Theoretical and Applied Genetics

, Volume 126, Issue 7, pp 1885–1896 | Cite as

Cloning and characterization of chromosomal markers in alfalfa (Medicago sativa L.)

  • Feng Yu
  • Yunting Lei
  • Yuan Li
  • Quanwen Dou
  • Haiqing Wang
  • Zhiguo Chen
Original Paper


Eleven tandemly repetitive sequences were identified from a Cot-1 library by FISH and sequence analysis of alfalfa (Medicago sativa). Five repetitive sequences (MsCR-1, MsCR-2, MsCR-3, MsCR-4, and MsCR-5) were centromeric or pericentromeric, of which three were satellite DNAs and two were minisatellite DNAs. Monomers of 144, 148, and 168 bp were identified in MsCR-1, MsCR-2, and MsCR-3, respectively, while 15 and 39 bp monomers were identified in MsCR-4 and MsCR-5, respectively. Three repetitive sequences were characterized as subtelomeric; one repetitive sequence, MsTR-1, had a 184 bp monomer, and two repetitive sequences had fragments of 204 and 327 bp. Sequence analysis revealed homology (70–80 %) between MsTR-1 and a highly repeated sequence (C300) isolated from M. ssp. caerulea. Three identified repetitive sequences produced hybridization signals at multiple sites in a few of the chromosomes; one repetitive sequence was identified as the E180 satellite DNA previously isolated from M. sativa, while the other 163 and 227 bp fragments had distinct sequences. Physical mapping of the repetitive sequences with double-target FISH revealed different patterns. Thus, nine novel tandemly repetitive sequences that can be adopted as distinct chromosome markers in alfalfa were identified in this study. Furthermore, the chromosome distribution of each sequence was well described. Though significant chromosome variations were detected within and between cultivars, a molecular karyotype of alfalfa was suggested with the chromosome markers we identified. Therefore, these novel chromosome markers will still be a powerful tool for genome composition analysis, phylogenetic studies, and breeding applications.


Repetitive Sequence Chromosome Marker Strong Hybridization Signal E180 Signal Minisatellite DNAs 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank Professor Tao Wang (China Agriculture University) for providing the seeds of some alfalfa materials. This study was financially supported by the “Joint Scholars” program of the “Lights in the Western Region” talent cultivation plan of the Chinese Academy of Sciences and partly supported by the Main Direction Program for Knowledge Innovation of the Chinese Academy of Sciences (KSCX2-EW-Q-23).

Supplementary material

122_2013_2103_MOESM1_ESM.doc (119 kb)
Supplementary material 1 (DOC 119 kb)


  1. Allen G, Flores-Vergara M, Krasynanski S, Kumar S, Thompson W (2006) A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc 1:2320–2325PubMedCrossRefGoogle Scholar
  2. Barnes D, Bingham E, Murphy R, Hunt O, Beard D, Skrdla W, Teuber L (1977) Alfalfa germplasm in the United States: genetic vulnerability, use, improvement and maintenance. USDA Technical Bulletin 1571. ARS, Washington DC, p 21Google Scholar
  3. Bauchan G, Hossain M (1997) Identification of Alfalfa chromosomes using Geimsa banding and image analysis techniques. In: Proceedings of the XVIII International Rangeland Congress, Canada, pp 61–62Google Scholar
  4. Bauchan GR, Hossain MA (2001) Distribution and characterization of heterochromatic DNA in the tetraploid African population alfalfa genome. Crop Sci 41:1921–1926CrossRefGoogle Scholar
  5. Begum R, Alam SS, Menzel G, Schmidt T (2009) Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh. Ann Bot 104:863–872PubMedCrossRefGoogle Scholar
  6. Blondon F, Marie D, Brown S, Kondorosi A (1994) Genome size and base composition in Medicago sativa and Medicago truncatula species. Genome 37:264–270PubMedCrossRefGoogle Scholar
  7. Calderini O, Pupilli F, Paolocci F, Arcioni S (1997) A repetitive and species-specific sequence as a tool for detecting the genome contribution in somatic hybrids of the genus Medicago. Theor Appl Genet 95:734–740CrossRefGoogle Scholar
  8. Cheng Z, Dong F, Langdon T, Ouyang S, Buell CR, Gu M, Blattner FR, Jiang J (2002) Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon. Plant Cell 14:1691–1704PubMedCrossRefGoogle Scholar
  9. Cleveland R, Stanford E (1959) Chromosome pairing in hybrids between tetraploid Medicago sativa L. and diploid Medicago falcata L. Agron J 51:488–492CrossRefGoogle Scholar
  10. Dou QW, Chen ZG, Liu YA, Tsujimoto H (2009) High frequency of karyotype variation revealed by sequential FISH and GISH in plateau perennial grass forage Elymus nutans. Breed Sci 59:651–656CrossRefGoogle Scholar
  11. Falistocco E (2000) Brief communication. Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization. J Hered 91:256–260PubMedCrossRefGoogle Scholar
  12. Falistocco E, Falcinelli M, Veronesi F (1995) Karyotype and C-banding pattern of mitotic chromosomes in alfalfa, Medicago sativa L. Plant Breed 114:451–453CrossRefGoogle Scholar
  13. Fonsêca A, Ferreira J, dos Santos TR, Mosiolek M, Bellucci E, Kami J, Gepts P, Geffroy V, Schweizer D, dos Santos KGB, Pedrosa-Harand A (2010) Cytogenetic map of common bean (Phaseolus vulgaris L.). Chromosome Res 18:487–502PubMedCrossRefGoogle Scholar
  14. Fukui K, Kamisugi Y, Sakai F (1994) Physical mapping of 5S rDNA loci by direct-cloned biotinylated probes in barley chromosomes. Genome 37:105–111PubMedCrossRefGoogle Scholar
  15. Galasso I, Schmidt T, Pignone D (2001) Identification of Lens culinaris ssp. culinaris chromosomes by physical mapping of repetitive DNA sequences. Chromosome Res 9(3):199–209PubMedCrossRefGoogle Scholar
  16. Gillies CB (1970) Alfalfa Chromosomes 1. Pachytene karyotype of diploid Medicago falcata L. and its relationship to Medicago sativa L. Crop Sci 10:169–171CrossRefGoogle Scholar
  17. Han Y, Zhang Z, Huang S, Jin W (2011) An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2. BMC Genet 12:18PubMedCrossRefGoogle Scholar
  18. Ho KM, Kasha KJ (1972) Chromosome homology at pachytene in diploid Medicago sativa, Medicago falcata and their hybrids. Can J Genet Cytol 14:829–838Google Scholar
  19. Jiang JM, Gill BS (2006) Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research. Genome 49:1057–1068PubMedCrossRefGoogle Scholar
  20. Jin W, Melo JR, Nagaki K, Talbert PB, Henikoff S, Dawe RK, Jiang J (2004) Maize centromeres: organization and functional adaptation in the genetic background of oat. Plant Cell 16:571–581PubMedCrossRefGoogle Scholar
  21. Kato A, Lamb JC, Birchler JA (2004) Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize. Proc Natl Acad Sci USA 101:13554PubMedCrossRefGoogle Scholar
  22. Kulikova O, Geurts R, Lamine M, Kim DJ, Cook DR, Leunissen J, De Jong H, Roe BA, Bisseling T (2004) Satellite repeats in the functional centromere and pericentromeric heterochromatin of Medicago truncatula. Chromosoma 113:276–283PubMedCrossRefGoogle Scholar
  23. Lesins K (1957) Cytogenetic study on a tetraploid plant at the diploid chromosome level. Can J Bot 35:181–196CrossRefGoogle Scholar
  24. Masoud SA, Gill BS, Johnson LB (1991) C-Banding of alfalfa chromosomes standard karyotype and analysis of a somaclonal variant. J Hered 82:335–338Google Scholar
  25. McCoy TJ, Bingham ET (1988) Cytology and cytogenetics of alfalfa. In: alfalfa and alfalfa improvement. Agron. Monogr. 29. ASA, CSSA and SSSA. Madison, WI, pp 737–776Google Scholar
  26. Paesold S, Borchardt D, Schmidt T, Dechyeva D (2012) A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome-arm identification, integration of genetic linkage groups and analysis of major repeat family distribution. Plant J 72:600–611PubMedCrossRefGoogle Scholar
  27. Richards EJ, Ausubel FM (1988) Isolation of a higher eukaryotic telomere from Arabidopsis thaliana. Cell 53:127–136PubMedCrossRefGoogle Scholar
  28. Rosato M, Galian JA, Rossello JA (2012) Amplification, contraction and genomic spread of a satellite DNA family (E180) in Medicago (Fabaceae) and allied genera. Ann Bot 109:773–782PubMedCrossRefGoogle Scholar
  29. Schmidt T, Heslop-Harrison JS (1998) Genomes, genes and junk: a model of the large-scale organization of plant chromosomes. Trends Plant Sci 3:195–199CrossRefGoogle Scholar
  30. Winicov I, Maki DH, Waterborg JH, Riehm MR, Harrington RE (1988) Characterization of the alfalfa (Medicago sativa) genome by DNA reassociation. Plant Mol Biol 10:369–371CrossRefGoogle Scholar
  31. Xia X, Erickson L (1993) An AT-rich satellite DNA sequence, E180, in alfalfa (Medicago sativa). Genome 36:427–432PubMedCrossRefGoogle Scholar
  32. Zakrzewski F, Wenke T, Holtgrawe D, Weisshaar B, Schmidt T (2010) Analysis of a Cot-1 library enables the targeted identification of minisatellite and satellite families in Beta vulgaris. BMC Plant Bio 10:8CrossRefGoogle Scholar
  33. Zhang L, Xu C, Yu W (2012) Cloning and characterization of chromosomal markers from a Cot-1 library of peanut (Arachis hypogaea L.). Cytogenet Genome Res 137:31–41PubMedCrossRefGoogle Scholar
  34. Zwick MS, Hanson RE, Islam-Faridi MN, Stelly DM, Wing RA, Price HJ, McKnight TD (1997) A rapid procedure for the isolation of Cot-1 DNA from plants. Genome 40:138–142PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Feng Yu
    • 1
    • 2
  • Yunting Lei
    • 1
  • Yuan Li
    • 1
    • 2
  • Quanwen Dou
    • 1
  • Haiqing Wang
    • 1
  • Zhiguo Chen
    • 1
  1. 1.Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Plateau Institute of BiologyChinese Academy of SciencesXiningChina
  2. 2.Graduate University of Chinese Academy of SciencesBeijingChina

Personalised recommendations