Transcriptome sequencing of Eucalyptus camaldulensis seedlings subjected to water stress reveals functional single nucleotide polymorphisms and genes under selection.. Eucalyptus camaldulensis

Purpose: Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq) to identify the candidate genes and alleles and to explore the evolutionary signatures of selection Methods: We studied the effect of water stress on gene expression in Eucalyptus camaldulensis seedlings derived from three natural populations. We used reference-guided transcriptome mapping to study gene expression. Reads mapping to predicted transcripts were analysed with edgeR pacakge to study differntial expression of genes under control and water-stress conditions. SNPs within the genes were obtained using pileup file generated with samtools and the SNP frequencies were analysed with VarScan package. SNPs were further characterised as sysnonymous and non-synonymous using popoolation2 pacakge. Results: Several genes showed differential expression between control and stress conditions. Gene ontology (GO) enrichment tests revealed up-regulation of 140 stress-related gene categories and down-regulation of 35 metabolic and cell wall organisation gene categories. More than 190,000 single nucleotide polymorphisms (SNPs) were detected and 2737 of these showed differential allelic expression. Allelic expression of 52% of these variants was correlated with total gene expression. Signatures of selection patterns were studied by estimating the proportion of nonsynonymous to synonymous substitution rates (Ka/Ks). The average Ka/Ks ratio among the 13,719 genes was 0.39 indicating that most of the genes are under purifying selection. Among the positively selected genes (Ka/Ks > 1.5) apoptosis and cell death categories were enriched. Of the positively selected genes, ninety genes showed differential expression and 27 SNPs from 17 positively selected genes showed differential allelic expression between treatments. Conclusions: Correlation of allelic expression of several SNPs with total gene expression indicates that these variants may be the cis-acting variants or in linkage disequilibrium with such variants. Enrichment of apoptosis and cell death gene categories among the positively selected genes reveals the past selection pressures experienced by the populations used in this study. Overall design: Leaf mRNA of 12 samples were used to study the gene expression under control and water-stress conditions. Three samples under water-stress conditions and three samples under control conditions were used as biological replicates for differenctial gene expression between the treatments. High-throughput sequence reads from mRNA samples were generated by deep sequencing with Illumina GAIIx.

研究背景与目的：水分胁迫在全球诸多区域限制植物存活与生产。鉴定响应水分胁迫的基因与等位基因，对培育更适应干旱的植物品种具有重要意义。目前尚无研究系统解析水分胁迫下全转录组范围的基因及等位基因表达模式。本研究采用RNA测序(RNA-seq)技术筛选候选基因与等位基因，并探索选择的进化特征。

研究方法：以源自3个自然种群的赤桉(Eucalyptus camaldulensis)幼苗为材料，探究水分胁迫对基因表达的影响。采用参考引导转录组映射(reference-guided transcriptome mapping)分析基因表达情况。利用edgeR包对比对至预测转录本的测序读段进行分析，以鉴定对照组与水分胁迫组间的差异表达基因。借助samtools生成的pileup文件获取基因内的单核苷酸多态性(SNP)，并通过VarScan包分析SNP频率；进一步利用popoolation2包将SNP划分为同义突变与非同义突变两类。

研究结果：对照组与胁迫组间存在多个差异表达基因。基因本体(GO)富集分析显示，140个与胁迫相关的基因类别呈现上调表达，35个涉及代谢与细胞壁组织的基因类别呈现下调表达。共检测到超过190,000个单核苷酸多态性(SNP)，其中2737个位点表现出差异等位基因表达。52%的变异位点的等位基因表达与总基因表达呈显著相关。通过估算非同义替换率与同义替换率的比值(Ka/Ks)，解析选择模式的进化特征。13,719个基因的平均Ka/Ks比值为0.39，表明多数基因处于纯化选择压力之下。在正向选择基因(Ka/Ks >1.5)中，细胞凋亡与细胞死亡类别显著富集。在正向选择基因中，90个基因表现出差异表达；其中17个正向选择基因携带的27个SNP位点在两组处理间呈现差异等位基因表达。

研究结论：多个SNP位点的等位基因表达与总基因表达的相关性表明，这些变异位点可能为顺式作用变异，或与此类变异存在连锁不平衡。正向选择基因中细胞凋亡与细胞死亡类别的富集，反映了本研究使用的种群曾经历的选择压力。

实验设计：本研究共采用12个样本的叶片mRNA，分析对照组与水分胁迫组的基因表达情况。其中水分胁迫组与对照组各3个样本作为生物学重复，用于鉴定两组间的差异表达基因。所有mRNA样本的高通量测序读段均通过Illumina GAIIx平台完成深度测序。