Using E. grandis gene annotations we classified the SNPs as three prime, synonymous, nonsynonymous, five prime and intronic SNPs. Synonymous and nonsy nonymous SNPs were annotated using PoPoolation package. While most of the SNPs were from coding regions, there were however several SNPs from intron regions suggesting that some of these SNPs may be from unspliced pre mRNA. The intronic SNPs may also represent incomplete annotations of E. grandis. Ten of the intronic SNPs were within the splice sites. GO analysis of genes showing differential allelic expression We used GO enrichment analysis to identify the func tional categories enriched among the genes that showed significant differential allelic expression.
GO enrichment tests were performed separately for genes Inhibitors,Modulators,Libraries that showed sig nificant differential allelic expression as well as total gene expression Inhibitors,Modulators,Libraries between control and stress treat ments and genes that showed only significant differential allelic expression but similar total gene expression be tween control and stress treatments. Genes that showed both allelic and total gene expression were enriched in stress and metabolic process gene categories as identified previously. Interestingly, sev eral stress related gene categories were also enriched among the genes that showed differential allelic expression but no change in total gene expression. Identification of genes under selection To study the evolutionary selection patterns among the genes we analysed the nonsynonymous to synonymous substitution ratios. To estimate the Ka Ks ratios we combined the reads from all the populations before and after the stress treatment.
We identified 194855 SNPs from coding regions of 13,719 genes using PoPoo lationpackage. These SNPs were annotated Drug_discovery as non synonymous or synonymous using the PoPoolation package. Annotations of these variants were further con firmed Inhibitors,Modulators,Libraries by visually inspecting the tracks in integrative genomics viewer IGV. The proportion of nonsynon ymous to synonymous mutation Inhibitors,Modulators,Libraries rates among the genes has ranged from 0. 05 to 5. 9 with a mean of 0. 39 among 13,719 genes. Genes with Ka Ks ratios below 0. 5 were treated as under purifying selection while gene with Ka Ks ratios above 1. 5 were treated as under positive selection. Most of the genes were under negative se lection with the Ka Ks ratios below 0. 50. In contrast the number of genes under positive selection or under diver sifying selection was small. Only 2% of the genes were under positive selection with Ka Ks ratios above 1. 5. To identify the gene categories enriched among the genes we conducted GO enrichment tests separately for negatively and positively selected genes.