Danielle A. Faria

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.

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

BackgroundHigh-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.ResultsWe have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico 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 in silico 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.SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased.ConclusionsThis study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity ≥2%. The development of a much larger array of informative SNPs across multiple Eucalyptus 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 Eucalyptus.

High synteny and colinearity among Eucalyptus genomes revealed by high-density comparative genetic mapping

Understanding genome differentiation is important to compare and transfer genomic information between taxa, such as from model to non-model organisms. Comparative genetic mapping can be used to assess genome differentiation by identifying similarities and differences in chromosome organization. Following release of the assembled Eucalyptus grandis genome sequence (January 2011; http://www.phytozome.net/), a better understanding of genome differentiation between E. grandis and other commercially important species belonging to the subgenus Symphyomyrtus is required. In this study, comparative genetic mapping analyses were conducted between E. grandis, Eucalyptus urophylla, and Eucalyptus globulus using high-density linkage maps constructed from Diversity Array Technology and microsatellite molecular markers. There were 236–393 common markers between maps, providing the highest resolution yet achieved for comparative mapping in Eucalyptus. In two intra-section comparisons (section Maidenaria–E. globulus and section Latoangulatae–E. grandis vs. E. urophylla), ∼1% of common markers were non-syntenic and within chromosomes 4.7–6.8% of markers were non-colinear. Consistent with increasing taxonomic distance, lower synteny (6.6% non-syntenic markers) was observed in an inter-section comparison between E. globulus and E. grandis × E. urophylla consensus linkage maps. Two small chromosomal translocations or duplications were identified in this comparison representing possible genomic differences between E. globulus and section Latoangulatae species. Despite these differences, the overall high level of synteny and colinearity observed between section Maidenaria–Latoangulatae suggests that the genomes of these species are highly conserved indicating that sequence information from the E. grandis genome will be highly transferable to related Symphyomyrtus species.

Genotyping systems for Eucalyptus based on tetra-, penta-, and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests

Eucalypts are keystone species in their natural ranges and are extensively planted worldwide for high-quality woody biomass. A novel set of 21 polymorphic and interspecifically transferable microsatellite markers based on tetra-, penta- and hexanucleotide repeats were developed and tested for high-precision genotyping of species of Eucalyptus. These microsatellites were characterized in population samples of four species, Eucalyptus grandis, Eucalyptus globulus, Eucalyptus urophylla, and Eucalyptus camaldulensis, representing three phylogenetic sections of subgenus Symphyomyrtus. These markers provide a clear advantage for accurate allele calling due to their larger allele size difference. Two multiplexed microsatellite combinations, a 14-locus/four-dye and an 18-locus/five-dye set, analyzable in single lanes were designed, providing resolution and throughput analogous to those routinely used in human DNA profiling. This set of microsatellites was shown to have high resolution for clone fingerprinting, inter-individual genetic distance estimation, species distinction, and assignment of hybrid individuals to their most likely ancestral species. These systems will be particularly useful for comparative population genetics and molecular breeding applications that require consistent allele calling across different points in time or laboratories.

A Selected Set of EST-Derived Microsatellites, Polymorphic and Transferable across 6 Species of Eucalyptus

Species of Eucalyptus are keystone species for ecological studies in their natural ranges and are extensively planted in the tropical and subtropical regions of the world to supply high-quality woody biomass for various applications. We report the development of a selected set of 20 dinucleotide and trinucleotide repeat microsatellites derived from Eucalyptus expressed sequence tags (ESTs). These microsatellites were selected for full transferability and homogeneous rate of polymorphism across species. They were evaluated for individual fingerprinting, parentage testing, and intraspecific population structure analyses in 6 of the most extensively studied and planted species worldwide, representing key phylogenetic sections of the largest subgenus Symphyomyrtus. This set of markers provides exceptional resolution for population genetics and molecular breeding applications in the genus Eucalyptus. As they were developed from conserved transcribed regions, the transferability and polymorphism of these microsatellites will most likely extend to the other 300 or more species within the same subgenus.

Positioning of the major locus for Puccinia psidii rust resistance (Ppr1) on the Eucalyptus reference map and its validation across unrelated pedigrees

In this report the major locus for Puccinia psidii rust resistance, Ppr1, was positioned on the reference genetic map for Eucalyptus. Additionally, its position was validated by association genetics in a related and two unrelated pedigrees involving different Eucalyptus grandis resistant trees crossed to individuals of two other species, Eucalyptus tereticornis and Eucalyptus camaldulensis. These results are consistent with the hypothesis that Ppr1 controls a large proportion of the variation for rust resistance, strengthening its role as a major locus in Eucalyptus and providing its unequivocal genomic position on linkage group 3. A localized map with 19 microsatellite loci was built around Ppr1. Multiallelic profiles were observed at several mapped microsatellites suggesting recent tandem duplications in the genomic landscape surrounding Ppr1. Markers EMBRA125 and EMBRA1071 flank Ppr1 at 9.5% and 7% recombination, respectively, and were found to be in linkage equilibrium in a E. grandis breeding population, consistent with the expectations in outcrossed Eucalyptus. Their potential use for MAS will specifically be directed to identifying resistant offspring of P. psidii resistant parent trees that are heterozygous at Ppr1. In these circumstances, a significant amount of LD is expected to occur between specific alleles at flanking microsatellites and the resistance allele at Ppr1. Moreover, the positional information of Ppr1 paves the way for prospective undertakings in this genomic region with the upcoming availability of a draft genome for E. grandis.

Genetic mapping provides evidence for the role of additive and non-additive QTLs in the response of inter-specific hybrids of Eucalyptus to Puccinia psidii rust infection

Eucalypts are susceptible to a wide range of diseases. One of the most important diseases that affect Eucalyptus plantations worldwide is caused by the rust fungus Puccinia psidii. Here, we provide evidence on the complex genetic control of rust resistance in Eucalyptus inter-specific hybrids, by analyzing a number of full-sib families that display different patterns of segregation for rust resistance. These families are totally unrelated to those previously used in other inheritance studies of rust resistance. By using a full genome scan with 114 genetic markers (microsatellites and expressed sequence tag derived microsatellites) we also corroborated the existence and segregation of a resistance locus, explaining 11.5% of the phenotypic variation, on linkage group 3, corresponding to Ppr1. This find represents an additional validation of this locus in totally unrelated pedigree. We have also detected significant additive × additive digenic interactions with LOD >10.0 on several linkage groups. The additive and epistatic QTLs identified explain between 29.8 and 44.8% of the phenotypic variability for rust resistance. The recognition that both additive and non-additive genetic variation (epistasis) are important contributors to rust resistance in eucalypts reveals the complexity of this host-pathogen interaction and helps explain the success that breeding has achieved by selecting rust-resistant clones, where all the additive and non-additive effects are readily captured. The positioning of epistatic QTLs also provides starting points to look for the underlying genes or genomic regions controlling this phenotype on the upcoming E. grandis genome sequence.

A novel genome-wide microsatellite resource for species of Eucalyptus with linkage-to-physical correspondence on the reference genome sequence

Keystone species in their native ranges, eucalypts, are ecologically and genetically very diverse, growing naturally along extensive latitudinal and altitudinal ranges and variable environments. Besides their ecological importance, eucalypts are also the most widely planted trees for sustainable forestry in the world. We report the development of a novel collection of 535 microsatellites for species of Eucalyptus, 494 designed from ESTs and 41 from genomic libraries. A selected subset of 223 was evaluated for individual identification, parentage testing, and ancestral information content in the two most extensively studied species, Eucalyptus grandis and Eucalyptus globulus. Microsatellites showed high transferability and overlapping allele size range, suggesting they have arisen still in their common ancestor and confirming the extensive genome conservation between these two species. A consensus linkage map with 437 microsatellites, the most comprehensive microsatellite‐only genetic map for Eucalyptus, was built by assembling segregation data from three mapping populations and anchored to the Eucalyptus genome. An overall colinearity between recombination‐based and physical positioning of 84% of the mapped microsatellites was observed, with some ordering discrepancies and sporadic locus duplications, consistent with the recently described whole genome duplication events in Eucalyptus. The linkage map covered 95.2% of the 605.8‐Mbp assembled genome sequence, placing one microsatellite every 1.55 Mbp on average, and an overall estimate of physical to recombination distance of 618 kbp/cM. The genetic parameters estimates together with linkage and physical position data for this large set of microsatellites should assist marker choice for genome‐wide population genetics and comparative mapping in Eucalyptus.

Fine mapping and single nucleotide polymorphism effects estimation on pig chromosomes 1, 4, 7, 8, 17 and X

Fine mapping of quantitative trait loci (QTL) from previous linkage studies was performed on pig chromosomes 1, 4, 7, 8, 17, and X which were known to harbor QTL. Traits were divided into: growth performance, carcass, internal organs, cut yields, and meat quality. Fifty families were used of a F2 population produced by crossing local Brazilian Piau boars with commercial sows. The linkage map consisted of 237 SNP and 37 microsatellite markers covering 866 centimorgans. QTL were identified by regression interval mapping using GridQTL. Individual marker effects were estimated by Bayesian LASSO regression using R. In total, 32 QTL affecting the evaluated traits were detected along the chromosomes studied. Seven of the QTL were known from previous studies using our F2 population, and 25 novel QTL resulted from the increased marker coverage. Six of the seven QTL that were significant at the 5% genome-wide level had SNPs within their confidence interval whose effects were among the 5% largest effects. The combined use of microsatellites along with SNP markers increased the saturation of the genome map and led to smaller confidence intervals of the QTL. The results showed that the tested models yield similar improvements in QTL mapping accuracy.

Comparative assessment of SNPs and microsatellites for fingerprinting, parentage and assignment testing in species of Eucalyptus

Background Brazil stands on the international scenario by having one of the largest natural forest heritage and extensive sustainable planted forests. Brazilian planted forestry is based mainly on fast growing eucalyptus and pines, with the pulp, paper and steel industries as the major consumers. Introduced commercially in Brazil in the early twentieth century, the eucalypts have experienced increasing levels of genetic improvement over the years. Along with the classical breeding, the use of genetic markers has positively influenced breeding programs, contributing to quality control processes of clonal forestry and advanced breeding. Microsatellite markers have been the main tool used to date. They are multiallelic, highly polymorphic and thus very efficient for several applications that require identification and discrimination of elite clones and determination of parentage. While di and tri-nucleotide repeat microsatellites tend to be more polymorphic, tetra and higher order repeats allow a more robust allele calling [1]. SNP markers genotyped by a high-throughput system have been recently developed for Eucalyptus [2] but not yet routinely incorporated in Eucalyptus breeding programs. In spite of their lower genetic information content, many more SNPs can be simultaneously typed using automated systems, an appealing feature to operational breeding programs. The aim of this study was to comparatively evaluate the resolving power and precision of different sets of molecular markers (microsatellites and SNPs assayed by the Golden Gate technology) for the most common operational applications in Eucalyptus breeding programs.