Project description:Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, ?-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants.

Project description:BACKGROUND:F1 hybrid clones of Eucalyptus grandis and E. urophylla are widely grown for pulp and paper production in tropical and subtropical regions. Volume growth and wood quality are priority objectives in Eucalyptus tree improvement. The molecular basis of quantitative variation and trait expression in eucalypt hybrids, however, remains largely unknown. The recent availability of a draft genome sequence (http://www.phytozome.net) and genome-wide genotyping platforms, combined with high levels of genetic variation and high linkage disequilibrium in hybrid crosses, greatly facilitate the detection of quantitative trait loci (QTLs) as well as underlying candidate genes for growth and wood property traits. In this study, we used Diversity Arrays Technology markers to assess the genetic architecture of volume growth (diameter at breast height, DBH) and wood basic density in four-year-old progeny of an interspecific backcross pedigree of E. grandis and E. urophylla. In addition, we used Illumina RNA-Seq expression profiling in the E. urophylla backcross family to identify cis- and trans-acting polymorphisms (eQTLs) affecting transcript abundance of genes underlying QTLs for wood basic density. RESULTS:A total of five QTLs for DBH and 12 for wood basic density were identified in the two backcross families. Individual QTLs for DBH and wood basic density explained 3.1 to 12.2% of phenotypic variation. Candidate genes underlying QTLs for wood basic density on linkage groups 8 and 9 were found to share trans-acting eQTLs located on linkage groups 4 and 10, which in turn coincided with QTLs for wood basic density suggesting that these QTLs represent segregating components of an underlying transcriptional network. CONCLUSION:This is the first demonstration of the use of next-generation expression profiling to quantify transcript abundance in a segregating tree population and identify candidate genes potentially affecting wood property variation. The QTLs identified in this study provide a resource for identifying candidate genes and developing molecular markers for marker-assisted breeding of volume growth and wood basic density. Our results suggest that integrated analysis of transcript and trait variation in eucalypt hybrids can be used to dissect the molecular basis of quantitative variation in wood property traits.

Project description:BackgroundWood formation affects the chemical and physical properties of wood, and thus affects its utility as a building material or a feedstock for biofuels, pulp and paper. To obtain genome-wide insights on the transcriptome changes and regulatory networks in wood formation, we used high-throughput RNA sequencing to characterize cDNA libraries of mature xylem from tension wood (TW), opposite wood (OW), and normal wood (NW), in the industrial tree species Populus tomentosa.ResultsOur sequencing generated 140,978,316 (TW), 128,972,228 (OW), and 117,672,362 (NW) reads, corresponding to 10,127 (TW), 10,129 (OW), and 10,129 (NW) unique genes. Of these, 361 genes were differentially transcribed between TW and OW (log2FC ≥ 1 or ≤ -1, FDR < 0.05), 2,658 differed between OW and NW, and 2,417 differed between TW and NW. This indicates that NW differs significantly from the wood in branches; GO term analysis also indicated that OW experienced more transcriptome remodeling. The differentially expressed genes included 97 encoding transcription factors (TFs), 40 involved in hormone signal transduction, 33 in lignin biosynthesis, 21 in flavonoid biosynthesis, and 43 in cell wall metabolism, including cellulose synthase, sucrose synthase, and COBRA. More than half of the differentially expressed TF showed more than 4-fold lower transcript levels in NW compared with TW or OW, indicating that TF abundances differed dramatically in different wood types and may have important roles in the formation of reaction wood. In addition, transcripts of most of the genes involved in lignin biosynthesis were more abundant in OW compared with TW, consistent with the higher lignin content of OW. We constructed two transcriptomic networks for the regulation of lignin and cellulose biosynthesis, including TFs, based on the co-expression patterns of different genes. Lastly, we used reverse transcription quantitative PCR to validate the differentially expressed genes identified.ConclusionsHere, we identified the global patterns and differences in gene expression among TW, OW, and NW, and constructed two transcriptomic regulatory networks involved in TW formation in P. tomentosa. We also identified candidate genes for molecular breeding of wood quality, and provided a starting point to decipher the molecular mechanisms of wood formation in Populus.

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.

Project description:BACKGROUND: In a context of climate change, phenotypic plasticity provides long-lived species, such as trees, with the means to adapt to environmental variations occurring within a single generation. In eucalyptus plantations, water availability is a key factor limiting productivity. However, the molecular mechanisms underlying the adaptation of eucalyptus to water shortage remain unclear. In this study, we compared the molecular responses of two commercial eucalyptus hybrids during the dry season. Both hybrids differ in productivity when grown under water deficit. RESULTS: Pyrosequencing of RNA extracted from shoot apices provided extensive transcriptome coverage - a catalog of 129,993 unigenes (49,748 contigs and 80,245 singletons) was generated from 398 million base pairs, or 1.14 million reads. The pyrosequencing data enriched considerably existing Eucalyptus EST collections, adding 36,985 unigenes not previously represented. Digital analysis of read abundance in 14,460 contigs identified 1,280 that were differentially expressed between the two genotypes, 155 contigs showing differential expression between treatments (irrigated vs. non irrigated conditions during the dry season), and 274 contigs with significant genotype-by-treatment interaction. The more productive genotype displayed a larger set of genes responding to water stress. Moreover, stress signal transduction seemed to involve different pathways in the two genotypes, suggesting that water shortage induces distinct cellular stress cascades. Similarly, the response of functional proteins also varied widely between genotypes: the most productive genotype decreased expression of genes related to photosystem, transport and secondary metabolism, whereas genes related to primary metabolism and cell organisation were over-expressed. CONCLUSIONS: For the most productive genotype, the ability to express a broader set of genes in response to water availability appears to be a key characteristic in the maintenance of biomass growth during the dry season. Its strategy may involve a decrease of photosynthetic activity during the dry season associated with resources reallocation through major changes in the expression of primary metabolism associated genes. Further efforts will be needed to assess the adaptive nature of the genes highlighted in this study.

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RNA-seq analysis of lignocellulose-related genes in hybrid Eucalyptus with contrasting wood basic density.

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RNA-seq analysis of lignocellulose-related genes in hybrid Eucalyptus with contrasting wood basic density.

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