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Genetic diversity in the core subset of the U.S. red clover germplasm.

Article Excerpt
RED CLOVER is one of the most important forage species in the USA and the world. It is grown alone or in mixture with grasses in a wide range of soil and environmental conditions. The adaptability of red clover and its capacity to fix nitrogen in association with Rhizobium makes it useful for hay, silage, pasture, and soil improvement (Smith et al., 1985).

Red clover originated in Eurasia where wild-type populations are found in the Caucasus Mountains. In its natural habitat it is found in meadows, forest margins, and field borders (Gillett and Taylor, 2001). The U.S. National Germplasm System (NPGS) has a collection of about 1300 accessions of this species. A core subset was developed for red clover using morphological and physiological descriptors (Kouame and Quesenberry, 1993). This core subset is expected to represent the different patterns of phenotypic diversity present in the collection. However, the biochemical diversity present in the core subset is unknown. Characterization of the NPGS red clover core subset using isozymes would facilitate the study of relationship among accessions and assist in the identification of unique accessions. This information will help in the detection of genetic differences among accessions and in the identification of duplicate accessions (Bretting and Wilrlechner, 1995). Furthermore, information on genetic diversity serves as a basis to design strategies that capture and maintain a high proportion of the variability of the species (Hamrick and Godt, 1997).

Several diversity studies have been conducted in red clover using isozymes in recent years. Hagen and Hamrick (1998) measured high levels of genetic diversity within nine naturalized red clover populations and low levels of genetic divergence among the red clover populations. Yu et al. (2001) conducted a more extensive study on diversity of North American red clover cultivars. Percentage polymorphic loci within cultivars ranged from 61.5 to 84.6% with an overall mean of 74.0%. At the species level, the number of alleles per polymorphic locus was 2.55 and effective number of alleles per locus was 1.64. Within-cultivar averages were 2.71 and 1.59, respectively. Genetic diversity was 0.292 at the species level and 0.285 for within cultivars. Most of the genetic diversity (98.4-99.7%) was distributed within the cultivars. Mosjidis et al. (2004) assessed genetic diversity in 15 wild red clover populations from Russia. They reported that 90% of the loci were polymorphic in at least one population. Percent polymorphic loci within populations ranged between 50 and 90%. At the species level, the number of alleles per polymorphic locus was 2.89 and effective number of alleles per locus was 1.84. Within-population averages were 2.75 and 1.70, respectively. Genetic diversity was 0.353 at the species level and the mean value for the populations was 0.323. The objective of this study was to assess genetic diversity in the core subset (Available online at http://www. ars-grin.gov/cgi-bin/npgs/html/eval.pl?424001) (verified 28 November 2005) of the USA red clover germplasm based on isozyme data.

MATERIAL AND METHODS

Seeds of 81 red clover accessions (Table 1) were obtained from the USDA-ARS Plant Introduction Stations at Pullman (W-6), Washington. Eighteen plants of each accession were grown in pots filled with potting soil in a greenhouse.

Isozyme assays were conducted on young leaf tissue from each individual plant. The youngest fully expanded leaf (about 230 mg) was homogenized with 90 [micro]L extraction buffer [sucrose 16.7% (w/v) and sodium ascorbate 8.3% (w/v) in 50 mM Tris-HCl, pH 7.4] at -20[degrees]C. Crude extracts were centrifuged for 5 min at 8160 x g. Supernatant (5.5 [degrees]L/well) was loaded onto precast, agarose isoelectric focusing (IEF) gels (Isolab, Akron, OH). Gels with pH gradients 3 to 5 (50%), 3 to...