High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

<p>Abstract</p> <p>Background</p> <p>High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera.</p> <p>Results</p> <p>We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of <it>Eucalyptus </it>from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for <it>E. grandis</it>. A systematic assessment of <it>in silico </it>SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous <it>in silico </it>constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species.</p> <p>SNP reliability was high across nine <it>Eucalyptus </it>species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased.</p> <p>Conclusions</p> <p>This study indicates that the GGGT performs well both within and across species of <it>Eucalyptus </it>notwithstanding its nucleotide diversity ≥2%. The development of a much larger array of informative SNPs across multiple <it>Eucalyptus </it>species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in <it>Eucalyptus</it>.</p

Species Discrimination, Population Structure and Linkage Disequilibrium in Eucalyptus camaldulensis and Eucalyptus tereticornis Using SSR Markers

Eucalyptus camaldulensis and E. tereticornis are closely related species commonly cultivated for pulp wood in many tropical countries including India. Understanding the genetic structure and linkage disequilibrium (LD) existing in these species is essential for the improvement of industrially important traits. Our goal was to evaluate the use of simple sequence repeat (SSR) loci for species discrimination, population structure and LD analysis in these species. Investigations were carried out with the most common alleles in 93 accessions belonging to these two species using 62 SSR markers through cross amplification. The polymorphic information content (PIC) ranged from 0.44 to 0.93 and 0.36 to 0.93 in E. camaldulensis and E. tereticornis respectively. A clear delineation between the two species was evident based on the analysis of population structure and species-specific alleles. Significant genotypic LD was found in E. camaldulensis, wherein out of 135 significant pairs, 17 pairs showed r2≥0.1. Similarly, in E. tereticornis, out of 136 significant pairs, 18 pairs showed r2≥0.1. The extent of LD decayed rapidly showing the significance of association analyses in eucalypts with higher resolution markers. The availability of whole genome sequence for E. grandis and the synteny and co-linearity in the genome of eucalypts, will allow genome-wide genotyping using microsatellites or single nucleotide polymorphims

A high-density transcript linkage map with 1,845 expressed genes positioned by microarray-based Single Feature Polymorphisms (SFP) in Eucalyptus

<p>Abstract</p> <p>Background</p> <p>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 <it>Eucalyptus </it>using Single Feature Polymorphism (SFP) markers.</p> <p>Results</p> <p>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. <it>In silico </it>validation showed that 87% of the SFPs map to the expected location on the 4.5X draft sequence of the <it>Eucalyptus grandis </it>genome.</p> <p>Conclusions</p> <p>The <it>Eucalyptus </it>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 <it>Eucalyptus </it>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.</p

Transcriptome profiling of Pinus radiata juvenile wood with contrasting stiffness identifies putative candidate genes involved in microfibril orientation and cell wall mechanics

<p>Abstract</p> <p>Background</p> <p>The mechanical properties of wood are largely determined by the orientation of cellulose microfibrils in secondary cell walls. Several genes and their allelic variants have previously been found to affect microfibril angle (MFA) and wood stiffness; however, the molecular mechanisms controlling microfibril orientation and mechanical strength are largely uncharacterised. In the present study, cDNA microarrays were used to compare gene expression in developing xylem with contrasting stiffness and MFA in juvenile <it>Pinus radiata </it>trees in order to gain further insights into the molecular mechanisms underlying microfibril orientation and cell wall mechanics.</p> <p>Results</p> <p>Juvenile radiata pine trees with higher stiffness (HS) had lower MFA in the earlywood and latewood of each ring compared to low stiffness (LS) trees. Approximately 3.4 to 14.5% out of 3, 320 xylem unigenes on cDNA microarrays were differentially regulated in juvenile wood with contrasting stiffness and MFA. Greater variation in MFA and stiffness was observed in earlywood compared to latewood, suggesting earlywood contributes most to differences in stiffness; however, 3-4 times more genes were differentially regulated in latewood than in earlywood. A total of 108 xylem unigenes were differentially regulated in juvenile wood with HS and LS in at least two seasons, including 43 unigenes with unknown functions. Many genes involved in cytoskeleton development and secondary wall formation (cellulose and lignin biosynthesis) were preferentially transcribed in wood with HS and low MFA. In contrast, several genes involved in cell division and primary wall synthesis were more abundantly transcribed in LS wood with high MFA.</p> <p>Conclusions</p> <p>Microarray expression profiles in <it>Pinus radiata </it>juvenile wood with contrasting stiffness has shed more light on the transcriptional control of microfibril orientation and the mechanical properties of wood. The identified candidate genes provide an invaluable resource for further gene function and association genetics studies aimed at deepening our understanding of cell wall biomechanics with a view to improving the mechanical properties of wood.</p

Identification of quantitative trait loci for wood and fibre properties in two full-sib pedigrees of Eucalyptus globulus

Regions of the genome influencing wood and fibre traits in Eucalyptus globulus Labill. have been identified in two full-sib pedigrees that share a common male parent. The first pedigree, cross A, contains 148 progeny, and the second pedigree, cross B, contains 135 progeny. Subsets of progeny of these two controlled crosses were planted at seven sites throughout Australia in 1990. Wood cores were taken at 0.9 m above ground in 1997, and wood and fibre traits were analysed for each individual. Three quantitative trait loci (QTL) affecting wood density, one QTL affecting pulp yield and one QTL affecting microfibril angle have been located in both pedigrees, using single-factor analysis of variance. Other QTLs affecting these traits, as well as fibre length and cellulose content were located in cross A only