Scientific Commission,IHGC, held 24–27 June 2003, Dobrna, Slovenia. Edited by E.Seigner. Bayerische Landesanstalt für Landwirtschaft, Institutfür Pflanzenbau und Pflanzenzüchtung, Arbeitsbereich Hopfen,Hüll, Germany. pp. 92–96.

Murray, B.W. 1996. The estimation of genetic distance and popula-tion structure from microsatellite allele frequency data. Avail-able from http://helix.biology.mcmaster.ca/brent/brent.html.

Neve, R.A. 1991. Hops. Chapman and Hall, London, UK.Ohta, T., and Kimura, M. 1973. A model of mutation appropriate

to estimate the number of electrophoretically detectable allelesin a finite population. Genet. Res. 22: 201–204.

Patzak, J. 2001. Comparison of RAPD, STS, ISSR and AFLP mo-lecular methods used for assessment of genetic diversity in hop(Humulus lupulus L.). Euphytica, 121: 9–18.

Pillay, M., and Kenny, S.T. 1996. Structure and inheritance of ribo-somal DNA variants in cultivated and wild hop, Humulus lupulusL. Theor. Appl. Genet. 93: 333–340.

Powell, W., Morgante, M., Andre, C., Hanafey, M., Vogel, J.,Tingey, S., and Raffalski, A. 1996. The comparison of RFLP,RAPD, AFLP and SSR (microsatellite) markers for germplasmanalysis. Mol. Breed. 2: 225–238.

Rallo, P., Dorado, G., and Martin, A. 2000. Development of simplesequence repeats (SSRs) in olive tree (Olea europaea L.). Theor.Appl. Genet. 101: 984–989.

Rohlf, F.J. 1998. NTSYS: numerical taxonomy and multivariate anal-ysis system, version 2.0. Applied Biostatistics Inc., Setauket, N.Y.

Schlötterer, C. 2000. Evolutionary dynamics of microsatellite DNA.Chromosoma, 109: 365–371.

Schlötterer, C., and Tautz, D. 1992. Slippage synthesis of simplesequence DNA. Nucleic Acids Res. 20: 211–215.

Seefelder, S., Ehrmaier, H., Schweizer, G., and Seigner, E. 2000.Genetic diversity and phylogenetic relationships among acces-sions of hops, Humulus lupulus, as determined by amplifiedfragment length polymorphism fingerprinting compared withpedigree data. Plant Breed. 119: 257–263.

Shriver, M.D., Jin, L., Boerwinkle, E., Deka, R., Ferrell, R.E., andChakraborty, R. 1995. A novel measure of genetic distance forhighly polymorphic tandem repeat loci. Mol. Biol. Evol. 12:914–920.

Small, E. 1978. A numerical and nomenclatural analysis of morph-geographic taxa of Humulus. Syst. Bot. 3: 37–76.

Stevens, J.F., Taylor, A.W., Nickerson, G.B., Ivancic, M., Henning,J., Haunold, A., and Deinzer, M.L. 2000. Prenylflavonoid varia-tion in Humulus lupulus: distribution and taxonomic significanceof xanthogalenol and 4′-O-methylxanthohumol. Phytochemistry,53: 759–775.

Sustar-Vozlic, J., and Javornik, B. 1999. Genetic relationships incultivars of hops, Humulus lupulus L., determined by RAPDanalysis. Plant Breed. 118: 175–181.

Tang, S.X., and Knapp, S.J. 2003. Microsatellites uncover extraordi-nary diversity in native American land races and wild populationsof cultivated sunflower. Theor. Appl. Genet. 106: 990–1003.

Testolin, R., Marrazzo, T., Cipriani, G., Quarta, R., Verde, I., Dettori,M.T., Pancaldi, M., and Sansavini, S. 2000. Microsatellite DNA inpeach (Prunus persica L. Batsch) and its use in fingerprinting andtesting the genetic origin of cultivars. Genome, 43: 512–520.

Thomas, M.R., and Scott, N.S. 1993. Microsatellite repeats ingrapevine reveal DNA polymorphisms when analyzed as se-quence-tagged sites (STSS). Theor. Appl. Genet. 86: 985–990.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., andHiggins, D.G. 1997. The ClustalX windows interface: flexiblestrategies for multiple sequence alignment aided by quality anal-ysis tools. Nucleic Acids Res. 25: 4876–4882.

Udupa, S.M., Robertson, L.D., Weigand, F., Baum, M., and Kahl,G. 1999. Allelic variation at (TAA)(n) microsatellite loci in aworld collection of chickpea (Cicer arletinum L.) germplasm.Mol. Gen. Genet. 261: 354–363.

Vigouroux, Y., Matsuoka, Y., and Doebley, J. 2003. Directionalevolution for microsatellite size in maize. Mol. Biol. Evol. 20:1480–1483.

Weber, J.L., and Wong, C. 1993. Mutation of human short tandemrepeats. Hum. Mol. Genet. 2: 1123–1128.

© 2004 NRC Canada

Jakse et al. 899