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Agronomy & Horticulture -- Faculty Publications Agronomy and Horticulture Department

1984

Identification of with Secondary Constrictions in Melilotus Species

S. E. Schlarbaum Kansas State University

L. B. Johnson United States Department of Agriculture

Herman J. Gorz United States Department of Agriculture

Francis A. Haskins University of Nebraska-Lincoln, [email protected]

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

This Article is brought to you for free and open access by the Agronomy and Horticulture Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Agronomy & Horticulture -- Faculty Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. The Journal of Heredity 75:23-26. 1984.

Identification of chromosomes with secondary constrictions in Me/ilotuB species

ABSTRACT: Secondary constrictions were determined in one pair in the com­ plements of M. intesta, M. macrocarpa, M. italica (subgenus Micromeli/otus), and M. alba and M. officinalis (subgenus Meli/otus). The of M. intesta and M. macrocarpa were found to be similar. Chromosomes of M. italica were larger than the chromosomes of the other four Melilotus species. The chromosome size in M. italica suggests the pres­ ence of large chromosomes in an ancestral or pro-Melilotus prototype. The chromosomes with satellites of M. intesta and M. macrocarpa appear to differ in morphology from the satellite chromosomes of M. alba and M. officinalis by a paracentric inversion. The mor­ phology of the satellite chromosomes in M. italica is thought to rep resent a more primitive type than in the other Meli/otus species studied .

S. E. Schla r baum T HE SW EETCLOVER GEN US, Melilotu s and M . italica. Addit ionally, a comparison Ada ns., contains forage and nitrogen fixing was made between the satellite chromosomes LB. Johnson species of agricultural importance. Taxo­ of these species and the satellite chromosomes of M . alba and M . officinalis for evidence of H.J.Gorz nomically, Melilot us is divided into two subgenera 10, (E ll )Melilotus Schulz and Mi­ kar yotypic evolution. F.A. Haskin s cromelilotus Schulz. Isely' considers the subgenus Melilotus to consist of nine biennial Materials and Methods species, including the commercially important M . alba Desr. and M. officinalis (L.) Larn., Seed of each species was obtained from the while the subgenus Mi crom elilotus contains collection of Melilotus germplasm that is kept II annual species, fewof which have economic in cold storage at the University of Nebraska. value as crops!". Other taxonomists, most M . infest a (Bdn. 61-98) and M . macrocarpa notably Suvorov!", consider the genus to (Bdn. 6 1-97) were from seed of Algerian ac­ contain a greater number of species. cessions. Seed of M. italica (Bdn. 523) came The chromosome number of many Me li­ from a collection made in Portugal. Seed of Theauthors are. respectively. former postdoctoral lotus species, 2n = 2x = 16, was determined commercially available cultivars of M . alba researchassociate. and professor. Department of Plant ea rly in this century-'U . analyses (Evergreen-Lot 14C) and M. officinalis Pathology. Throckmorton Hall. Kansas State Univer­ of all Melilotu s species, with the exception of (Y ukon-F.e. 40594) were used in the study. sity. Munhattun, KS 66506: supervisory research geneticist. USDA-ARS. and George Holmes Professor the du bious M . bicolor Boiss & Bal., were Actively growing root tips from one­ of Agronomy. Departm ent of Agronomy. University reported by Kita5.6. Chromosomes with sat­ mont h-old seedlings grown in clay pots were or Nebraska. Lincoln. NE 68583. Dr. Schlarbaum's ellites were found in all species except M. in­ utilized. The root tips were placed in ice water present address: Depart ment of Forestry. Wildlife and Fisheries. University of Tennessee. Knoxville. TN festa Guss., M. macrocarpa Goss. et Dur., and for 24 hours (for a detailed descr iption, see 37901. Research supported by National Science M . ita lica ( L.) Larn., which belong to Mi­ Singh and Tsuchiyal-) and then fixed in a 3:1 Foundation grant PCM 8022556. Contribu tion no. cromelilotus. Secondary constrictions could mixture of ethanol:propionic acid in which a 82-57 5·). Department of Plant Pathology. Kansas State not be defined in the chromosomes of these small amount of ferric chloride had been dis­ University. Agricultural Experiment Station. Man­ hauan, Kansas 66506. Research was done in coopera­ three species. solved as a mordant. After 3- 4 days in a fix­ tionwiththe Nebraska Agricultural Experiment Sta­ The present study was undert aken to de­ ative, the root tips were hydrolyzed in IN HCI tionand the U.S. Department of Agriculture. Agri­ lineate the occurrence and positions of sec­ at 60°C for 6 minutes and stained in aceto­ cultural Research Service. Please address reprint re­ queststoDr. S. E. Schlar baum. ondary constrictions in the chromosome carmine for at least 3 days. After sufficient i£ 1984. Ameriean Genetic Association. complements of M . infesta, M . macrocarpa , staining, the meristematic tissue was squashed

23 in 45 percent acetic acid. Dividing cells in the meristematic region were isolated and ana­ lyzed. For the Micromelilotu s species, the karyological interpretations were made on the chromosome complement of a representative cell from each species in which the chromo­ somes were well spread and the secondary constrictions were obvious. From these cells, karyotypes were constructed by arranging the I chromosomes in pairs considering the total \ length, position, and gross mor­ phology. The chromosome pairs were gener­ ally arranged in gradation from the longest to A the shortest pair of the complement. Satellite 5p.m chromosome morphology in M. alba and M. officinalis cells was studied in a number of preparations. II 1 11 , I Results 'I'. B The chromosome number of 2n = 2x = 16, as previously rep orted-P-U, was confirmed in FIGU RE 1 A shows somatic chro mosomes of Melilotus infesta, 2n = 2x = 16; all species. Numerous cellswere observed (ca. arrows indicate sate llite chromosomes. B-kar yotype of M . infesta; chromosome s 20) in each species in which chromosome cut from A . morphology was able to be characterized. Secondary constrictions in one chromosome pair were found to exist in M. infe sta , M . macrocarpa, and M. italica (Figures I, 2, 3, yotype were metacentric while the smaller Both species have small satellite bode and 4A -C). The existence of satellite chro­ chromosomes tended to have nonmedian tached to the short ar ms (Figure 4£ E) . mosomes in M. alba 3 and M. offici nalis I also (Figure 3B) . The chromosomes of M. italica stained very deeply, suggesting was reaffirmed (Figure 4D and E). Discussion The chromosome complement of M. inf esta a heterochromatic natur e. is shown in Figure IA. A secondary constric­ The satellite chromosomes of M. alba and Comparisons of the karyotypes of,\ tion was found in the largest chromosomepair M. officinalis are different in morphology festa , M. macrocarpa, and M. itali: near the centromere in the short arm (Figures from the preceding species (Figure 4A - E ). served in the present study with thos; IB and 4A). At somatic prophase, this chro­ mosome pair appeared to be heterochromatic in contrast to the majority of other chromo­ somes that appeared to stain normally (Figure 5). The karyotypeshows the presence of many nonmetacentric chromosomes (Figure IB). . .. Figure 2A showsthe somatic chromosomes of M . macrocarpa. The chromosomes with satellites in this species were the largest chromosomes in the complement (Figure 2B) and appear to be heterochromatic, as in M . •-, infesta. In general, the karyotype of M. ma­ crocarpa closely resembles the M. infesta karyotype. However, theshort arm bearing the satellite body in M. macrocarpa was shorter, A relatively, than the corresponding short arm in M. infesta (Figure 4A and B). The chro­ mosomes of M . macrocarpa, excluding the satellite chromosome pair, did not stain as deeply as chromosomes of the other species. Overall, the chromosomes of M. italica , were the largest of the speciesstudied (Figure 3A). The satellite bodies in this species were B the same or of greater length than the long arms of the satellite chromosomes(Figures 3B FIG URE 2 A shows somatic chromosomes of Melilotus macrocarpa, 2n = 2x = 16; arro» and 4C) . The larger chromosomes in the kar- sate llite chromosomes. B- karyotype of M. macrocarpa; chromosomes cut from A.

24 The Journal of Heredit y Schl arb aum et al.: Seconda ry constrictions in ,~ e4 ) II .... I A •• .. B" .. ' .. I I 5p.m C

I'II 'I II B 111111111111 o

,FIGURE 3 A shows somatic chromosomes of Melilotus italica, 2n = 2x = 16;arrows indicate satellite chromosomes. B-karyotype of M. italica; chromosomes cut from A. I E 5,um

sen ted by Kita" show a general agreement each species and to artifacts normally en­ FIGURE 4 Satellite chromosomes of Melilotus species isolated from different somatic cells. A--M. with the exception of the satellite chromo­ countered with the squash technique for slide infesta; B-M. macrocarpa;C-M. italica; D-M. somes. In each species, it appears that Kita preparations. alba; E-M. officinalis. misidentified the as the The differences in size between chromo­ pc;"!':!) constriction. The small size of the somes of M. italica and those of other Meli­ shoTt armof the satellite chromosomes in M. lotus species have been reported previously 1,5. infesta and M. macrocarpa causes a close In many plant species, it has been observed proximity between the primary and secondary that the evolution of karyotypes proceeds from constrictions making distinction very difficult large chromosomes in primitive species to (Figure 4A and B). Also, excessive chromo­ smaller chromosomes in specialized species'>, some contractionand deep staining often ob­ Chromosome studies in two leguminous gen­ scured theprimary constriction in many cells. era, Trifolium" and Medicago (Schlarbaurn, In the caseof M. italica, overstaining of the unpub.), indicate karyotypic evolution has presumably heterochromatic satellite chro­ occurred in this manner. This suggests that the mosomes probably confounded Kita's obser­ chromosome size of M. italica may be indic­ vations. ative of the chromosome size in an ancestral The chromosomes of M. macrocarpa are Melilotus or pro-Melilotus species. This hy­ similar to those of M. infesta, yet appear pothesis is supported by the morphological • substantially larger. This is in contrast with observations of Suvorov16, who considered M. the results of Kita 5.6 who observed no signifi­ italica to characterize the features of an an­ cant difference in chromosome size between cestral Melilotus type, although he recognized the two species. Comparison of the chromo­ that paleobotanic information was lacking for FIGURE 5 Chromosomes of Melilotus infesta at somes of M. infesta and M. macrocarpa by the genus. There is some doubt that the somatic prophase: arrows indicate heterochromatic cbromosome index (short arm/long arm ratio) present karyotype of M. italica should be satellite chromosomes. an~relative length, followingthe methodology considered as a basikaryotype- ' for a Meli­ of Tjio and Hagberg 17, reveals no critical lotus prototype, as it is semi-asymmetrical, dlferenee between the karyotypes. The size indicating an advanced evolutionary state/-!'. The satellite chromosomes of the species differential found in the present study is The functional basis of the large size and studied can be grouped into three types: type probably due to differences in chromosome heterochromatic nature of the M. italica I-satellite large, short arm large as in M. contraction between the two cells shown of chromosomes is not known. 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. ma­ paracentric inversion in a chromosome with 8. MATSUURA, H. Chromosome studies on Trili crocarpa 4A B ), a terminal NOR present in a primitive Meli­ kamtschaticum Pall. XVI. Alterations or (Figure and and type 111 ­ nucleolus-chromosome system due to irradiat satellite small, short arm large as in M. alba lotus karyotype. Cytologia 12:271- 288. 1942 . and M. officinalis (Figure 4D and E ). The 9.PRITCHARD, J. A. Numbe r and rnorpholog differences between types II and III could chromosomes in African species in the genus: References folium L. AUSI. J. Agric. Res. 13:1023- 1029. I' result from a paracentric inversion with 10. SCHULZ, O. E. Monographie der Gattung M breakage sites in the short ar m and satellite I. CLARKE, A. E. The number and morphology or lotu s. Bot. Jah rb. 29:660 -735. 190 I. chromosomes in the genus Melilotus. Univ. Calif II. SENN, H. A. Chromosome number relations body. The satellite chromosome morphology PubI. Bot. 17:435 -444. 1934. of type I possibly represents a more primitive in the Leguminosae. Bibliog. Genetica 12:175­ 2. DARLINGTON , C. D. and A. P. WYLI E. Chro­ 1938. state than satellite chromosomes of types II mosome Atlas or Flowering Plants. George Allen 12. SINGH, R. J. and T. TSUCHIYA. An imprr and III. and Unwin, London. 1955. Giemsa N-banding technique for the identifiea 3. FRYER, J. R. Cytological studies in Medi cago . or barleychromosomes. J. Hered. 73:227-229. I' After observing alteration of the nucleolus Melilotus and Trigonella. Call. J. Res . 3:3-50. 13. StNOTO, Y. and D. SATO. Basikaryotype an organizer chromosome system due to irra­ 1930. ana lysis. Cytologia 10:529- 538. 1940. diation in Trillium kamtschaticum Pall., 4. ISELY, D. Keys to sweet clovers (Melilotus ). lowa Acad . Sci . 6 1:119-1 31. 1954. 14. SMITH, W. K. and H. J. GORZ. Sweetclover Matsuura- proposed that one method of sat­ 5. KITA, F. St udies on the genus Melil otus provcrncnt. Adv. Agron . 17:164- 231. 1965. ellite chromosome origin was a paracentric (swcetclovcr) with special reference to interrela­ 15. STEBBtNS, G. L. Chromosomal Evolutior inversion of a terminally located nucleolus tionships among species from a cytological point Higher Plants. Addison-Wesley, Reading, MA. organizer region (NO R) to an intercalary or view.J. Fac. Agri . Hokk aido Univ. 54:25- 122. 16. S UVOROV , V. V. Sweetclover-Melilolus (To 1965. Adans.Em. I II Flora or Cultivated Plants 01 position. If the origin of satellitechromosomes 6. - - . Studi es on morphology or the somatic U.S.S.R. E. N. Sinskaya, Ed. 13:426-' in Melilotus proceeded according to this hy­ chromosomes of the genus Melilotus (sweetclover). Tra nslation from Russian as OTS 60-5 1198.' pothesis, the morphology of the satellite Jpn. 1. Bot. 19:149-1 74. 1966. Dept. Commerce, Washington, D.C. 1950. 7. LEVITSKY, G. A. The karyotype in systematics. 17. TJIO, J. H. and A. HAGBERG. Cytologicalsu chromosomes in M. italica (type I) appears Bull. API'. Bot. Genet. Plant Breed. 27:220-2 40. on some x-ray mutants or barley. An . Esuu: i similar to what would be expected from a 1931. Au/a Dei 2:149- 167. 1951.

26 Th e Journ al of Heredity