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Sample size for diversity studies in tetraploid alfalfa (Medicago sativa) based on codominantly coded SSR markers


The number of genotypes investigated per population is important for the reliability of diversity studies. The objective of this study was to determine the sample size for the identification of differences among populations of an outcrossing autotetraploid species, alfalfa (Medicago sativa), using codominantly coded SSR markers. One hundred and twenty genotypes from each of two closely related populations were analysed with two markers. Twenty random subsamples for each of three sample sizes (10, 20 and 40 genotypes) were built. Compared to the populations with 120 genotypes, alleles that were no longer present in subsamples with 40 genotypes were mainly rare, whereas abundant alleles were also excluded in subsamples with 10 genotypes. F ST values for pairs of subsamples between the two populations were always significantly different based on 40 genotypes, whereas for 10 genotypes more than half of the pairs were not significantly different. We concluded that 40 genotypes are a reasonable sample size for diversity studies with closely related populations of tetraploid alfalfa investigated with SSR markers. Twenty genotypes may be an economical alternative for large scale studies, but 10 genotypes were a too low number for reliable results.

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  1. Ayadi R, Barre P, Huyghe C, Julier B (2005) Estimation of the coefficient of double-reduction in autotetraploid lucerne. Wageningen. July 2005

  2. Becker H (1993) Plant Breeding. Stuttgart

  3. Bolaric S, Barth S, Melchinger AE, Posselt UK (2005) Genetic diversity in European perennial ryegrass cultivars investigated with RAPD markers. Plant Breed 124:161–166

    Article  CAS  Google Scholar 

  4. Cockerham CC (1973) Analyses of gene frequencies. Genetics 74:679–700

    PubMed  Google Scholar 

  5. Crossa J (1989) Methodologies for estimating the sample size required for genetic conservation of outbreeding crops. Theor Appl Genet 77:153–161

    Google Scholar 

  6. Estoup A, Angers B (1998) Microsatellites and minisatellites for molecular ecology: theoretical and empirical considerations. Adv Mol Ecol 306:55–86

    CAS  Google Scholar 

  7. Fjellheim S, Rognli OA (2005) Molecular diversity of local Norwegian meadow fescue (Festuca pratensis Huds.) populations and Nordic cultivars—consequences for management and utilisation. Theor Appl Genet 111:640–650

    Article  CAS  PubMed  Google Scholar 

  8. Flajoulot S, Ronfort J, Baudouin P, Barre P, Huguet T, Huyghe C, Julier B (2005) Genetic diversity among alfalfa (Medicago sativa) cultivars coming from a breeding program, using SSR markers. Theor Appl Genet 111:1420–1429

    Article  CAS  PubMed  Google Scholar 

  9. Gherardi M, Mangin B, Goffinet B, Bonnet D, Huguet T (1998) A method to measure genetic distance between allogamous populations of alfalfa (Medicago sativa) using RAPD molecular markers. Theor Appl Genet 96:406–412

    Article  CAS  Google Scholar 

  10. Herrmann D, Boller B, Widmer F, Kölliker R (2005) Optimization of bulked AFLP analysis and its application for exploring diversity of natural and cultivated populations of red clover. Genome 48:474–486

    Article  CAS  PubMed  Google Scholar 

  11. Herrmann D, Flajoulot S, Barre B, Huyghe C, Ronfort J, Julier B (2008) Comparison of morphological traits and SSR markers to analyze genetic diversity of alfalfa cultivars. North American Alfalfa Improvement Conference. North American Alfalfa Improvement Conference,

  12. Jenczewski E, Prosperi JM, Ronfort J (1999) Differentiation between natural and cultivated populations of Medicago sativa (Leguminosae) from Spain: analysis with random amplified polymorphic DNA (RAPD) markers and comparison to allozymes. Mol Ecol 8:1317–1330

    Article  CAS  PubMed  Google Scholar 

  13. Julier B, Flajoulot S, Barre P, Cardinet G, Santoni S, Huguet T, Huyghe C (2003) Construction of two genetic linkage maps in cultivated tetraploid alfalfa (Medicago sativa) using microsatellite and AFLP markers. BMC Plant Biol 3:9

    Article  PubMed  Google Scholar 

  14. Kölliker R, Stadelmann FJ, Reidy B, Nösberger J (1999) Genetic variability of forage grass cultivars: a comparison of Festuca pratensis Huds., Lolium perenne L., and Dactylis glomerata L. Euphytica 106:261–270

    Article  Google Scholar 

  15. Labombarda P, Pupilli F, Arcioni S (2000) Optimal population size for RFLP-assisted cultivar identification in alfalfa (Medicago sativa L.). Agronomie 20:233–240

    Article  Google Scholar 

  16. Lynch M, Milligan BG (1994) Analysis of population genetic-structure with RAPD markers. Mol Ecol 3:91–99

    Article  CAS  PubMed  Google Scholar 

  17. Patto MCV, Moreira PM, Almeida N, Satovic Z, Pego S (2008) Genetic diversity evolution through participatory maize breeding in Portugal. Euphytica 161:283–291

    Article  Google Scholar 

  18. Ronfort JL, Jenczewski E, Bataillon T, Rousset F (1998) Analysis of population structure in autotetraploid species. Genetics 150:921–930

    CAS  PubMed  Google Scholar 

  19. Sardaro MLS, Atallah M, Tavakol E, Russi L, Porceddu E (2008) Diversity for AFLP and SSR in natural populations of Lotus corniculatus L. from Italy. Crop Sci 48:1080–1089

    Article  CAS  Google Scholar 

  20. Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 68:1358–1370

    Article  Google Scholar 

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The authors thank F. Durand for her technical assistance in the lab. D Herrmann received a grant from the Plant Breeding department of INRA. The study was supported by the French Ministry of Agriculture, in the “Contrat de branches CB47” with ACVF (Association des Créateurs de Variétés Français).

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Correspondence to Bernadette Julier.

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Herrmann, D., Flajoulot, S. & Julier, B. Sample size for diversity studies in tetraploid alfalfa (Medicago sativa) based on codominantly coded SSR markers. Euphytica 171, 441 (2010).

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  • Diversity
  • Medicago sativa
  • Sample size
  • SSR marker
  • Tetraploidy