Cesar D. Petroli

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.

Population genetic analysis and phylogeny reconstruction in Eucalyptus (Myrtaceae) using high-throughput, genome-wide genotyping

A set of over 8000 Diversity Arrays Technology (DArT) markers was tested for its utility in high-resolution population and phylogenetic studies across a range of Eucalyptus taxa. Small-scale population studies of Eucalyptus camaldulensis, Eucalyptus cladocalyx, Eucalyptus globulus, Eucalyptus grandis, Eucalyptus nitens, Eucalyptus pilularis and Eucalyptus urophylla demonstrated the potential of genome-wide genotyping with DArT markers to differentiate species, to identify interspecific hybrids and to resolve biogeographic disjunctions within species. The population genetic studies resolved geographically partitioned clusters in E. camaldulensis, E. cladocalyx, E. globulus and E. urophylla that were congruent with previous molecular studies. A phylogenetic study of 94 eucalypt species provided results that were largely congruent with traditional taxonomy and ITS-based phylogenies, but provided more resolution within major clades than had been obtained previously. Ascertainment bias (the bias introduced in a phylogeny from using markers developed in a small sample of the taxa that are being studied) was not detected. DArT offers an unprecedented level of resolution for population genetic, phylogenetic and evolutionary studies across the full range of Eucalyptus species.

Diversity Arrays Technology (DArT) and next- generation sequencing combined: genome-wide, high throughput, highly informative genotyping for molecular breeding of Eucalyptus

Background Wider genome coverage and higher throughput genotyping methods have become increasingly important to meet the resolution and speed necessary for a variety of applications in genomics and molecular breeding of forest trees. Developed more than 10 years ago [1], the Diversity Arrays Technology (DArT) has experienced an increasing interest worldwide for it has efficiently satisfied the requirements of throughput, genome coverage and inter-specific transferability for over 40 different plant species to date, including Eucalyptus[2] and recently Pinus (Dione Alves-Freitas, this meeting). DArT is based on genome complexity reduction using restriction enzymes, followed by hybridization to microarrays to simultaneously assay hundreds to thousands of markers across a genome. Genome complexity reduction for genotyping has now been taken to another level when combined to next generation sequencing (NGS) technologies. Such a strategy has been used for rapid SNP discovery in different organisms [3], and proposed as a way to genotype with RAD (Restriction-associated DNA) sequencing [4]and recently by a similar method generally termed GbS (Genotyping-by-Sequencing)[5]. In this work we assessed the power of the now well established DArT marker platform in combination with Illumina short read sequencing to generate a linkage map for a segregating outcrossed F1 population derived from E. grandis BRASUZ1, the donor of the Eucalyptus reference genome.

A high-density Diversity Arrays Technology (DArT) microarray for genome-wide genotyping in Eucalyptus

BackgroundA number of molecular marker technologies have allowed important advances in the understanding of the genetics and evolution of Eucalyptus, a genus that includes over 700 species, some of which are used worldwide in plantation forestry. Nevertheless, the average marker density achieved with current technologies remains at the level of a few hundred markers per population. Furthermore, the transferability of markers produced with most existing technology across species and pedigrees is usually very limited. High throughput, combined with wide genome coverage and high transferability are necessary to increase the resolution, speed and utility of molecular marker technology in eucalypts. We report the development of a high-density DArT genome profiling resource and demonstrate its potential for genome-wide diversity analysis and linkage mapping in several species of Eucalyptus.FindingsAfter testing several genome complexity reduction methods we identified the Pst I/Taq I method as the most effective for Eucalyptus and developed 18 genomic libraries from Pst I/Taq I representations of 64 different Eucalyptus species. A total of 23,808 cloned DNA fragments were screened and 13,300 (56%) were found to be polymorphic among 284 individuals. After a redundancy analysis, 6,528 markers were selected for the operational array and these were supplemented with 1,152 additional clones taken from a library made from the E. grandis tree whose genome has been sequenced. Performance validation for diversity studies revealed 4,752 polymorphic markers among 174 individuals. Additionally, 5,013 markers showed segregation when screened using six inter-specific mapping pedigrees, with an average of 2,211 polymorphic markers per pedigree and a minimum of 859 polymorphic markers that were shared between any two pedigrees.ConclusionsThis operational DArT array will deliver 1,000-2,000 polymorphic markers for linkage mapping in most eucalypt pedigrees and thus provide high genome coverage. This array will also provide a high-throughput platform for population genetics and phylogenetics in Eucalyptus. The transferability of DArT across species and pedigrees is particularly valuable for a large genus such as Eucalyptus and will facilitate the transfer of information between different studies. Furthermore, the DArT marker array will provide a high-resolution link between phenotypes in populations and the Eucalyptus reference genome, which will soon be completed.

Genomic characterization of DArT markers based on high-density linkage analysis and physical mapping to the Eucalyptus genome

Diversity Arrays Technology (DArT) provides a robust, high throughput, cost-effective method to query thousands of sequence polymorphisms in a single assay. Despite the extensive use of this genotyping platform for numerous plant species, little is known regarding the sequence attributes and genome-wide distribution of DArT markers. We investigated the genomic properties of the 7,680 DArT marker probes of a Eucalyptus array, by sequencing them, constructing a high density linkage map and carrying out detailed physical mapping analyses to the Eucalyptus grandis reference genome. A consensus linkage map with 2,274 DArT markers anchored to 210 microsatellites and a framework map, with improved support for ordering, displayed extensive collinearity with the genome sequence. Only 1.4 Mbp of the 75 Mbp of still unplaced scaffold sequence was captured by 45 linkage mapped but physically unaligned markers to the 11 main Eucalyptus pseudochromosomes, providing compelling evidence for the quality and completeness of the current Eucalyptus genome assembly. A highly significant correspondence was found between the locations of DArT markers and predicted gene models, while most of the 89 DArT probes unaligned to the genome correspond to sequences likely absent in E. grandis, consistent with the pan-genomic feature of this multi-Eucalyptus species DArT array. These comprehensive linkage-to-physical mapping analyses provide novel data regarding the genomic attributes of DArT markers in plant genomes in general and for Eucalyptus in particular. DArT markers preferentially target the gene space and display a largely homogeneous distribution across the genome, thereby providing superb coverage for mapping and genome-wide applications in breeding and diversity studies. Data reported on these ubiquitous properties of DArT markers will be particularly valuable to researchers working on less-studied crop species who already count on DArT genotyping arrays but for which no reference genome is yet available to allow such detailed characterization.

A reference linkage map for Eucalyptus

BackgroundGenetic linkage maps are invaluable resources in plant research. They provide a key tool for many genetic applications including: mapping quantitative trait loci (QTL); comparative mapping; identifying unlinked (i.e. independent) DNA markers for fingerprinting, population genetics and phylogenetics; assisting genome sequence assembly; relating physical and recombination distances along the genome and map-based cloning of genes. Eucalypts are the dominant tree species in most Australian ecosystems and of economic importance globally as plantation trees. The genome sequence of E. grandis has recently been released providing unprecedented opportunities for genetic and genomic research in the genus. A robust reference linkage map containing sequence-based molecular markers is needed to capitalise on this resource. Several high density linkage maps have recently been constructed for the main commercial forestry species in the genus (E. grandis, E. urophylla and E. globulus) using sequenced Diversity Arrays Technology (DArT) and microsatellite markers. To provide a single reference linkage map for eucalypts a composite map was produced through the integration of data from seven independent mapping experiments (1950 individuals) using a marker-merging method.ResultsThe composite map totalled 1107 cM and contained 4101 markers; comprising 3880 DArT, 213 microsatellite and eight candidate genes. Eighty-one DArT markers were mapped to two or more linkage groups, resulting in the 4101 markers being mapped to 4191 map positions. Approximately 13% of DArT markers mapped to identical map positions, thus the composite map contained 3634 unique loci at an average interval of 0.31 cM.ConclusionThe composite map represents the most saturated linkage map yet produced in Eucalyptus. As the majority of DArT markers contained on the map have been sequenced, the map provides a direct link to the E. grandis genome sequence and will serve as an important reference for progressing eucalypt research.

Genomic Selection for growth traits in Eucalyptus : accuracy within and across breeding populations

Background Genomic selection (GS) involves selection decisions based on genomic breeding values estimated as the sum of the effects of genome-wide markers capturing most QTLs for the target trait(s). GS is revolutionizing breeding practice for complex trait in domestic animals. The same approach and concepts can be readily applied to forest tree breeding. Trees also have long generation times and late expressing traits. Differently from association genetics that aims at dissecting complex traits in their discrete components, GS precludes the discovery of individual marker-trait associations and focuses on prediction of performance. By capturing the “missing heritability” of complex quantitative traits beyond the few effect variants that association genetics has so far typically identified, GS might soon cause a paradigm shift in forest tree breeding. In a prior deterministic study we assessed the impact of linkage disequilibrium (modeled by Ne and inter-marker distance), the size of the training set, trait heritability and the number of QTL on the predicted accuracy of GS [1]. Results indicate that GS has the potential to radically improve the efficiency of tree breeding. The benchmark accuracy of conventional BLUP-based phenotypic selection (0.68) was reached by GS even at a marker density ~2 markers/cM when Ne ≤30, while up to 10 markers/cM are necessary for larger Ne. Shortening the breeding cycle by 50% with GS provides an expected increase ≥100% in selection efficiency. To validate these simulation results we carried out a large multi-population proof-of-concept study of GS in tropical Eucalyptus. In this report we present results of this on-going study for two populations and three different quantitative traits.

Genetic monitoring of a Santa Ines herd using microsatellite markers near or linked to the sheep MHC

This study aimed to analyze genetic diversity in a conservation nucleus of Santa Ines sheep using thirteen microsatellite loci on chromosome 20 (where the Sheep Major Histocompatibility Complex - Ovar-MHC - is found). Seventy three animals from one herd born from 2004 to 2006 were evaluated as a principal nucleus. Seventy one animals from two other herds were used as control comparison. There was a reduction in heterozygosity over the years in relation to the whole population. This may be due to the repeated use of the same sires. The estimates of molecular coancestrality also indicated an increase in genetic similarity between individuals with the herd over the years. A high number of alleles occurred exclusively in the principal nucleus herd, but with a frequency lower than 10%. The Ovar-MHC region of chromosome 20 was shown to be highly polymorphic. Monitoring of the herd over time should be implemented as additional tool for genetic management within the herd.

Genomic characterization, high-density mapping and anchoring of DArT markers to the reference genome of Eucalyptus

Background Genetic linkage maps have been essential tools to examine the inheritance of qualitative and quantitative traits, to carry out comparative mapping and to provide markers for molecular breeding applications. Linkage maps for species of Eucalyptus have been reported for several pedigrees using different molecular marker technologies [1]. However improved marker density, throughput and transferability across species are necessary to increase resolution of current maps for a variety of genomic applications. We report the development of a high density linkage map for Eucalyptus based on microsatellites and DArT (Diversity Arrays Technology) markers generated by a standardized genotyping microarray [2]. DNA probes that constitute the DArT microarray were sequenced and positioned on the reference Eucalyptus genome providing information about their sequence content, their distribution relative to annotated genes as well as the relationship between physical and recombination distance in the Eucalyptus genome.

A genetic linkage map for a Full sib population of Eucalyptus grandis using SSR, DArT, CG-SSR and EST-SSR markers

Background Eucalypts are the most widely planted hardwood trees in the world, occupying globally more than 18 million hectares, as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Intensive planting programs of Eucalyptus grandis have been carried out in the Argentinian Mesopotamia. Linkage maps are useful tools for quantitative trait loci (QTL) analyses and detection. Several maps for QTL analyses of growth and wood quality have been developed in this genus [1,2] and most of the E. grandis maps have been carried out in interspecific crosses. Improved marker density in genetic maps, highthroughput techniques and transferability across species are key aspects to increase resolution and speed for a variety of genomic applications in Eucalyptus.In this context, an important issue in association studies is the selection of appropriate mapped candidate genes that co-localize with QTL of interest. As part of the Biotech MERCOSUR project (Marcucci et al., this journal), we here report the construction of a genetic linkage map for E. grandis in the context of a QTL study of this specie in an effort to understand the molecular basis for quantitative trait variation in wood quality. This map includes Diversity Arrays Technology (DArT) [3], microsatellite (SSR) markers [4], Candidate Genes-SSR (CG-SSR) for wood quality traits and stress responses functions and Expressed Sequence Tag-SSR (EST-SSR) for putative function related to stress responses and other functions (Acuna et al., this journal). These CG-SSR and EST-SSR were not mapped in Eucalyptus previously.