Identification of DEGs and Gene Modules Analysis
Because more floral characteristics showed local adaptation, combined with genomic and phenotypic variation, transcriptome analysis of the flower buds of ten populations was performed. A total of 1,323,834,776 raw reads (456 Gb) were obtained from 30 samples from ten populations. The ten populations were also divided into four groups (NE, EL, CN and NW) for differential expression analysis. The greatest total numbers of DEGs were found between NE vs. EL, EL vs. NW and CN vs. NW (928, 1716 and 855 DEGs, respectively). Fewer DEGs were found for EL vs. CN, NE vs. CN and NE vs. NW (428, 666 and 513 DEGs, respectively). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted for the DEGs identified between different groups. The DEGs identified from EL vs. CN and NE vs. CN were not significantly enriched in any KEGG pathway. The DEGs identified from other groups were related to glutathione metabolism (ko00480), cytochrome P450 (ko00199), cyanoamino acid metabolism (ko00460), metabolism of xenobiotics by cytochrome P450 (ko00980), drug metabolism – cytochrome P450 (ko00983), carotenoid biosynthesis (ko00906), starch and sucrose metabolism (ko00500), amino sugar and nucleotide sugar metabolism (ko00520), photosynthesis – antenna proteins (ko00196), galactose metabolism (ko00052), CD molecules (ko04090), phenylpropanoid biosynthesis (ko00940), pentose and glucuronate interconversions (ko00040), and fructose and mannose metabolism (ko00051) (Figure S6 A - D).
Because the greatest number of DEGs and Fst was found between EL and NW, we used 23,418 genes expressed in both EL and NW for WGCNA to identify gene modules related to adaptive floral characteristics (including the corolla diameter, petal length, angle, spur length, pistil length and inflorescence number). The analysis identified 33 gene modules, the number of genes in each module ranged from 58 (darkolivegreen) to 3229 (turquoise), and 24 genes were not classified into any modules. Among the six adaptive floral characteristics, only spur length presented a significant correlation with four modules — namely, magenta (640 genes), darkgray (257 genes), saddlebrown (134 genes) and orange (201 genes). No gene modules were associated with other traits (Figure 5). KEGG enrichment analysis showed that the genes in the magenta module were related to DNA replication proteins (ko03032), cytoskeletal proteins (ko04812), the cell cycle (ko04110), protein kinases (ko01001), DNA replication (ko03030), homologous recombination (ko03440), and gap junctions (ko04540). The genes in the darkgray module were related to ribosomes (ko03008) and spliceosomes (ko03040) (Figure S6 E, F). The genes in the saddle brown and orange modules were not significantly enriched in any KEGG pathway.