Project description: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. 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.

Project description:Purpose: Kraft pulp yield (KPY) is a key determinant of plantation profitability and increasing the KPY of trees grown in plantations is a major breeding objective. To speed up the breeding process, molecular markers that can predict KPY are desirable. To achieve this goal, we carried out RNA-Seq studies on trees at extremes of KPY in two different trials to identify genes and alleles whose expression correlated with KPY. Methods: We analyzed samples from the extremes of the distribution of KPY in two Eucalyptus nitens trials which also differed in growth to identify genes having differential expression between high and low KPY samples. We used reference-guided transcriptome mapping to study gene expression. Results: Several genes showed differential expression between low and high KPY samples. Gene ontology (GO) enrichment tests revealed up-regulation of stress-related gene categories and down-regulation of gene categories related to wood formation and growth in low KPY samples. More than 110,000 single nucleotide polymorphisms (SNPs) were detected in both the trials and 2103 of these showed differential allelic expression. Allelic expression of 30% of these variants was correlated with total gene expression. To identify the genes showing patterns of positive selection among the genes expressed in the cambial tissue we compared Ka/Ks ratios. The Ka/Ks ratios compare the rate of nonsynonymous substitutions (Ka) to synonymous substitutions (Ks) which can help identifying genes under selection. By comparing the two trials we observed in total 196 genes which had Ka/Ks ratios of more than 1.5 in both the trials strongly suggesting that these genes are under positive selection. A total of six GO categories were enriched in both trials for genes showing signatures of positive selection . All six categories include genes involved in apoptosis, cell death and defense responses. Conclusions: By conducting RNA-Seq analysis in two trials we identified a number of candidate genes and alleles whose expression is correlated with KPY and growth traits in E. nitens. Most of the down-regulated genes in low KPY samples are cell wall-related genes, suggesting that the identified candidate genes are biologically relevant. A number of potential functional polymorphisms were also identified that showed DAE. We detected positive selection signatures in numerous genes that are consistent with the results from RNA-Seq study in E. camaldulensis. The genes and alleles identified in this study form a valuable resource for association and genomic selection studies. Xylem mRNA profiles of Eucalyptus nitens from low and high KPY samples were generated by deep sequencing, in two trials, using Illumina HiSeq.

Project description:Purpose: Kraft pulp yield (KPY) is a key determinant of plantation profitability and increasing the KPY of trees grown in plantations is a major breeding objective. To speed up the breeding process, molecular markers that can predict KPY are desirable. To achieve this goal, we carried out RNA-Seq studies on trees at extremes of KPY in two different trials to identify genes and alleles whose expression correlated with KPY. Methods: We analyzed samples from the extremes of the distribution of KPY in two Eucalyptus nitens trials which also differed in growth to identify genes having differential expression between high and low KPY samples. We used reference-guided transcriptome mapping to study gene expression. Results: Several genes showed differential expression between low and high KPY samples. Gene ontology (GO) enrichment tests revealed up-regulation of stress-related gene categories and down-regulation of gene categories related to wood formation and growth in low KPY samples. More than 110,000 single nucleotide polymorphisms (SNPs) were detected in both the trials and 2103 of these showed differential allelic expression. Allelic expression of 30% of these variants was correlated with total gene expression. To identify the genes showing patterns of positive selection among the genes expressed in the cambial tissue we compared Ka/Ks ratios. The Ka/Ks ratios compare the rate of nonsynonymous substitutions (Ka) to synonymous substitutions (Ks) which can help identifying genes under selection. By comparing the two trials we observed in total 196 genes which had Ka/Ks ratios of more than 1.5 in both the trials strongly suggesting that these genes are under positive selection. A total of six GO categories were enriched in both trials for genes showing signatures of positive selection . All six categories include genes involved in apoptosis, cell death and defense responses. Conclusions: By conducting RNA-Seq analysis in two trials we identified a number of candidate genes and alleles whose expression is correlated with KPY and growth traits in E. nitens. Most of the down-regulated genes in low KPY samples are cell wall-related genes, suggesting that the identified candidate genes are biologically relevant. A number of potential functional polymorphisms were also identified that showed DAE. We detected positive selection signatures in numerous genes that are consistent with the results from RNA-Seq study in E. camaldulensis. The genes and alleles identified in this study form a valuable resource for association and genomic selection studies.

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

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