Isolation and sequence analysis of DREB2A homologues in three cereal and two legume species

The transcription factor, DREB2A, is one of the promising candidate genes involved in dehydration
tolerance in crop plants. In order to isolate DREB2A homologues across cereals (rice, barley and sorghum)
and legumes (common bean and chickpea), specific or degenerate primers were used. Gene/phylogenetic
trees were constructed using a non-redundant set of 19 DREB1A and 27 DREB2A amino acid sequences and
were combined with taxonomic/species tree to prepare reconciled phylogenetic trees. In total, 86
degenerate primers were designed for different clades and 295 degenerate primer combinations were used
to amplify DREB homologues in targeted crop species. Successful amplification of DREB2A was obtained in
case of sorghum.In parallel, gene-specific primerswere used toamplifyDREB2Ahomologues inrice, barley,
common bean and chickpea. Seven to eight diverse genotypes from targeted species were used for
sequence analysis at DREB2A locus identified/isolated. A maximum of eight SNPs were found in the
commonbean DREB2A, indicating two distinct haplotypes, three SNPs with five haplotypes were observed
in barley whereas a single SNP was observed in rice, sorghumand chickpea. Parsimony based phylogenetic
tree revealed distinct clustering of cereals and legumes. Furthermore, alignment of corresponding amino
acid sequences showed conservation of AP2 domain across the targeted species.