Targeted re-sequencing and genome-wide association analysis for wood property traits in breeding population of Eucalyptus tereticornis × E. grandis.

Globally, Eucalyptus plantations occupy 22 million ha area and is one of the preferred hardwood species due to their short rotation, rapid growth, adaptability and wood properties. In this study, we present results of GWAS in parents and 100 hybrids of Eucalyptus tereticornis × E. grandis using 762 genes presumably involved in wood formation. Comparative analysis between parents predicted 32,202 polymorphic SNPs with high average read depth of 269-562× per individual per nucleotide. Seventeen wood related traits were phenotyped across three diverse environments and GWAS was conducted using 13,610 SNPs. A total of 45 SNP-trait associations were predicted across two locations. Seven large effect markers were identified which explained more than 80% of phenotypic variation for fibre area. This study has provided an array of candidate genes which may govern fibre morphology in this genus and has predicted potential SNPs which can guide future breeding programs in tropical Eucalyptus.

Bacterial-induced pH shifts link individual cell physiology to macroscale collective behavior.

Bacteria have evolved a diverse array of signaling pathways that enable them to quickly respond to environmental changes. Understanding how these pathways reflect environmental conditions and produce an orchestrated response is an ongoing challenge. Herein, we present a role for collective modifications of environmental pH carried out by microbial colonies living on a surface. We show that by collectively adjusting the local pH value, Paenibacillus spp., specifically, regulate their swarming motility. Moreover, we show that such pH-dependent regulation can converge with the carbon repression pathway to down-regulate flagellin expression and inhibit swarming in the presence of glucose. Interestingly, our results demonstrate that the observed glucose-dependent swarming repression is not mediated by the glucose molecule per se, as commonly thought to occur in carbon repression pathways, but rather is governed by a decrease in pH due to glucose metabolism. In fact, modification of the environmental pH by neighboring bacterial species could override this glucose-dependent repression and induce swarming of Paenibacillus spp. away from a glucose-rich area. Our results suggest that bacteria can use local pH modulations to reflect nutrient availability and link individual bacterial physiology to macroscale collective behavior.

Co-expression network of transcription factors reveal ethylene-responsive element-binding factor as key regulator of wood phenotype in Eucalyptus tereticornis.

Suitability of wood biomass for pulp production is dependent on the cellular architecture and composition of secondary cell wall. Presently, systems genetics approach is being employed to understand the molecular basis of trait variation and co-expression network analysis has enabled holistic understanding of complex trait such as secondary development. Transcription factors (TFs) are reported as key regulators of meristematic growth and wood formation. The hierarchical TF network is a multi-layered system which interacts with downstream structural genes involved in biosynthesis of cellulose, hemicelluloses and lignin. Several TFs have been associated with wood formation in tree species such as Populus, Eucalyptus, Picea and Pinus. However, TF-specific co-expression networks to understand the interaction between these regulators are not reported. In the present study, co-expression network was developed for TFs expressed during wood formation in Eucalyptus tereticornis and ethylene-responsive element-binding factor, EtERF2, was identified as the major hub transcript which co-expressed with other secondary cell wall biogenesis-specific TFs such as EtSND2, EtVND1, EtVND4, EtVND6, EtMYB70, EtGRAS and EtSCL8. This study reveals a probable role of ethylene in determining natural variation in wood properties in Eucalyptus species. Understanding this transcriptional regulation underpinning the complex bio-processing trait of wood biomass will complement the Eucalyptus breeding program through selection of industrially suitable phenotypes by marker-assisted selection.

Identification of PEG-induced water stress responsive transcripts using co-expression network in Eucalyptus grandis.

Ecophysiological studies in Eucalyptus have shown that water is the principal factor limiting stem growth. Effect of water deficit conditions on physiological and biochemical parameters has been extensively reported in Eucalyptus. The present study was conducted to identify major polyethylene glycol induced water stress responsive transcripts in Eucalyptus grandis using gene co-expression network. A customized array representing 3359 water stress responsive genes was designed to document their expression in leaves of E. grandis cuttings subjected to -0.225MPa of PEG treatment. The differentially expressed transcripts were documented and significantly co-expressed transcripts were used for construction of network. The co-expression network was constructed with 915 nodes and 3454 edges with degree ranging from 2 to 45. Ninety four GO categories and 117 functional pathways were identified in the network. MCODE analysis generated 27 modules and module 6 with 479 nodes and 1005 edges was identified as the biologically relevant network. The major water responsive transcripts represented in the module included dehydrin, osmotin, LEA protein, expansin, arabinogalactans, heat shock proteins, major facilitator proteins, ARM repeat proteins, raffinose synthase, tonoplast intrinsic protein and transcription factors like DREB2A, ARF9, AGL24, UNE12, WLIM1 and MYB66, MYB70, MYB 55, MYB 16 and MYB 103. The coordinated analysis of gene expression patterns and coexpression networks developed in this study identified an array of transcripts that may regulate PEG induced water stress responses in E. grandis.

Construction of co-expression network based on natural expression variation of xylogenesis-related transcripts in Eucalyptus tereticornis.

Natural genetic variation is randomly distributed and gene expression patterns vary widely in natural populations. These variations are an effect of multifactorial genetic perturbations resulting in different phenotypes. Genome-wide analysis can be used to comprehend the genetic basis governing this naturally occurring developmental variation. Secondary growth is a highly complex trait and systems genetics models are presently being applied to understand the molecular architecture of wood formation. In the present study, the natural variation in expression patterns of 18,987 transcripts expressed in the developing xylem tissues were documented across four phenotypes of Eucalyptus tereticornis with distinct holocellulose/klason lignin content. The differentially expressed genes across all the phenotypes were used to construct co-expression networks and sub-network 2 with 380 nodes and 17,711 edges was determined as the network of relevance, including 30 major cell wall biogenesis related transcripts with 2394 interactions and 10 families of transcription factors with 3360 interactions. EYE [EMBRYO YELLOW] was identified as major hub transcript with 173 degrees which interacted with known cell wall biogenesis genes. K-mean clustering was also performed for differentially expressed transcripts and two clusters discriminated the phenotypes based on their holocellulose/klason lignin content. The cluster based networks were enriched with GOs related to cell wall biogenesis and sugar metabolism. The networks developed in the present study enabled identification of critical regulators and novel transcripts whose expression variation could presumably govern the phenotypic variation in wood properties across E. tereticornis.

Development of genetic markers in Eucalyptus species by target enrichment and exome sequencing.

The advent of next-generation sequencing has facilitated large-scale discovery, validation and assessment of genetic markers for high density genotyping. The present study was undertaken to identify markers in genes supposedly related to wood property traits in three Eucalyptus species. Ninety four genes involved in xylogenesis were selected for hybridization probe based nuclear genomic DNA target enrichment and exome sequencing. Genomic DNA was isolated from the leaf tissues and used for on-array probe hybridization followed by Illumina sequencing. The raw sequence reads were trimmed and high-quality reads were mapped to the E. grandis reference sequence and the presence of single nucleotide variants (SNVs) and insertions/ deletions (InDels) were identified across the three species. The average read coverage was 216X and a total of 2294 SNVs and 479 InDels were discovered in E. camaldulensis, 2383 SNVs and 518 InDels in E. tereticornis, and 1228 SNVs and 409 InDels in E. grandis. Additionally, SNV calling and InDel detection were conducted in pair-wise comparisons of E. tereticornis vs. E. grandis, E. camaldulensis vs. E. tereticornis and E. camaldulensis vs. E. grandis. This study presents an efficient and high throughput method on development of genetic markers for family- based QTL and association analysis in Eucalyptus.