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Environmental stresses such as low temperature, drought, and high salinity significantly affect plant growth and yield. As selective forces, these adverse factors play essential roles in shaping phenotypic variation in plant populations. Black poplar ( Populus nigra ) is an economically and ecologically important forest tree species with widely distributed populations and is thus suitable for experiments detecting evolutionary footprints left by stress. Here, we performed expression and evolutionary analysis of two duplicated DREB A1-subgroup (DREB1) genes,  PnDREB68  and  PnDREB69 , encoding transcription factors that are involved in stress responses. The two genes showed partially overlapping but distinct expression patterns in response to stresses. These genes were strongly and rapidly induced by cold stress in leaves, stems, and roots. In leaf tissue, dehydration stress induced the expression of  PnDREB68  but not  PnDREB69 .  PnDREB69  displayed more rapid responses and longer expression durations than  PnDREB68  under salt and ABA stress, respectively. Based on single nucleotide polymorphism (SNP) analysis, we found significant population genetic differentiation, with a greater  F   ST  value (0.09189) for  PnDREB69  than for  PnDREB68  (0.07743). Nucleotide diversity analysis revealed a two-fold higher π T  for  PnDREB68  than for  PnDREB69  (0.00563 vs. 0.00243), reflecting strong purifying selection acting on the former. The results suggest that positive selection acted on  PnDREB69 , as evidenced by neutral testing using Tajima’s  D  statistic. The distinct selective forces to which each of the genes was subjected may be associated with expression divergence. Linkage disequilibrium (LD) was low for the sequenced region, with a higher level for  PnDREB68  than for  PnDREB69 . Additionally, analysis of the relationship among carbon isotope ratios, SNP classes and gene expression, together with motif and domain analysis, suggested that 14 polymorphisms within the two genes may be candidates for an association study of important traits such as water use efficiency/drought tolerance in black poplar.

Expression and Molecular Evolution of Two DREB1 Genes in Black Poplar (Populus nigra)