Development and characterization of SSR markers and their use to assess genetic relationships among alfalfa germplasms

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Simple sequence repeat (SSR), or microsatellite markers, are codominant, abundant and hypervariable molecular markers from eukaryotic genomes that are being widely used in genetic mapping, phylogenetic studies, and marker-assisted selection. Currently, the number of SSR markers available from alfalfa (Medicago sativa L.) genomic libraries is limited. This study was conducted to identify additional SSR markers in the alfalfa genome and to evaluate their ability to separate the nine historically recognized progenitors of North American cultivated alfalfa (African, Chilean, Falcata, Flemish, Indian, Ladak, Peruvian, Turkistan, and Varia), as well as seven additional accessions of M. sativa ssp. sativa, falcata, and coerulea and the model legume M. truncatula. Genomic DNA from the autotetraploid alfalfa germplasm W10 was used to develop 81 primer pairs, which amplified SSRs containing AC, AT, CT, CTT, GAT, and GGT motifs. The majority (96%) of the primer pairs were functional and 61 (78.2%) detected 2 to 11 polymorphic fragments among the accessions. A dendrogram was constructed using cluster analysis from these data, representing three main clusters: (i) diploid ssp. falcata; (ii) M. truncatula; and (iii) all remaining entries. Additional separation of some accessions [M. truncatula (‘Jemalong’), Ladak (‘Ladak’), Fall dormancy 11 (UC-1465), Indian (Sirsa Type 9), Flemish (‘Dupuit’), Peruvian (‘Hairy Peruvian’), and African 2 (‘Moapa’)] was obtained through multiple correspondence analysis. These genomic alfalfa SSRs have excellent utility for polymorphic assessment with potential application for phylogenetic and genetic mapping studies of alfalfa.

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