Trifolium polymorphum Poir. is an amphicarpic forage legume from southern Brazil, Uruguay, Argentina, Paraguay and Chile. Information on the genetic diversity of natural populations in natural grasslands in southern Brazil is limited. In order to increase the knowledge about this species, an analysis of the genetic diversity was carried out in 10 natural populations of T. polymorphum with the use of 20 microsatellite markers. The expected heterozygosity in T. polymorphum populations ranged from 0.40 to 0.43, with a mean of 0.42. A total of 193 alleles were detected with a mean of 9.3 alleles per locus and polymorphic information content (PIC) for these markers of 0.62 to 0.89 with a mean of 0.84.The grouping based on the Jaccard's coefficient of similarity classified populations, regardless of their regions of origin, into two groups with a mean similarity coefficient of 0.32, reflecting the high genetic variability of the populations, especially those located in the Campanha phytogeographic region. This information on diversity can be used to plan future germplasm collection strategies for conservation purposes and also for the breeding of the species.
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