Identification of transcripts associated with the acquisition of superior freezing tolerance in recurrently-selected populations of alfalfa

Abstract

The identification of transcripts that vary during the acquisition of superior tolerance to environmental stress contribute to the understanding of the molecular bases of adaptation to adverse growth conditions. SRAP-cDNA was used to identify genes that were differentially expressed between plants of alfalfa (Medicago sativa L.) that were non-acclimated (NA) or hardened to a non-lethal freezing temperature (HF). Search of polymorphism was performed within the cultivar Apica (ATF0) and population ATF5 obtained after five cycles of recurrent selection for superior tolerance to freezing (TF) within ATF0. Analysis of bulked cDNA from 50 non-acclimated and 50 cold-acclimated plants in each population identified transcripts that vary in abundance in response to acclimation to sub-zero temperature and recurrent selection. Sequencing of purified fragments revealed significant homologies with genes with key physiological functions that are distributed across the Medicago truncatula genome. Regulatory genes including receptor-like kinases, a phytochrome-interacting factor and a phosphoinositide phosphatase are among potential candidates involved in the improvement of freezing tolerance by RS. RT-qPCR analysis of samples from populations recurrently selected within the cultivars Apica and Evolution confirmed the common cold-induced responses in unrelated genetic backgrounds. Our results show that random amplification of cDNA in combination with bulk segregant analysis of pooled samples from recurrently selected populations is an effective strategy to identify functional sequences putatively associated with superior acclimation to environmental stress in open-pollinated species.