Marker-assisted selection (MAS) accelerates conventional breeding approaches in the improvement of multigenic traits. We used a bulk segregant analysis (BSA) approach to identify genetic polymorphisms closely associated to cold adaptation among populations of alfalfa (Medicago sativa L.) recurrently selected for increased tolerance to freezing (TF). Using bulk DNA samples from cultivar Apica (A-TFO) and populations (A-TF2 and A-TF5) derived from that initial background, we observed both the intensification and the disappearance of several DNA fragments in response to selection pressure. Subsequent assessment of freezing tolerance of individual genotypes confirmed a close relationship between some of these polymorphisms and freezing tolerance. Our results illustrate that the combination of BSA and populations recurrently selected for the improvement of polygenic traits are effective tools to develop MAS applications in alfalfa.
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