Identification of Transcript-Derived Sequences That Vary in Abundance during Cold Acclimation in Red Clover (Trifolium pratense L.) By SRAP-cDNA

Red clover freezing tolerance (FT) is a major limitation to its persistence under cold-climate. We demonstrated that reed clover FT increases markedly in response to cold acclimation and can be improved by indoor recurrent phenotypic selection. The identification of genes associated to cold acclimation will help understanding the genetic basis of that trait, and could increase efficiency of breeding for superior FT in red clover.

We used SRAP-cDNA to identify genes differentially expressed between a red clover cultivar, Christie (C0), and three populations obtained after five to seven recurrent selection cycles for superior FT within this cultivar (C5, C6 and C7). Twenty-five combinations of SRAP primers identified 63 differentially amplified sequences. Sequence analysis revealed homology of 39 differentially amplified sequences with known genes in NCBI databases. A subset of 17 candidate genes was selected for analysis of their expression before cold acclimation (in October) and during winter (monthly between November and March). Expression of these 17 genes was analysed by RT-qPCR in two red clover cultivars, Christie (C0) and Endure (E0) and in populations obtained after several cycles of recurrent selection within each cultivar (C5, C6, C7 and E5, E6, E7). The 17 genes showed differential expression during cold acclimation, and similar profiles were observed in the two genetic backgrounds. Significant differences of gene expression were observed between populations within each genetic background.

Our results shows that differentially expressed genes can be identified by SRAP-cDNA, and that recurrent selection for superior FT affects the expression of genes during cold acclimation.