Assembly, annotation and polymorphism analysis of a draft transcriptome sequence for a fast-growing Eucalyptus plantation tree

Ultra-high throughput DNA sequencing technologies have rapidly changed the face of genomic research projects. Technologies such as mRNA-Seq have the potential to rapidly profile the expressed gene-catalog of non-model organisms, albeit with significant bioinformatics related costs and support required. This study developed automated data analysis workflows focused on the quality evaluation of mRNA-Seq reads, de novo transcriptome assembly, transcriptome annotation and digital gene expression profiling making use of data analysis tools available in the public domain and novel tools developed for this purpose. The developed workflows were made available in a private instance of the Galaxy workflow management system. The developed workflows were used to perform the de novo assembly of a gene-catalog of a Eucalyptus plantation tree. The fast growing and good wood properties of Eucalyptus tree species and their hybrids make them excellent renewable resources of fiber for pulp and paper, and woody biomass for bioenergy production. We produced an expressed gene-catalog of 18 894 de novo assembled contigs from Illumina deep mRNA-Seq of six sampled plant tissues. Using a novel coverage-assisted re-assembly approach, we were able to assemble near full-length biologically relevant transcripts. The assembly was evaluated in terms of contig quality and contiguity, and functional annotations were assigned. Digital expression profiling (FPKM values) of each contig across the tissues were calculated, which was used to identify of tissue-specific sets of expressed genes. Polymorphism analysis of 13 806 high-confidence contigs revealed a combined exon and untranslated region SNP density of 0.534 SNPs/100 bp, which provides a good opportunity for designing high-density SNP assays in the expressed regions of the Eucalyptus genome. The assembled and annotated gene catalog was made available for public use in a user-friendly, web-based interface as the Eucspresso database (http://eucspresso.bi.up.ac.za). The developed database acts as a prelude to a more comprehensive mRNA-Seq whole-transcriptome repository, the Eucalyptus Genome Intergrative Explorer (EucGenIE), a resource that will focus on identifying transcriptional networks active during woody biomass development. Results from the study proved that current bioinformatics software tools and approaches can be used to successfully assemble and characterize a large proportion of the transcriptome of a complex eukaryotic organism. This approach can be used to characterise the gene catalog of a wide range of non-model organisms using only data derived from uHTS experiments.Thesis (PhD)--University of Pretoria, 2011.Biochemistryunrestricte

Comparative interrogation of the developing xylem transcriptomes of two wood-forming species : Populus trichocarpa and Eucalyptus grandis

Wood formation is a complex developmental process governed by genetic and environmental stimuli. Populus and Eucalyptus are fast-growing, high-yielding tree genera that represent ecologically and economically important species suitable for generating significant lignocellulosic biomass. Comparative analysis of the developing xylem and leaf transcriptomes of Populus trichocarpa and Eucalyptus grandis together with phylogenetic analyses identified clusters of homologous genes preferentially expressed during xylem formation in both species. A conserved set of 336 single gene pairs showed highly similar xylem preferential expression patterns, as well as evidence of high functional constraint. Individual members of multigene orthologous clusters known to be involved in secondary cell wall biosynthesis also showed conserved xylem expression profiles. However, species-specific expression as well as opposite (xylem versus leaf) expression patterns observed for a subset of genes suggest subtle differences in the transcriptional regulation important for xylem development in each species. Using sequence similarity and gene expression status, we identified functional homologs likely to be involved in xylem developmental and biosynthetic processes in Populus and Eucalyptus. Our study suggests that, while genes involved in secondary cell wall biosynthesis show high levels of gene expression conservation, differential regulation of some xylem development genes may give rise to unique xylem properties.Genome Canada Large-Scale Applied Research Project (Project 168BIO) South African Department of Science and Technology (DST), Mondi and Sappi through the Forest Molecular Genetics (FMG) Programme, the Technology and Human Resources for Industry Programme (THRIP,UID 80118), and the National Research Foundation (NRF, UID 71255 and 86936) of South Africa.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1469-81372016-06-30hb201

Structural, evolutionary and functional analysis of the NAC domain protein family in Eucalyptus

NAC domain transcription factors regulate many developmental processes and stress responses in plants and vary widely in number and family structure. We analysed the characteristics and evolution of the NAC gene family of Eucalyptus grandis, a fastgrowing forest tree in the rosid order Myrtales. NAC domain genes identified in the E. grandis genome were subjected to amino acid sequence, phylogenetic and motif analyses. Transcript abundance in developing tissues and abiotic stress conditions in E. grandis and E. globulus was quantified using RNA-seq and RT-qPCR.189 E. grandis NAC (EgrNAC) proteins, arranged into 22 subfamilies, are extensively duplicated in subfamilies associated with stress response. Most EgrNAC genes form tandem duplicate arrays that frequently carry signatures of purifying selection. Sixteen amino acid motifs were identified in EgrNAC proteins, eight of which are enriched in, or unique to, Eucalyptus. New candidates for the regulation of normal and tension wood development and cold responses were identified.This first description of a Myrtales NAC domain family reveals a unique history of tandem duplication in stress-related subfamilies that has likely contributed to the adaptation of eucalypts to the challenging Australian environment. Several new candidates for the regulation of stress, wood formation and tree-specific development are reported.ANR (Project “Tree For Joules” ANR-2010-KBBE-007-01; Labex Tulip ANR-10-LABX-41), the CNRS, and the University Toulouse III (UPS). Bioinformatics and Functional Genomics Programme of the National Research Foundation, South Africa (UID 71255).http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1469-81372016-06-30hb201

Genetic dissection of growth, wood basic density and gene expression in interspecific backcrosses of Eucalyptus grandis and E. urophylla

Sappi through the Forest Molecular Genetics Program and by the Technology and Human Resources for Industry Program (THRIP), the National Research Foundation (NRF) and the Department of Science and Technology (DST) of South Africa.http://www.biomedcentral.com/1471-2156/13/6

Investigating the molecular underpinnings underlying morphology and changes in carbon partitioning during tension wood formation in Eucalyptus

Tension wood has distinct physical and chemical properties, including altered fibre properties, cell wall composition and ultrastructure. It serves as a good system for investigating the genetic regulation of secondary cell wall biosynthesis and wood formation. The reference genome sequence for Eucalyptus grandis allows investigation of the global transcriptional reprogramming that accompanies tension wood formation in this global wood fibre crop. We report the first comprehensive analysis of physicochemical wood property changes in tension wood of Eucalyptus measured in a hybrid (E. grandis 9 Eucalyptus urophylla) clone, as well as genome-wide gene expression changes in xylem tissues 3wk post-induction using RNA sequencing. We found that Eucalyptus tension wood in field-grown trees is characterized by an increase in cellulose, a reduction in lignin, xylose and mannose, and a marked increase in galactose. Gene expression profiling in tension wood-forming tissue showed corresponding down-regulation of monolignol biosynthetic genes, and differential expression of several carbohydrate active enzymes. We conclude that alterations of cell wall traits induced by tension wood formation in Eucalyptus are a consequence of a combination of down-regulation of lignin biosynthesis and hemicellulose remodelling, rather than the often proposed up-regulation of the cellulose biosynthetic pathway.South African Department of Science and Technology (DST), Sappi and Mondi, through the Forest Molecular Genetics Programme, the Technology and Human Resources for Industry Programme (THRIP, UID 80118) and the Bioinformatics and Functional Genomics Programme of the National Research Foundation (NRF, UID 18312) of South Africa.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1469-81372016-06-30hb201

The complete genome sequence of the nitrile biocatalyst Rhodococcus rhodochrous ATCC BAA-870

BACKGROUND : Rhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst. RESULTS : The genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase. Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci. CONCLUSIONS : The sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.Additional file 1: SuppInfo Frederick et al. BAA-870 genome. Table S1. All sequenced Rhodococcus strains (353) according to the NCBI database (accessed 13/03/2019). Table S2. All complete sequenced Rhodococcus species ranked by release date according to the NCBI Genome database (accessed 11/03/2019). Table S3. Whole genome distance statistics between Rhodococcus rhodochrous ATCC BAA-870 and two closely matched strains. Table S4. Rhodococcus rhodochrous ATCC BAA-870 protein function breakdown based on BASys annotation COG classifications.The Department of Science and Technology (DST) Biocatalysis Initiative (Grant 0175/2013), South Africa, the CSIR Thematic Programme, the Welch Foundation (grant E-1264) and the European Science Foundation COST Action CM0701.https://bmcgenomics.biomedcentral.comam2020BiochemistryGeneticsMicrobiology and Plant Patholog

Genetic diversity and population structure of locally adapted South African chicken lines : implications for conservation

In this study microsatellite markers were applied to investigate the genetic diversity and population structure of the six local chicken lines kept in the "Fowls for Africa" program, for better clarification of parameters for breed differentiation and genetic conservation of this valuable resource. The lines included the Black Australorp, Potchefstroom Koekoek, New Hampshire, Ovambo, Lebova- Venda and a Naked Neck line. Unbiased estimates for heterozygosity ranged from 50% in the Potchefstroom Koekoek to as high as 65% in the Naked Neck chickens. FIS values varied from as low as 0.16 for the Black Australorp line to as high as 0.35 for the Ovambo chickens. The FST values indicated moderate to high genetic differentiation between the Naked Neck and New Hampshire (0.11); Ovambo and Naked Neck lines (0.12), and Naked Neck and Lebowa- Venda (0.14). A total of 13% of the total genetic variation observed was between the chicken lines and 87% within the lines, supporting moderate genetic differentiation. Population structure was assessed using STRUCTURE where the Black Australorp was genetically best defined. Although six clusters for the different populations could be distinguished, the other lines were not as clearly defined, with individual birds tending to share more than one cluster. Results support a broad classification of these lines and further investigation of unique alleles is recommended for conservation of the lines within the program

Genetic diversity and population structure of locally adapted South African chicken lines : implications for conservation

In this study microsatellite markers were applied to investigate the genetic diversity and population structure of the six local chicken lines kept in the "Fowls for Africa" program, for better clarification of parameters for breed differentiation and genetic conservation of this valuable resource. The lines included the Black Australorp, Potchefstroom Koekoek, New Hampshire, Ovambo, Lebova- Venda and a Naked Neck line. Unbiased estimates for heterozygosity ranged from 50% in the Potchefstroom Koekoek to as high as 65% in the Naked Neck chickens. FIS values varied from as low as 0.16 for the Black Australorp line to as high as 0.35 for the Ovambo chickens. The FST values indicated moderate to high genetic differentiation between the Naked Neck and New Hampshire (0.11); Ovambo and Naked Neck lines (0.12), and Naked Neck and Lebowa- Venda (0.14). A total of 13% of the total genetic variation observed was between the chicken lines and 87% within the lines, supporting moderate genetic differentiation. Population structure was assessed using STRUCTURE where the Black Australorp was genetically best defined. Although six clusters for the different populations could be distinguished, the other lines were not as clearly defined, with individual birds tending to share more than one cluster. Results support a broad classification of these lines and further investigation of unique alleles is recommended for conservation of the lines within the program