Martin Ranik

The genome of Eucalyptus grandis

Eucalypts are the world’s most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

De novo assembled expressed gene catalog of a fast-growing Eucalyptus tree produced by Illumina mRNA-Seq

BackgroundDe novo assembly of transcript sequences produced by short-read DNA sequencing technologies offers a rapid approach to obtain expressed gene catalogs for non-model organisms. A draft genome sequence will be produced in 2010 for a Eucalyptus tree species (E. grandis) representing the most important hardwood fibre crop in the world. Genome annotation of this valuable woody plant and genetic dissection of its superior growth and productivity will be greatly facilitated by the availability of a comprehensive collection of expressed gene sequences from multiple tissues and organs.ResultsWe present an extensive expressed gene catalog for a commercially grown E. grandis × E. urophylla hybrid clone constructed using only Illumina mRNA-Seq technology and de novo assembly. A total of 18,894 transcript-derived contigs, a large proportion of which represent full-length protein coding genes were assembled and annotated. Analysis of assembly quality, length and diversity show that this dataset represent the most comprehensive expressed gene catalog for any Eucalyptus tree. mRNA-Seq analysis furthermore allowed digital expression profiling of all of the assembled transcripts across diverse xylogenic and non-xylogenic tissues, which is invaluable for ascribing putative gene functions.ConclusionsDe novo assembly of Illumina mRNA-Seq reads is an efficient approach for transcriptome sequencing and profiling in Eucalyptus and other non-model organisms. The transcriptome resource (Eucspresso, http://eucspresso.bi.up.ac.za/) generated by this study will be of value for genomic analysis of woody biomass production in Eucalyptus and for comparative genomic analysis of growth and development in woody and herbaceous plants.

Comparative analysis of orthologous cellulose synthase promoters from Arabidopsis, Populus and Eucalyptus : evidence of conserved regulatory elements in angiosperms

* The cellulose synthase (CesA) gene family encodes the catalytic subunits of a large protein complex responsible for the deposition of cellulose into plant cell walls. Early in vascular plant evolution, the gene family diverged into distinct members with conserved structures and functions (e.g. primary or secondary cell wall biosynthesis). Although the functions and expression domains of CesA genes have been extensively studied in plants, little is known about transcriptional regulation and promoter evolution in this gene family. * Here, comparative sequence analysis of orthologous CesA promoters from three angiosperm genera, Arabidopsis, Populus and Eucalyptus, was performed to identify putative cis-regulatory sequences. The promoter sequences of groups of Arabidopsis genes that are co-expressed with the primary or secondary cell wall-related CesA genes were also analyzed. * Reporter gene analysis of newly isolated promoter regions of six E. grandis CesA genes in Arabidopsis revealed the conserved functionality of the promoter sequences. Comparative sequence analysis identified 71 conserved sequence motifs, of which 66 were significantly over-represented in either primary or secondary wall-associated promoters. * The presence of conserved cis-regulatory elements in the evolutionary distant CesA promoters of Arabidopsis, Populus and Eucalyptus suggests an ancient transcriptional network regulating cellulose biosynthesis in vascular plants.

Diversity and cis-element architecture of the promoter regions of cellulose synthase genes in Eucalyptus.

Lignocellulosic biomass from fast-growing plantation trees is composed of carbohydrate-rich materials deposited into plant cell walls in a coordinated manner during wood formation. The diversity and evolution of the transcriptional networks regulating this process have not been studied extensively. We investigated patterns of species-level nucleotide diversity in the promoters of cellulose synthase (CesA) genes from different Eucalyptus tree species and assessed the possible roles of DNA sequence polymorphism in the gain or loss of cis-elements harboured within the promoters. Promoter regions of three primary and three secondary cell wall-associated CesA genes were isolated from 13 Eucalyptus species and were analysed for nucleotide and cis-element diversity. Species-level nucleotide diversity (π) ranged from 0.014 to 0.068, and different CesA promoters exhibited distinct patterns of sequence conservation. A set of 22 putative cis-elements were mapped to the CesA promoters using in silico methods. Forty-two percent of the mapped cis-element occurrences contained singleton polymorphisms which resulted in either gain or loss of a cis-element in a particular Eucalyptus species. The promoters of Eucalyptus CesA genes contained regions that are highly conserved at the species (Eucalyptus) and genus (with Arabidopsis and Populus) level, suggesting the presence of regulatory modules imposing functional constraint on such regions. Nucleotide polymorphisms in the CesA promoters more frequently created new cis-element occurrences than disrupted existing cis-element occurrences, a process which may be important for the maintenance and evolution of cellulose gene regulation in plants.

Forest and fibre genomics: biotechnology tools for applied tree improvement

Eucalyptus tree breeders and geneticists stand to benefit tremendously from a recently announced effort to produce the first complete genome sequence for a eucalypt tree by 2010. A milestone for eucalypt research, the project will facilitate the development of new biotechnology tools that will accelerate the domestication, improvement and conservation of eucalypt tree species. We have embarked on a research venture in South Africa to study the molecular genetics of wood formation in fast-growing Eucalyptus trees and to develop biotechnology tools that can be integrated into applied tree-breeding programmes. The Wood and Fibre Molecular Genetics (WFMG) Programme, initiated in 2003, is a joint research programme of the University of Pretoria and the two largest pulp and paper companies in South Africa, Sappi and Mondi South Africa. Early objectives of the programme included the identification and isolation of candidate genes for cellulose production in Eucalyptus trees and the assessment of allelic diversity in candidate wood-formation genes. The application of DNA fingerprinting in eucalypt breeding programmes represented an early technology delivery to industry with practical, short-term benefits, which have served to offset the long-term nature of the research programme. Current research projects in the WFMG programme focus on the genetic control of carbon allocation, the transcriptional regulation of cellulose biosynthesis and the role of miRNAs and other small RNAs in wood formation in Eucalyptus. This paper provides a brief overview of research progress in the WFMG programme and discusses realistic time frames for the delivery of genome-based tree improvement tools.

Development of Eucalyptus tissue culture conditions for improved in vitro plant health and transformability

Background Despite its importance as a widely-planted crop tree, eucalypt species and hybrids are relatively difficult to micropropagate, culture and genetically transform in vitro. Compared to other plant species, few non-commercial laboratories are proficient at Eucalyptus tissue culture and transformation. We have undertaken to establish and transform several eucalypt clones in the laboratory. Our main aims include the identification of clones amenable to culturing and transformation, and the development of robust and transferable micropropagation, organogenesis and transformation protocols to enable routine production of transgenic eucalypts for public sector research. Efficient transformation protocols are essential to take full value of the eucalypt genome for functional genomics, ecophysiology, and biotechnology.

In silico and functional characterization of the promoter of a Eucalyptus secondary cell wall associated cellulose synthase gene (EgCesA1)

Background Cellulose is an important biopolymer produced by all plants and is used in a number of different industries, including for pulp and paper production. Cellulose is deposited into the plant cell wall by a large membranebound protein complex, which is composed of different cellulose synthase (CESA) proteins. The cellulose content and pattern of deposition in plant cell walls is highly variable depending on the function of the cell. All plant cells have a thin primary cell wall, but a number of plant cell types, including xylem cells, also deposit a secondary cell wall to give these tissues mechanical strength required to perform their function. Different cellulose synthase (CesA) genes have been shown to be involved in the deposition of primary and secondary walls. In Arabidopsis, three CesA (AtCesA4, 7 and 8) genes have consistently been associated with cells depositing secondary cell walls, while a different set of CesA genes have been shown to function during primary cell wall formation [Reviewed in 1]. These findings have been mirrored by studies of CesA gene orthologs in Populus and Eucalyptus[2-4]. While there have been a number of studies on CesA genes and their functions, much less is known about the regulation of these genes. In a previous study, we investigated the promoters of CesA genes involved in primary and secondary cell wall formation by performing a phylogenetic footprinting analysis to identify cis-elements conserved in the promoters from orthologous Arabidopsis, Populus and Eucalyptus cellulose synthase genes [5]. We identified a number of putative cis-regulatory elements that may play a role in the regulation of cellulose biosynthesis during primary and secondary cell wall formation. In the current study our aim is to further validate the ciselements identified in the CesA gene promoters by investigating their conservation across different Eucalyptus species and to determine the regulatory function of these promoter regions and the proteins which bind to them.