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Agronomy & Horticulture -- Faculty Publications Agronomy and Horticulture Department

1984

Identification of Chromosomes with SecondaryConstrictions inMelilotus SpeciesS. E. SchlarbaumKansas State University

L. B. JohnsonUnited States Department of Agriculture

Herman J. GorzUnited States Department of Agriculture

Francis A. HaskinsUniversity of Nebraska - Lincoln, fhaskins@neb.rr.com

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Schlarbaum, S. E.; Johnson, L. B.; Gorz, Herman J.; and Haskins, Francis A., "Identification of Chromosomes with SecondaryConstrictions in Melilotus Species" (1984). Agronomy & Horticulture -- Faculty Publications. 307.http://digitalcommons.unl.edu/agronomyfacpub/307

The Journal of Heredity 75:23-26. 1984.

Identification of chromosomes withsecondary constrictions inMe/ilotuB species

ABSTRACT: Secondary constrictions were determined in one chromosome pair in the com­plements of M. intesta, M. macrocarpa, M. italica (subgenus Micromeli/otus), and M. albaand M. officinalis (subgenus Meli/otus). The karyotypes of M. intesta and M. macrocarpawere found to be similar. Chromosomes of M. italica were larger than the chromosomesof the other four Melilotus species. The chromosome size in M. italica suggests the pres­ence of large chromosomes in an ancestral or pro-Melilotus prototype. The chromosomeswith satellites of M. intesta and M. macrocarpa appear to differ in morphology from thesatellite chromosomes of M. alba and M. officinalis by a paracentric inversion. The mor­phology of the satellite chromosomes in M. italica is thought to rep resent a more primitivetype than in the other Meli/otus species studied .

S. E.Schla rbaum

LB. Johnson

H.J.Gorz

F.A. Haskin s

Theauthors are. respectively. former postdoctoralresearchassociate. and professor. Department of PlantPathology. Throckmorton Hall. Kansas State Univer­sity. Munhattun, KS 66506: supervisory researchgeneticist. USDA-ARS. and George Holmes Professorof Agronomy. Departm ent of Agronomy. Universityor Nebraska. Lincoln. NE 68583. Dr. Schlarbaum'spresent address: Depart ment of Forestry. Wildlife andFisheries. University of Tennessee. Knoxville. TN37901. Research supported by Nat ional ScienceFoundation grant PCM 8022556. Contribu tion no.82-575·). Department of Plant Pathology. Kansas StateUniversity. Agricultural Experiment Station. Man­hauan, Kansas 66506. Research was done in coopera­tionwiththe Nebraska Agricultural Experiment Sta­tionand the U.S. Department of Agriculture. Agri­cultural Research Service. Please address reprint re­queststoDr. S. E. Schlar baum.i£ 1984. Ameriean Genetic Association.

T HE SW EETCLOVER GEN US, Melilotu sAda ns., contains forage and nitrogen fixingspecies of agricultural importance. Taxo­nomica lly, Melilot us is divided into twosubgenera 10, (E ll )Melilotus Schulz and Mi­cromelilotus Schulz. Isely' considers thesubgenus Melilotu s to consist of nine biennialspecies, including the commercially importantM . alba Desr. and M. officinalis (L. ) Larn.,while the subgenus Mi crom elilotus conta insII annual species, fewof which have economicvalue as crops!". Other taxonomists, mostnotably Suvorov!", consider the genus tocontain a greater number of species.

The chromosome number of many Me li­lotus species, 2n = 2x = 16, was determinedea rly in this century-'U . Karyotype analysesof all Melilotu s species, with the exception ofthe du bious M . bicolor Boiss & Bal., werereported by Kita5.6. Chromosomes with sat­ellites were found in all species except M. in ­festa Guss., M. macrocarpa Goss. et Dur., andM . ita lica ( L.) Larn., which belong to Mi­cromelilotus. Secondary constrictions couldnot be defined in the chromosomes of thesethree species.

The present study was undert aken to de­lineate the occurrence and positions of sec­onda ry constric tions in the chromosomecomplements of M . infesta , M . macrocarpa ,

23

and M . italica. Addit ionally, a compari sonwas made between the satellite chromosomesof these species and the satellite chromosomesof M . alba and M . officinalis for evidence ofkar yotypic evolution.

Mat erials and Methods

Seed of each species was obtained from thecollection of Melilotus germplasm that is keptin cold storage at the University of Nebraska.M . infe st a ( Bdn. 61-98) and M . macrocarpa(Bdn. 6 1-97) were from seed of Algerian ac­cessions. Seed of M. italica (Bdn. 523) camefrom a collection made in Portugal. Seed ofcommercia lly available cultivars of M . alba(Evergreen- Lot 14C) and M. officinalis(Y ukon-F .e. 40594) were used in the study.

Actively growing root tips from one­mont h-old seedlings grown in clay pots wereutilized. The root tips were placed in ice waterfor 24 hours (for a detailed descr iption, seeSingh and Tsuchiyal -) and then fixed in a 3:1mixture of ethanol:propionic acid in which asmall amount of ferric chloride had been dis­solved as a mordant. After 3- 4 days in a fix­ative, the root tips were hydrolyzed in IN HCIat 60°C for 6 minutes and stained in aceto­carmine for at least 3 days. After sufficientstaining, the meristematic tissue was squashed

,

5p.m

I 'I'.

Discussion

. ..

Both species have small satellite bodetached to the short ar ms (Figure 4£E) .

Comparisons of the karyotypes of,\festa , M. macrocarpa , and M. itali:served in the present study with thos;

\I

1 11 ,

•-,

II

A

B

FIGU RE 1 A shows somatic chro mosomes of Melilotus infesta, 2n = 2x = 16;arrows indicate sate llite chromosomes. B-kar yotype of M . infesta; chromosome scut from A .

FIG URE 2 A shows somatic chromosomes of Melilotus macrocarpa, 2n = 2x = 16; arro»sate llite chromosomes. B- karyotype of M. macrocarpa; chromosomes cut from A.

B

A

yotype were metacentric while the smallerchromosomes tended to have nonmediancentromeres (Figure 3B) . The chromosomesof M. ital ica stained very deeply, suggestinga heterochromatic natur e.

The satellite chromosomes of M. alba andM. officinalis are different in morphologyfrom the preceding species (Figure 4A - E).

Results

The chromosome number of 2n = 2x = 16,as previously rep orted -P-U, was confirmed inall species. Numerous cellswere observed (ca.20) in each species in which chromosomemorphology was able to be characterized.Secondary constr ictions in one chromosomepair were found to exist in M. infe sta , M .macrocarpa, and M. italica (Figures I, 2, 3,and 4A -C). The existence of sate llite chro­mosomes in M. alba3 and M. offici nalis I alsowas reaffirmed (Figure 4D and E).

The chromosome complement of M. inf estais shown in Figure IA. A secondary constric­tion was found in the largest chromosome pairnear the centromere in the short arm (FiguresIB and 4A ). At somatic prophase, this chro­mosome pair appeared to be heterochromaticin contrast to the majority of other chromo­somes that appeared to stain normally (Figure5). The karyotypeshows the presence of manynonmetacentric chromosomes (Figure IB ).

Figure 2A shows the somatic chromosomesof M . macrocarpa. The chromosomes withsatellites in this species were the largestchromosomes in the complement (Figure 2B)and appear to be heterochromatic, as in M .infes ta. In general, the karyotype of M. ma­crocarpa closely resembles the M. infestakaryotype. However, theshort arm bearing thesate llite body in M. macrocarpa was shorter,relatively, than the corresponding short armin M. infes ta (Figure 4A and B). The chro­mosomes of M . macrocarpa, excluding thesate llite chromosome pair, did not sta in asdeeply as chromosomes of the other species.

Overall, the chromosomes of M. italicawere the largest of the species studied (Figure3A ). The satellite bodies in this species werethe same or of greater length than the longarms of the satellite chromosomes(Figures 3Band 4C) . The larger chromosomes in the kar-

in 45 percent acetic acid. Dividing cells in themeristematic region were isolated and ana­lyzed.

For the Micromelilotu s species, thekaryological interpretations were made on thechromosome complement of a representat ivecell from each species in which the chromo­somes were well spread and the secondaryconstrictions were obvious. From these cells,karyotypes were constructed by arranging thechromosomes in pairs considering the totallength, centromere position, and gross mor­phology. The chromosome pairs were gener­ally arranged in gradation from the longest tothe shortest pair of the complement. Sate llitechromosome morphology in M. alba and M.officinalis cells was studied in a number ofpreparations.

24 Th e Journal of Heredit y Schl arb aum et al.: Seconda ry constrictions in ,~

..I

II

II)

I

A

'Io

••"B

I

5p.m C' ..

e4......

111111111111 I' IIB

,FIGURE 3 A shows somatic chromosomes of Melilotus italica, 2n = 2x = 16; arrows indicate satellitechromosomes. B-karyotype of M. italica; chromosomes cut from A. I

E 5,um

sen ted by Kita" show a general agreementwith the exception of the satellite chromo­somes. In each species, it appears that Kitamisidentified the secondary constriction as thepc;"!':!) constriction. The small size of the

shoTt armof the satellite chromosomes in M.infesta and M. macrocarpa causes a closeproximity between the primary and secondaryconstrictions making distinction very difficult(Figure 4A and B). Also, excessive chromo­some contractionand deep staining often ob­scured theprimary constriction in many cells.In the caseof M. italica, overstaining of thepresumably heterochromatic satellite chro­mosomes probably confounded Kita's obser­vations.

The chromosomes of M. macrocarpa aresimilar to those of M. infesta, yet appearsubstantially larger. This is in contrast withthe results of Kita 5.6 who observed no signifi­cant difference in chromosome size betweenthe two species. Comparison of the chromo­somes of M. infesta and M. macrocarpa bycbromosome index (short arm/long arm ratio)an~relative length, followingthe methodologyof Tjio and Hagberg 17, reveals no criticaldlferenee between the karyotypes. The sizedifferential found in the present study isprobably due to differences in chromosomecontraction between the two cells shown of

each species and to artifacts normally en­countered with the squash technique for slidepreparations.

The differences in size between chromo­somes of M. italica and those of other Meli­lotus species have been reported previously1,5.

In many plant species, it has been observedthat the evolution of karyotypes proceeds fromlarge chromosomes in primitive species tosmaller chromosomes in specialized species'>,Chromosome studies in two leguminous gen­era, Trifolium" and Medicago (Schlarbaurn,unpub.), indicate karyotypic evolution hasoccurred in this manner. This suggests that thechromosome size of M. italica may be indic­ative of the chromosome size in an ancestralMelilotus or pro-Melilotus species. This hy­pothesis is supported by the morphologicalobservations of Suvorov16, who considered M.italica to characterize the features of an an­cestral Melilotus type, although he recognizedthat paleobotanic information was lacking forthe genus. There is some doubt that thepresent karyotype of M. italica should beconsidered as a basikaryotype- ' for a Meli­lotus prototype, as it is semi-asymmetrical,indicating an advanced evolutionary state/-!'.The functional basis of the large size andheterochromatic nature of the M. italicachromosomes is not known.

FIGURE 4 Satellite chromosomes of Melilotusspecies isolated from different somatic cells. A--M.infesta; B-M. macrocarpa;C-M. italica; D-M.alba; E-M. officinalis.

•FIGURE 5 Chromosomes of Melilotus infesta atsomatic prophase: arrows indicate heterochromaticsatellite chromosomes.

The satellite chromosomes of the speciesstudied can be grouped into three types: typeI-satellite large, short arm large as in M.italica (Figure 4C); type II-satellite large,

&hlarbaum et al.: Secondary constrictions in Melilotus January / February 1984 25

short arm small as in M . infes ta and M . ma­crocarpa (Figure 4A and B ), and type 111 ­satellite small, short arm large as in M . albaand M. officinalis (Figure 4D and E ). Thedifferences between types II and III couldresult from a paracentric inversion withbreakage sites in the short ar m and satellitebody. The satellite chromosome morphologyof type I possibly represents a more primitivestate than sate llite chromosomes of types IIand III.

After observing altera tion of the nucleolusorganizer chromosome system due to irra­diat ion in Trill ium kamtschaticum Pall.,Matsuura- proposed that one method of sat­ellite chromosome origin was a paracentricinversion of a terminally located nucleolusorganizer region (NO R) to an intercalaryposition. If the origin of satellitechromosomesin Melilotus proceeded according to this hy­pothesis, the morphology of the satellitechromosomes in M . italica (type I) appearssimilar to what would be expected from a

26 Th e Journ al of Heredity

paracentric inversion in a chromosome witha terminal NOR present in a primitive Meli­lotus karyotype.

References

I. CLARKE, A. E. The number and morphology orchromosomes in the genus Melilotus. Univ. CalifPubI. Bot . 17:435 -444. 1934.

2. DARLINGTON , C. D. and A. P. WYLI E. Chro­mosome Atlas or Flowering Plants. George Allenand Unwin, London. 1955.

3. FRYER, J. R. Cytological studies in Medi cago .Melilotus and Trigonella. Call. J. Res . 3:3-50.1930.

4. ISELY, D. Keys to sweet clovers (Melilotus ). lowaAcad . Sci . 6 1:119-1 31. 1954.

5. KITA, F. St udies on the genus Melil ot us(swcetclovcr) with special reference to interrela­tionships among species from a cytological pointor view. J . Fac. Agri . Hokk aido Univ. 54:25- 122.1965.

6. - - . Studi es on morphology or the somaticchromosomes of the genus Melilotus (sweetclover).Jpn. 1. Bot. 19:149-1 74. 1966.

7. LEVITSKY, G. A. The karyotype in systematics.Bull. API'. Bot. Genet. Plant Breed. 27:220-2 40.1931.

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10. SCHULZ, O. E. Monographie der Gattung Mlotu s. Bot. Jah rb. 29:660 -735 . 190 I.

II. SENN, H. A. Chromosome number relationsin the Leguminosae. Bibliog. Genetica 12:175­1938.

12. SINGH, R. J . and T. TSUCHIYA. An imprrGiemsa N-banding technique for the identifieaor barleychromosomes. J. Hered. 73:227-229. I'

13. S tNOTO, Y. and D. SATO. Basikaryotype anana lysis. Cytologia 10:529- 538. 1940.

14. SMITH, W. K. and H. J. GORZ. Sweetcloverprovcrncnt. Adv. Agron . 17:164- 231. 1965.

15. STEBBtNS, G. L. Chromo somal EvolutiorHigher Plants. Addison-Wesley, Reading, MA.

16. S UVOROV , V. V. Sweetclover-Melilolus (ToAdans. Em. I II Flora or Cultivated Plants 01

U.S.S.R. E. N. Sinskaya, Ed. 13:426-'Tra nslation from Russian as OTS 60-5 1198.'Dept. Commerce , Washington, D.C. 1950.

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