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Several studies suggest the relation of DNA methylation to diseases in humans and important phenotypes in plants drawing attention to this epigenetic mark as an important source of variability. In the last decades, several methodologies were developed to assess the methylation state of a genome. However, there is still a lack of affordable and precise methods for genome wide analysis in large sample size studies. Methyl sensitive double digestion MS-DArT sequencing method emerges as a promising alternative for methylation profiling. We developed a computational pipeline for the identification of DNA methylation using MS-DArT-seq data and carried out a pilot study using the Eucalyptus grandis tree sequenced for the species reference genome. Using a statistic framework as in differential expression analysis, 72,515 genomic sites were investigated and 5,846 methylated sites identified, several tissue specific, distributed along the species 11 chromosomes. We highlight a bias towards identification of DNA methylation in genic regions and the identification of 2,783 genes and 842 transposons containing methylated sites. Comparison with WGBS, DNA sequencing after treatment with bisulfite, data demonstrated a precision rate higher than 95% for our approach. The availability of a reference genome is useful for determining the genomic context of methylated sites but not imperative, making this approach suitable for any species. Our approach provides a cost effective, broad and reliable examination of DNA methylation profile on MspI/HpaII restriction sites, is fully reproducible and the source code is available on GitHub (https://github.com/wendelljpereira/ms-dart-seq)., Competing Interests: Authors MRP and DG are employed by EMBRAPA, but this commercial affiliation does not alter our adherence to all PLOS ONE policies on sharing data and materials.

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.

• Genomic selection (GS) is expected to cause a paradigm shift in tree breeding by improving its speed and efficiency. By fitting all the genome-wide markers concurrently, GS can capture most of the 'missing heritability' of complex traits that quantitative trait locus (QTL) and association mapping classically fail to explain. Experimental support of GS is now required. • The effectiveness of GS was assessed in two unrelated Eucalyptus breeding populations with contrasting effective population sizes (N(e) = 11 and 51) genotyped with > 3000 DArT markers. Prediction models were developed for tree circumference and height growth, wood specific gravity and pulp yield using random regression best linear unbiased predictor (BLUP). • Accuracies of GS varied between 0.55 and 0.88, matching the accuracies achieved by conventional phenotypic selection. Substantial proportions (74-97%) of trait heritability were captured by fitting all genome-wide markers simultaneously. Genomic regions explaining trait variation largely coincided between populations, although GS models predicted poorly across populations, likely as a result of variable patterns of linkage disequilibrium, inconsistent allelic effects and genotype × environment interaction. • GS brings a new perspective to the understanding of quantitative trait variation in forest trees and provides a revolutionary tool for applied tree improvement. Nevertheless population-specific predictive models will likely drive the initial applications of GS in forest tree breeding., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

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.

Background: 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., Results: We 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., Conclusions: This 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.

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.

Background: Benefits from high-throughput sequencing using 454 pyrosequencing technology may be most apparent for species with high societal or economic value but few genomic resources. Rapid means of gene sequence and SNP discovery using this novel sequencing technology provide a set of baseline tools for genome-level research. However, it is questionable how effective the sequencing of large numbers of short reads for species with essentially no prior gene sequence information will support contig assemblies and sequence annotation., Results: With the purpose of generating the first broad survey of gene sequences in Eucalyptus grandis, the most widely planted hardwood tree species, we used 454 technology to sequence and assemble 148 Mbp of expressed sequences (EST). EST sequences were generated from a normalized cDNA pool comprised of multiple tissues and genotypes, promoting discovery of homologues to almost half of Arabidopsis genes, and a comprehensive survey of allelic variation in the transcriptome. By aligning the sequencing reads from multiple genotypes we detected 23,742 SNPs, 83% of which were validated in a sample. Genome-wide nucleotide diversity was estimated for 2,392 contigs using a modified theta (theta) parameter, adapted for measuring genetic diversity from polymorphisms detected by randomly sequencing a multi-genotype cDNA pool. Diversity estimates in non-synonymous nucleotides were on average 4x smaller than in synonymous, suggesting purifying selection. Non-synonymous to synonymous substitutions (Ka/Ks) among 2,001 contigs averaged 0.30 and was skewed to the right, further supporting that most genes are under purifying selection. Comparison of these estimates among contigs identified major functional classes of genes under purifying and diversifying selection in agreement with previous researches., Conclusion: In providing an abundance of foundational transcript sequences where limited prior genomic information existed, this work created part of the foundation for the annotation of the E. grandis genome that is being sequenced by the US Department of Energy. In addition we demonstrated that SNPs sampled in large-scale with 454 pyrosequencing can be used to detect evolutionary signatures among genes, providing one of the first genome-wide assessments of nucleotide diversity and Ka/Ks for a non-model plant species.

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. [ABSTRACT FROM AUTHOR]

An abstract of the conference paper "Genome-wide genotyping and SNP discovery by ultra-deep Restriction-Associated DNA (RAD) tag sequencing of pooled samples of E. grandis and E. globulus," by Dario Grattapaglia and colleagues is presented.

An abstract of the conference paper "Eucalyptus research in the post-genome era," by Georgios Pappas and colleagues is presented.

An abstract of the conference paper "How many genes might underlie QTLs for growth and wood quality traits in Eucalyptus?," by Carolina Sansaloni and colleagues is presented.

An abstract of the conference paper "Genomic characterization, high-density mapping and anchoring of DArT markers to the reference genome of Eucalyptus," by César Petroli and colleagues is presented.

An abstract of the conference paper "A new set of 182 microsatellites for Eucalyptus: Characterization and mapping in a four-species consensus linkage map," by Danielle Faria and colleagues is presented.

An abstract of the conference paper "Assessment of SNPs for linkage mapping in Eucalyptus: construction of a consensus SNP/ microsatellite map from two unrelated pedigrees," by Bruno Marco Lima and colleagues is presented.