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Abstract Background Genetic 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. Results The 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. Conclusion The 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.

Abstract 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.

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

Abstract Background Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. Results We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. Conclusions The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence.

Abstract Background A 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. Findings After testing several genome complexity reduction methods we identified the PstI/TaqI method as the most effective for Eucalyptus and developed 18 genomic libraries from PstI/TaqI 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. Conclusions This 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.

Abstract 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 (θ) 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.

Abstract Background Brazil holds the largest commercial cattle populations worldwide. Local cattle breeds can be classified according to their origin, as exotic or Creole. Exotic breeds imported in the last 100 years, both zebuine and taurine, currently make up the bulk of the intensively managed populations. Locally adapted Creole breeds, originated from cattle introduced by the European conquerors derive from natural selection and events of breed admixture. While historical knowledge exists on the Brazilian Creole breeds very little is known on their genetic composition. The objective of this study was to assess the levels of genetic diversity, phylogenetic relationships and patterns of taurine/zebuine admixture among ten cattle breeds raised in Brazil. Results Significant reduction of heterozygosity exists due both to within-population inbreeding and to breed differentiation in both subspecies (taurine and zebuine). For taurine breeds the number of markers that contribute to breed differentiation is larger than for zebuine. A consistently similar number of alleles was seen in both subspecies for all microsatellites. Four Creole breeds were the most genetically diverse followed by the zebuine breeds, the two specialized taurine breeds and the Creole Caracu. Pairwise genetic differentiation were all significant indicating that all breeds can be considered as genetically independent entities. A STRUCTURE based diagram indicated introgression of indicine genes in the local Creole breeds and suggested that occasional Creole introgression can be detected in some Zebuine animals. Conclusion This study reports on a comprehensive study of the genetic structure and diversity of cattle breeds in Brazil. A significant amount of genetic variation is maintained in the local cattle populations. The genetic data show that Brazilian Creole breeds constitute an important and diverse reservoir of genetic diversity for bovine breeding and conservation. The genetic data was able to shed light on a number of issues related to the local breeds origin and structure. The Brazilian Creole breeds are all important and viable targets for conservation for they display peculiar traits both phenotypic and of cultural and historical nature that deserve conservation efforts.

Abstract 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. Quantitative Trait Loci (QTLs) in Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus. Results The consensus map covers ~90% of the recombining genome of Eucalyptus, involves 234 mapped EMBRA loci on 11 linkage groups, an observed length of 1,568 cM and a mean distance between markers of 8.4 cM. A compilation of all microsatellite linkage information published in Eucalyptus allowed us to establish the homology among linkage groups between this consensus map and other maps published for E. globulus. Comparative mapping analyses also resulted in the linkage group assignment of other 41 microsatellites derived from other Eucalyptus species as well as candidate genes and QTLs for wood and flowering traits published in the literature. This report significantly increases the availability of microsatellite markers and mapping information for species of Eucalyptus and corroborates the high conservation of microsatellite flanking sequences and locus ordering between species of the genus. Conclusion This work represents an important step forward for Eucalyptus comparative genomics, opening stimulating perspectives for evolutionary studies and molecular breeding applications. The generalized use of an increasingly larger set of interspecific transferable markers and consensus mapping information, will allow faster and more detailed investigations of QTL synteny among species, validation of expression-QTL across variable genetic backgrounds and positioning of a growing number of candidate genes co-localized with QTLs, to be tested in association mapping experiments.