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:Technological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. High-density maps enriched for transcribed genes facilitate the discovery of connections between genes and phenotypes. We report the construction of a high-density linkage map of expressed genes for the heterozygous genome of Eucalyptus using Single Feature Polymorphism (SFP) markers. SFP discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. SFP genotyping was carried out by hybridizing complementary RNA prepared from 4.5 year-old trees xylem to an SFP array containing 103,000 25-mer oligonucleotide probes representing 20,726 unigenes derived from a modest size expressed sequence tags collection. An SFP-mapping microarray with 43,777 selected candidate SFP probes representing 15,698 genes was subsequently designed and used to genotype SFPs in a larger subset of the segregating population drawn by selective mapping. A total of 1,845 genes were mapped, with 884 of them ordered with high likelihood support on a framework map anchored to 180 microsatellites with average density of 1.2 cM. Using more probes per unigene increased by two-fold the likelihood of detecting segregating SFPs eventually resulting in more genes mapped. In silico validation showed that 87% of the SFPs map to the expected location on the 4.5X draft sequence of the Eucalyptus grandis genome. The Eucalyptus 1,845 gene map is the most highly enriched map for transcriptional information for any forest tree species to date. It represents a major improvement on the number of genes previously positioned on Eucalyptus maps and provides an initial glimpse at the gene space for this global tree genome. A general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by SFP genotyping with a concurrent objective of reducing microarray costs. HIgh-density gene-rich maps represent a powerful resource to assist gene discovery endeavors when used in combination with QTL and association mapping and should be especially valuable to assist the assembly of reference genome sequences soon to come for several plant and animal species.

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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Eucalyptus nitens

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