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Genomic Selection (GS) in tree breeding optimizes genetic gains by leveraging genomic data to enable early selection of seedlings without phenotypic data reducing breeding cycle and increasing selection intensity. Traditional assessments of the potential of GS in forest trees have typically focused on model performance using cross-validation within the same generation but evaluating effectively realized predictive ability (RPA) across generations is crucial. This study estimated RPAs for volume growth (VOL), wood density (WD), and pulp yield (PY) across four generations breeding of Eucalyptus grandis. The training set spanned three generations, including 34,461 trees with three-year growth data, 6,014 trees with wood quality trait data, and 1,918 trees with 12,695 SNPs (single nucleotide polymorphisms) data. Employing single-step genomic BLUP, we compared the genomic predictions of breeding values (GEBVs) for 1,153 fourth-generation full-sib seedlings in the greenhouse with their later-collected phenotypic estimated breeding values (EBVs) at age three years. RPAs were estimated using three GS targets (individual trees, trees within families, and families), two selection criteria (single- and multiple-trait), and training populations of either all 1,918 genotyped trees or the 67 direct ancestors of the selection candidates. RPAs were higher for wood quality traits (0.33 to 0.59) compared to VOL (0.14 to 0.19) and improved for wood traits (0.42 to 0.75) but not for VOL when trained only with direct ancestors, highlighting the challenges in accurately predicting growth traits. GS was more effective at excluding bottom-ranked candidates than selecting top-ranked ones. The between-family GS approach outperformed individual-tree selection for VOL (0.11 to 0.16) and PY (0.72 to 0.75), but not for WD (0.43 vs. 0.42). Furthermore, higher levels of relatedness and lower genotype by environment (G × E) interaction between training and testing populations enhanced RPAs for VOL (0.39). In summary, despite limited effectiveness in ranking top VOL individuals, GS effectively identified low-performing individuals and families. These multi-generational findings underscore GS's potential in tree breeding, stressing the importance of considering relatedness and G × E interaction for optimal performance. [ABSTRACT FROM AUTHOR]

Despite extensive introductions of Eucalyptus germplasm to Brazil in the last 50 years, relatively little is known about the genetic diversity within current breeding programs, particularly for non-mainstream species. A prime example is Eucalyptus benthamii, a subtropical species with a restricted natural range, endangered due to anthropogenic population fragmentation. We used microsatellite markers to reconstruct the pedigrees and estimate diversity within the two most representative pools of E. benthamii germplasm managed in separate, commercial Brazilian breeding populations. The analysis, together with evidence from Australian Tree Seed Centre (ATSC), historical germplasm export data confirmed that the genetic diversity within the two populations is modest. In the two breeding populations, the estimated status number was in the range of 8.5–22.0 per population considering marker-based co-ancestry. Extensive close relatedness and family structure were identified within and across the two populations, confirming that they share a high proportion of founder ancestors. This is likely a result of the narrow genetic base of the initial introductions and subsequent directional selection and breeding. With the increasing importance of E. benthamii in Brazilian plantation forestry, and as further breeding will likely reduce the status number, our study highlights the need for infusions to augment the existing genetic diversity. Complementary to sourcing infusions, implementing breeding strategies that take advantage of the marker-based information reported in this study to carefully manage the pedigree should help to minimise inbreeding. Exchanging unrelated individuals between the two Brazilian breeding populations would also be advantageous. [ABSTRACT FROM AUTHOR]

Introduction: The coconut tree crop (Cocos nucifera L.) provides vital resources for millions of people worldwide. Coconut germplasm is largely classified into 'Tall' (Typica) and 'Dwarf' (Nana) types. While Tall coconuts are outcrossing, stress tolerant, and late flowering, Dwarf coconuts are inbred and flower early with a high rate of bunch emission. Precocity determines the earlier production of a plantation and facilitates management and harvest. Methods: A unique outbred F2 population was used, generated by intercrossing F1 hybrids between Brazilian Green Dwarf from Jiqui (BGDJ) and West African Tall (WAT) cultivars. Single-nucleotide polymorphism (SNP) markers fixed for alternative alleles in the two varieties, segregating in an F2 configuration, were used to build a high-density linkage map with ~3,000 SNPs, anchored to the existing chromosome-level genome assemblies, and a quantitative trait locus (QTL) mapping analysis was carried out. Results: The linkage map established the chromosome numbering correspondence between the two reference genome versions and the relationship between recombination rate, physical distance, and gene density in the coconut genomes. Leveraging the strong segregation for precocity inherited from the Dwarf cultivar in the F2, a major effect QTL with incomplete dominance was mapped for flowering time. FLOWERING LOCUS T (FT) gene homologs of coconut previously described as putatively involved in flowering time by alternative splice variant analysis were colocalized within a ~200-kb window of the major effect QTL [logarithm of the odds (LOD) = 11.86]. Discussion: Our work provides strong phenotype-based evidence for the role of the FT locus as the putative underlying functional variant for the flowering time difference between Dwarf and Tall coconuts. Major effect QTLs were also detected for developmental traits of the palm, plausibly suggesting pleiotropism of the FT locus for other precocity traits. Haplotypes of the two SNPs flanking the flowering time QTL inherited from the Dwarf parent BGDJ caused a reduction in the time to flower of approximately 400 days. These SNPs could be used for high-throughput marker-assisted selection of early-flowering and higher-productivity recombinant lines, providing innovative genetic material to the coconut industry. [ABSTRACT FROM AUTHOR]

Eucalyptus dunnii is one of the most important Eucalyptus species for short-fiber pulp production in regions where other species of the genus are affected by poor soil and climatic conditions. In this context, E. dunnii holds promise as a resource to address and adapt to the challenges of climate change. Despite its rapid growth and favorable wood properties for solid wood products, the advancement of its improvement remains in its early stages. In this work, we evaluated the performance of two single nucleotide polymorphism, (SNP), genotyping methods for population genetics analysis and Genomic Selection in E. dunnii. Double digest restriction-site associated DNA sequencing (ddRADseq) was compared with the EUChip60K array in 308 individuals from a provenanceprogeny trial. The compared SNP set included 8,011 and 19,008 informative SNPs distributed along the 11 chromosomes, respectively. Although the two datasets differed in the percentage of missing data, genome coverage, minor allele frequency and estimated genetic diversity parameters, they revealed a similar genetic structure, showing two subpopulations with little differentiation between them, and low linkage disequilibrium. GS analyses were performed for eleven traits using Genomic Best Linear Unbiased Prediction (GBLUP) and a conventional pedigree-based model (ABLUP). Regardless of the SNP dataset, the predictive ability (PA) of GBLUP was better than that of ABLUP for six traits (Cellulose content, Total and Ethanolic extractives, Total and Klason lignin content and Syringyl and Guaiacyl lignin monomer ratio). When contrasting the SNP datasets used to estimate PAs, the GBLUP-EUChip60K model gave higher and significant PA values for six traits, meanwhile, the values estimated using ddRADseq gave higher values for three other traits. The PAs correlated positivelywith narrow sense heritabilities, with the highest correlations shown by the ABLUP and GBLUP-EUChip60K. The two genotyping methods, ddRADseq and EUChip60K, are generally comparable for population genetics and genomic prediction, demonstrating the utility of the former when subjected to rigorous SNP filtering. The results of this study provide a basis for future whole-genome studies using ddRADseq in non-model forest species for which SNP arrays have not yet been developed. [ABSTRACT FROM AUTHOR]

Introduction: Genomic selection (GS) experiments in forest trees have largely reported estimates of predictive abilities from cross-validation among individuals in the same breeding generation. In such conditions, no effects of recombination, selection, drift, and environmental changes are accounted for. Here, we assessed the effectively realized predictive ability (RPA) for volume growth at harvest age by GS across generations in an operational reciprocal recurrent selection (RRS) program of hybrid Eucalyptus. Methods: Genomic best linear unbiased prediction with additive (GBLUP_G), additive plus dominance (GBLUP_G+D), and additive single-step (HBLUP) models were trained with different combinations of growth data of hybrids and pure species individuals (N = 17,462) of the G1 generation, 1,944 of which were genotyped with ~16,000 SNPs from SNP arrays. The hybrid G2 progeny trial (HPT267) was the GS target, with 1,400 selection candidates, 197 of which were genotyped still at the seedling stage, and genomically predicted for their breeding and genotypic values at the operational harvest age (6 years). Seedlings were then grown to harvest and measured, and their pedigree-based breeding and genotypic values were compared to their originally predicted genomic counterparts. Results: Genomic RPAs ≥0.80 were obtained as the genetic relatedness between G1 and G2 increased, especially when the direct parents of selection candidates were used in training. GBLUP_G+D reached RPAs ≥0.70 only when hybrid or pure species data of G1 were included in training. HBLUP was only marginally better than GBLUP. Correlations ≥0.80 were obtained between pedigree and genomic individual ranks. Rank coincidence of the top 2.5% selections was the highest for GBLUP_G (45% to 60%) compared to GBLUP_G+D. To advance the pure species RRS populations, GS models were best when trained on pure species than hybrid data, and HBLUP yielded ~20% higher predictive abilities than GBLUP, but was not better than ABLUP for ungenotyped trees. Discussion: We demonstrate that genomic data effectively enable accurate ranking of eucalypt hybrid seedlings for their yet-to-be observed volume growth at harvest age. Our results support a two-stage GS approach involving family selection by average genomic breeding value, followed by within-top-families individual GS, significantly increasing selection intensity, optimizing genotyping costs, and accelerating RRS breeding. [ABSTRACT FROM AUTHOR]

Eucalyptus is an economically important genus comprising more than 890 species in different subgenera and sections. Approximately twenty species of subgenus Symphyomyrtus account for 95% of the world's planted eucalypts. Discrimination of closely related eucalypt taxa is challenging, consistent with their recent phylogenetic divergence and occasional hybridization in nature. Admixture, misclassification or mislabeling of Eucalyptus germplasm resources maintained as exotics have been suggested, although no reports are available. Moreover, hybrids with increased productivity and traits complementarity are planted worldwide, but little is known about their actual genomic ancestry. In this study we examined a set of 440 trees of 16 different Eucalyptus species and 44 interspecific hybrids of multi-species origin conserved in germplasm banks in Brazil. We used genome-wide SNP data to evaluate the agreement between the alleged phylogenetic classification of species and provenances as registered in their historical records, and their observed genetic clustering derived from SNP data. Genetic structure analyses correctly assigned each of the 16 species to a different cluster although the PCA positioning of E. longirostrata was inconsistent with its current taxonomy. Admixture was present for closely related species' materials derived from local germplasm banks, indicating unintended hybridization following germplasm introduction. Provenances could be discriminated for some species, indicating that SNP-based discrimination was directly proportional to geographical distance, consistent with an isolation-by-distance model. SNP-based genomic ancestry analysis showed that the majority of the hybrids displayed realized genomic composition deviating from the expected ones based on their pedigree records, consistent with admixture in their parents and pervasive genome-wide directional selection toward the fast-growing E. grandis genome. SNP data in support of tree breeding provide precise germplasm identity verification, and allow breeders to objectively recognize the actual ancestral origin of superior hybrids to more realistically guide the program toward the development of the desired genetic combinations. [ABSTRACT FROM AUTHOR]

Genomic selection (GS) is poised to revolutionize eucalypt tree improvement by shortening breeding cycles and increasing selection intensities. This could be particularly valuable for alternative, non-mainstream Eucalyptus species that are still in the initial stages of breeding. Eucalyptus benthamii is important for its adaptation to frost-prone subtropical regions. In this work, we compared seven genomic prediction models, six Bayesian and one frequentist GBLUP (Genomic Best Linear Unbiased Prediction) with the conventional pedigree-based ABLUP approach. Models were evaluated for their ability to estimate heritabilities and predict wood quality traits (wood density, extractives, lignin, and carbohydrates content) and volume growth in 77 open-pollinated families of Eucalyptus benthamii. We also evaluated predictive abilities and heritabilities using variable numbers of SNP in the models. Heritabilities ranged from 0.09 (extractives content) using Bayesian Lasso (BL) to 0.55 (wood density) using ABLUP. Predictive abilities (PA) ranged from 0.12 (for volume using ABLUP) to 0.44 (for wood density using three Bayesian models). All seven genomic models performed similarly well and better than the pedigree model for all traits, except extractives content. Subsets of 5000–7000 SNPs yielded heritabilities and PAs nearly as large as using all 15,293 SNPs. However, a low-density SNP panel might not be economically and technically advantageous compared to the current high-density multi-species Eucalyptus EUCHIP60k. Our results support a positive outlook to implement GS to accelerate Eucalyptus benthamii breeding for adaptation to frost-prone regions. [ABSTRACT FROM AUTHOR]

Tropical fruit tree species constitute a yet untapped supply of outstanding diversity of taste and nutritional value, barely developed from the genetics standpoint, with scarce or no genomic resources to tackle the challenges arising in modern breeding practice. We generated a de novo genome assembly of the Psidium guajava, the super fruit "apple of the tropics", and successfully transferred 14,268 SNP probesets from Eucalyptus to Psidium at the nucleotide level, to detect genomic loci linked to resistance to the root knot nematode (RKN) Meloidogyne enterolobii derived from the wild relative P. guineense. Significantly associated loci with resistance across alternative analytical frameworks, were detected at two SNPs on chromosome 3 in a pseudo-assembly of Psidium guajava genome built using a syntenic path approach with the Eucalyptus grandis genome to determine the order and orientation of the contigs. The P. guineense-derived resistance response to RKN and disease onset is conceivably triggered by mineral nutrients and phytohormone homeostasis or signaling with the involvement of the miRNA pathway. Hotspots of mapped resistance quantitative trait loci and functional annotation in the same genomic region of Eucalyptus provide further indirect support to our results, highlighting the evolutionary conservation of genomes across genera of Myrtaceae in the adaptation to pathogens. Marker assisted introgression of the resistance loci mapped should accelerate the development of improved guava cultivars and hybrid rootstocks. [ABSTRACT FROM AUTHOR]

Cacao is a globally important crop with a long history of domestication and selective breeding. Despite the increased use of elite clones by cacao farmers, worldwide plantations are established mainly using hybrid progeny material derived from heterozygous parents, therefore displaying high tree-to-tree variability. The deliberate development of hybrids from advanced inbred lines produced by successive generations of self-pollination has not yet been fully considered in cacao breeding. This is largely due to the self-incompatibility of the species, the long generation cycles (3–5 years) and the extensive trial areas needed to accomplish the endeavor. We propose a simple and accessible approach to develop inbred lines based on accelerating the buildup of homozygosity based on regular selfing assisted by genome-wide SNP genotyping. In this study we genotyped 90 clones from the Brazilian CEPEC´s germplasm collection and 49 inbred offspring of six S1 or S2 cacao families derived from self-pollinating clones CCN-51, PS-13.19, TSH-1188 and SIAL-169. A set of 3,380 SNPs distributed across the cacao genome were interrogated on the EMBRAPA multi-species 65k Infinium chip. The 90 cacao clones showed considerable variation in genome-wide SNP homozygosity (mean 0.727± 0.182) and 19 of them with homozygosity ≥90%. By assessing the increase in homozygosity across two generations of self-pollinations, SNP data revealed the wide variability in homozygosity within and between S1 and S2 families. Even in small families (<10 sibs), individuals were identified with up to ~1.5 standard deviations above the family mean homozygosity. From baseline homozygosities of 0.476 and 0.454, offspring with homozygosities of 0.862 and 0.879 were recovered for clones TSH-1188 and CCN-51 respectively, in only two generations of selfing (81–93% increase). SNP marker assisted monitoring and selection of inbred individuals can be a practical tool to optimize and accelerate the development of inbred lines of outbred tree species. This approach will allow a faster and more accurate exploitation of hybrid breeding strategies in cacao improvement programs and potentially in other perennial fruit and forest trees. [ABSTRACT FROM AUTHOR]

Twelve years have passed since the early outlooks of applying genomic selection (GS) to forest tree breeding, initially based on deterministic simulations, soon followed by empirical reports. Given its solid projections for causing a paradigm shift in tree breeding practice in the years to come, GS went from a hot, somewhat hyped, topic to a fast-moving area of applied research and operational implementation worldwide. The hype cycle curve of emerging technologies introduced by Gartner Inc. in 1995, models the path a technology takes in terms of expectations of its value through time. Starting with a sudden and excessively positive "peak of inflated expectations" at its introduction, a technology that survives the "valley of disappointment" moves into maturity to climb the "slope of enlightenment", to eventually reach the "plateau of productivity". Following the pioneering steps of GS in animal breeding, we have surpassed the initial phases of the Gartner hype cycle and we are now climbing the slope of enlightenment towards a wide application of GS in forest tree breeding. By merging modern high-throughput DNA typing, time-proven quantitative genetics and mixed-model analysis, GS moved the focus away from the questionable concept of dissecting a complex, polygenic trait in its individual components for breeding advancement. Instead of trying to find the needle in a haystack, i.e., the "magic" gene in the complex and fluid genome, GS more efficiently and humbly "buys the whole haystack" of genomic effects to predict complex phenotypes, similarly to an exchange-traded fund that more efficiently "buys the whole market". Tens of studies have now been published in forest trees showing that GS matches or surpasses the performance of phenotypic selection for growth and wood properties traits, enhancing the rate of genetic gain per unit time by increasing selection intensity, radically reducing generation interval and improving the accuracy of breeding values. Breeder-friendly and cost-effective SNP (single nucleotide polymorphism) genotyping platforms are now available for all mainstream plantation forest trees, but methods based on low-pass whole genome sequencing with imputation might further reduce genotyping costs. In this perspective, I provide answers to why GS will soon become the most efficient and effective way to carry out advanced tree breeding, and outline a simple pilot demonstration project that tree breeders can propose in their organization. While the fundamental properties of GS in tree breeding are now solidly established, strategic, logistics and financial aspects for the optimized adoption of GS are now the focus of attentions towards the plateau of productivity in the cycle, when this new breeding method will become fully established into routine tree improvement. [ABSTRACT FROM AUTHOR]

The unique adaptation of Eucalyptus benthamii to low temperatures coupled to fast growth and versatile wood quality has made it a valued plantation species in frost-prone areas worldwide, but little is known on its quantitative genetic parameters for key industrial traits. We used GBLUP additive (GA), additive-dominant (GAD), single-step (HBLUP), and pedigree-based predictive models to estimate lignin, extractives, carbohydrates, and wood density at age 4 and tree volume at age 6. By capturing hidden relatedness and correcting pedigree errors, SNP data disentangled non-additive from additive variance providing more realistic estimates of narrow-sense heritability than pedigrees, and more accurate predictions of trait values. Predictive abilities (PAs) ranged from 0.12 for volume (pedigree-based model) to 0.44 for wood density (models H, GA, and GAD). Considerable dominance variance was seen for all traits, growth was the trait most influenced by it, resulting in PAs 48.9% higher when this effect is considered, a result with important consequences both for clonal propagation and overall selection efficiency (Seff). Using a HBLUP model, phenotypes of non-genotyped trees increased PAs by increasing sample size and provided realized relationships with reduced genotyping cost. In a recurrent selection program, the preclusion of progeny testing provides an increase in Seff between 232% and 299%. In a clonal selection program, the elimination of both progeny and initial clonal trial may increase Seff between 134% and 277%. Increasing selection intensity by genomic prediction resulted in an additional impact on Seff. This study provides groundwork to implement genomic selection in E. benthamii breeding. [ABSTRACT FROM AUTHOR]

Corymbia citriodora is a member of the predominantly Southern Hemisphere Myrtaceae family, which includes the eucalypts (Eucalyptus, Corymbia and Angophora; ~800 species). Corymbia is grown for timber, pulp and paper, and essential oils in Australia, South Africa, Asia, and Brazil, maintaining a high-growth rate under marginal conditions due to drought, poor-quality soil, and biotic stresses. To dissect the genetic basis of these desirable traits, we sequenced and assembled the 408 Mb genome of Corymbia citriodora, anchored into eleven chromosomes. Comparative analysis with Eucalyptus grandis reveals high synteny, although the two diverged approximately 60 million years ago and have different genome sizes (408 vs 641 Mb), with few large intra-chromosomal rearrangements. C. citriodora shares an ancient whole-genome duplication event with E. grandis but has undergone tandem gene family expansions related to terpene biosynthesis, innate pathogen resistance, and leaf wax formation, enabling their successful adaptation to biotic/abiotic stresses and arid conditions of the Australian continent. Healey and colleagues presented a reference sequence assembly of Corymbia citriodora (spotted gum), a tree which is crucial for timber, pulp, and paper, as well as carbon sequestration and essential oil production. [ABSTRACT FROM AUTHOR]

Key message: We propose the application of enviromics to breeding practice, by which the similarity among sites assessed on an "omics" scale of environmental attributes drives the prediction of unobserved genotype performances. Genotype by environment interaction (GEI) studies in plant breeding have focused mainly on estimating genetic parameters over a limited number of experimental trials. However, recent geographic information system (GIS) techniques have opened new frontiers for better understanding and dealing with GEI. These advances allow increasing selection accuracy across all sites of interest, including those where experimental trials have not yet been deployed. Here, we introduce the term enviromics, within an envirotypic-assisted breeding framework. In summary, likewise genotypes at DNA markers, any particular site is characterized by a set of "envirotypes" at multiple "enviromic" markers corresponding to environmental variables that may interact with the genetic background, thus providing informative breeding re-rankings for optimized decisions over different environments. Based on simulated data, we illustrate an index-based enviromics method (the "GIS–GEI") which, due to its higher granular resolution than standard methods, allows for: (1) accurate matching of sites to their most appropriate genotypes; (2) better definition of breeding areas that have high genetic correlation to ensure selection gains across environments; and (3) efficient determination of the best sites to carry out experiments for further analyses. Environmental scenarios can also be optimized for productivity improvement and genetic resources management, especially in the current outlook of dynamic climate change. Envirotyping provides a new class of markers for genetic studies, which are fairly inexpensive, increasingly available and transferable across species. We envision a promising future for the integration of enviromics approaches into plant breeding when coupled with next-generation genotyping/phenotyping and powerful statistical modeling of genetic diversity. [ABSTRACT FROM AUTHOR]

Main conclusion: Bignoniaceae species have conserved chloroplast structure, with hotspots of nucleotide diversity. Several genes are under positive selection, and can be targets for evolutionary studies. Bignoniaceae is one of the most species-rich family of woody plants in Neotropical seasonally dry forests. Here we report the assembly of Handroanthus impetiginosus chloroplast genome and evolutionary comparative analyses of ten Bignoniaceae species representing the genera for which whole-genome chloroplast sequences were available. The chloroplast genome of H. impetiginosus is 159,462 bp in size and has a similar structure compared to the other nine species. The total number of genes was slightly variable amongst the Bignoniaceae, ranging from 124 in H. impetiginosus to 144 in Anemopaegma acutifolium. The inverted repeat (IR) size was variable, ranging from 24,657 bp (Tecomaria capensis) to 40,481 bp (A. acutifolium), due to the contraction and retraction at its boundaries. However, gene boundaries were very similar among the ten species. We found 98 forward and palindromic dispersed repeats, and 85 simple sequence repeats (SSRs). In general, chloroplast sequences were highly conserved, with few nucleotide diversity hotspots in the genes accD, clpP, rpoA, ycf1, ycf2. The phylogenetic analysis based on 77 coding genes was highly consistent with Angiosperm Phylogeny Group (APG) IV. Our results also indicate that most genes are under negative selection or neutral evolution. We found no evidence of branch-site selection, implying that H. impetiginosus is not evolving faster than the other species analyzed, notwithstanding we found site positive selection signal in several genes. These genes can provide targets for evolutionary studies in Bignoniaceae and Lamiales species. [ABSTRACT FROM AUTHOR]

High-throughput SNP genotyping has become a precondition to move to higher precision and wider genome coverage genetic analysis of natural and breeding populations of non-model species. We developed a 44,318 annotated SNP catalog for Araucaria angustifolia, a grandiose subtropical conifer tree, one of the only two native Brazilian gymnosperms, critically endangered due to its valuable wood and seeds. Following transcriptome assembly and annotation, SNPs were discovered from RNA-seq and pooled RAD-seq data. From the SNP catalog, an Axiom® SNP array with 3,038 validated SNPs was developed and used to provide a comprehensive look at the genetic diversity and structure of 15 populations across the natural range of the species. RNA-seq was a far superior source of SNPs when compared to RAD-seq in terms of conversion rate to polymorphic markers on the array, likely due to the more efficient complexity reduction of the huge conifer genome. By matching microsatellite and SNP data on the same set of A. angustifolia individuals, we show that SNPs reflect more precisely the actual genome-wide patterns of genetic diversity and structure, challenging previous microsatellite-based assessments. Moreover, SNPs corroborated the known major north-south genetic cline, but allowed a more accurate attribution to regional versus among-population differentiation, indicating the potential to select ancestry-informative markers. The availability of a public, user-friendly 3K SNP array for A. angustifolia and a catalog of 44,318 SNPs predicted to provide ~29,000 informative SNPs across ~20,000 loci across the genome, will allow tackling still unsettled questions on its evolutionary history, toward a more comprehensive picture of the origin, past dynamics and future trend of the species' genetic resources. Additionally, but not less importantly, the SNP array described, unlocks the potential to adopt genomic prediction methods to accelerate the still very timid efforts of systematic tree breeding of A. angustifolia. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

The current study investigated the association between vitamin-D-receptor (VDR) genotypes with bone-mineral density (BMD) and its interaction with physical activity level (PAL). Individuals in a sample of 192 volunteers (67.84 ± 5.23 years) underwent BMD evaluation and were genotyped for VDR ApaI, BsmI, FokI, and TaqI polymorphisms. Haplotypes were reconstructed through expectation-maximization algorithm, and regression-based haplotype-specific association tests were performed with studied phenotypes. None of the polymorphisms were associated with BMD at any site; however, haplotype was associated with femoral-neck and Ward's-triangle BMD. Interaction between PAL and VDR genotypes was significant for the FokI polymorphism at femoral-neck and Ward's-triangle BMD. The FokI T/T genotype was associated with higher BMD in active women. It was concluded that VDR haplotypes, but not genotypes, are associated with femoral-neck and Ward's-triangle BMD in postmenopausal women. Moreover, the results suggest that VDR FokI polymorphism might be a potential determinant of BMD response to physical activity. [ABSTRACT FROM AUTHOR]

A thorough understanding of the heritability, genetic correlations and additive and non-additive variance components of tree growth and wood properties is a requisite for effective tree breeding. This knowledge is essential to maximize genetic gain, that is, the amount of increase in trait performance achieved annually through directional selection. Understanding the genetic attributes of traits targeted by breeding is also important to sustain decade-long genetic progress, that is, the progress made by increasing the average genetic value of the offspring as compared to that of the parental generation. In this study, we report quantitative genetic parameters for fifteen growth, wood chemical and physical traits for the world-famous Eucalyptus urograndis hybrid (E. grandis × E. urophylla). These traits directly impact the optimal use of wood for cellulose pulp, paper, and energy production. A population of 1,000 trees sampled in a progeny trial was phenotyped directly or following the development and use of near-infrared spectroscopy calibration models. Trees were genotyped with 33,398 SNPs and 24,001 DArT-seq genome-wide markers and genomic realized relationship matrices (GRM) were used for parameter estimation with an individual-tree additive-dominant mixed model. Wood chemical properties and wood density showed stronger genetic control than growth, cellulose and fiber traits. Additive effects are the main drivers of genetic variation for all traits, but dominance plays an equally or more important role for growth, singularly in this hybrid. GRM´s with >10,000 markers provided stable relationships estimates and more accurate parameters than pedigrees by capturing the full genetic relationships among individuals and disentangling the non-additive from the additive genetic component. Low correlations between growth and wood properties indicate that simultaneous selection for wood traits can be applied with minor effects on genetic gain for growth. Conversely, moderate to strong correlations between wood density and chemical traits exist, likely due to their interdependency on cell wall structure such that responses to selection will be connected for these traits. Our results illustrate the advantage of using genome-wide marker data to inform tree breeding in general and have important consequences for operational breeding of eucalypt urograndis hybrids. [ABSTRACT FROM AUTHOR]

Lignin syringyl/guaiacyl (S/G) ratio is a key trait in the cellulose pulping industry. Near-infrared (NIR) spectroscopy data collected with a bench and a portable instrument were used to develop prediction models for S/G ratio from sawdust samples of 1220 trees of two contrasting Eucalyptus species, namely E. benthamii (n = 484), which is a temperate, cold-resistant species, and E. pellita (n = 736), which is a heat- and disease-resistant tropical species. For each species, samples were selected based on maximal NIR spectral variation and analyzed for S/G using a streamlined thioacidolysis method with minimal sample input. NIR models were developed for each species separately and jointly. Instead of just using the coefficient of determination (R2) and the ratio of performance to deviation (RPD), the Spearman's rank correlation (rs) and the average of the coefficient of correlation between the references and predicted values (CVRP) were taken into account to evaluate the models. The bench spectrometer had a better performance than the portable instrument (R2 from 0.77 to 0.86 versus 0.31 to 0.47). Species-specific NIR models were better for E. benthamii (rs = 0.89, CVRP = 2.75%, R2 = 0.86 and RPD = 1.8), while the joint-species model was better for E. pellita (rs = 0.97, CVRP = 3.98%, R2 = 0.82 and RPD = 2.1). These NIR models should prove useful for high-throughput wood phenotyping in advanced breeding programs. [ABSTRACT FROM AUTHOR]

Summary: Genome‐wide association studies (GWAS) in plants typically suffer from limited statistical power. An alternative to the logistical and cost challenge of increasing sample sizes is to gain power by meta‐analysis using information from independent studies.We carried out GWAS for growth traits with six single‐marker models and regional heritability mapping (RHM) in four Eucalyptus breeding populations independently and by Joint‐GWAS, using gene and segment‐based models, with data for 3373 individuals genotyped with a communal EUChip60KSNP platform.While single‐single nucleotide polymorphism (SNP) GWAS hardly detected significant associations at high‐stringency in each population, gene‐based Joint‐GWAS revealed nine genes significantly associated with tree height. Associations detected using single‐SNP GWAS, RHM and Joint‐GWAS set‐based models explained on average 3–20% of the phenotypic variance. Whole‐genome regression, conversely, captured 64–89% of the pedigree‐based heritability in all populations. Several associations independently detected for the same SNPs in different populations provided unprecedented GWAS validation results in forest trees. Rare and common associations were discovered in eight genes involved in cell wall biosynthesis and lignification.With the increasing adoption of genomic prediction of complex phenotypes using shared SNPs and much larger tree breeding populations, Joint‐GWAS approaches should provide increasing power to pinpoint discrete associations potentially useful toward tree breeding and molecular applications. [ABSTRACT FROM AUTHOR]

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Woody perennial angiosperms (i.e., hardwood trees) are polyphyletic in origin and occur in most angiosperm orders. Despite their independent origins, hardwoods have shared physiological, anatomical, and life history traits distinct from their herbaceous relatives. New high-throughput DNA sequencing platforms have provided access to numerous woody plant genomes beyond the early reference genomes of Populus and Eucalyptus , references that now include willow and oak, with pecan and chestnut soon to follow. Genomic studies within these diverse and undomesticated species have successfully linked genes to ecological, physiological, and developmental traits directly. Moreover, comparative genomic approaches are providing insights into speciation events while large-scale DNA resequencing of native collections is identifying population-level genetic diversity responsible for variation in key woody plant biology across and within species. Current research is focused on developing genomic prediction models for breeding, defining speciation and local adaptation, detecting and characterizing somatic mutations, revealing the mechanisms of gender determination and flowering, and application of systems biology approaches to model complex regulatory networks underlying quantitative traits. Emerging technologies such as single-molecule, long-read sequencing is being employed as additional woody plant species, and genotypes within species, are sequenced, thus enabling a comparative ("evo-devo") approach to understanding the unique biology of large woody plants. Resource availability, current genomic and genetic applications, new discoveries and predicted future developments are illustrated and discussed for poplar, eucalyptus, willow, oak, chestnut, and pecan. [ABSTRACT FROM AUTHOR]

Forest tree breeding has been successful at delivering genetically improved material for multiple traits based on recurrent cycles of selection, mating, and testing. However, long breeding cycles, late flowering, variable juvenile-mature correlations, emerging pests and diseases, climate, and market changes, all pose formidable challenges. Genetic dissection approaches such as quantitative trait mapping and association genetics have been fruitless to effectively drive operational marker-assisted selection (MAS) in forest trees, largely because of the complex multifactorial inheritance of most, if not all traits of interest. The convergence of high-throughput genomics and quantitative genetics has established two new paradigms that are changing contemporary tree breeding dogmas. Genomic selection (GS) uses large number of genome-wide markers to predict complex phenotypes. It has the potential to accelerate breeding cycles, increase selection intensity and improve the accuracy of breeding values. Realized genomic relationships matrices, on the other hand, provide innovations in genetic parameters' estimation and breeding approaches by tracking the variation arising from random Mendelian segregation in pedigrees. In light of a recent flow of promising experimental results, here we briefly review the main concepts, analytical tools and remaining challenges that currently underlie the application of genomics data to tree breeding. With easy and cost-effective genotyping, we are now at the brink of extensive adoption of GS in tree breeding. Areas for future GS research include optimizing strategies for updating prediction models, adding validated functional genomics data to improve prediction accuracy, and integrating genomic and multi-environment data for forecasting the performance of genetic material in untested sites or under changing climate scenarios. The buildup of phenotypic and genome-wide data across large-scale breeding populations and advances in computational prediction of discrete genomic features should also provide opportunities to enhance the application of genomics to tree breeding. [ABSTRACT FROM AUTHOR]

Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction. [ABSTRACT FROM AUTHOR]

Targeted sequence capture coupled to high-throughput sequencing has become a powerful method for the study of genome-wide sequence variation. Following our recent development of a genome assembly for the Pink Ipê tree (Handroanthus impetiginosus), a widely distributed Neotropical timber species, we now report the development of a set of 24,751 capture probes for single-nucleotide polymorphisms (SNPs) characterization and genotyping across 18,216 distinct loci, sampling more than 10 Mbp of the species genome. This system identifies nearly 200,000 SNPs located inside or in close proximity to almost 14,000 annotated protein-coding genes, generating quality genotypic data in populations spanning wide geographic distances across the species native range. To provide recommendations for future developments of similar systems for highly heterozygous plant genomes we investigated issues such as probe design, sequencing coverage and bioinformatics, including the evaluation of the capture efficiency and a reassessment of the technical reproducibility of the assay for SNPs recall and genotyping precision. Our results highlight the value of a detailed probe screening on a preliminary genome assembly to produce reliable data for downstream genetic studies. This work should inspire and assist the development of similar genomic resources for other orphan crops and forest trees with highly heterozygous genomes. [ABSTRACT FROM AUTHOR]

Background: Handroanthus impetiginosus (Mart. ex DC.) Mattos is a keystone Neotropical hardwood tree widely distributed in seasonally dry tropical forests of South and Mesoamerica. Regarded as the "new mahogany," it is the second most expensive timber, the most logged species in Brazil, and currently under significant illegal trading pressure. The plant produces large amounts of quinoids, specialized metabolites with documented antitumorous and antibiotic effects. The development of genomic resources is needed to better understand and conserve the diversity of the species, to empower forensic identification of the origin of timber, and to identify genes for important metabolic compounds. Findings: The genome assembly covers 503.7 Mb (N50 = 81 316 bp), 90.4% of the 557-Mbp genome, with 13 206 scaffolds. A repeat database with 1508 sequences was developed, allowing masking of ~31% of the assembly. Depth of coverage indicated that consensus determination adequately removed haplotypes assembled separately due to the extensive heterozygosity of the species. Automatic gene prediction provided 31 688 structures and 35 479 messenger RNA transcripts, while external evidence supported a well-curated set of 28 603 high-confidence models (90% of total). Finally, we used the genomic sequence and the comprehensive gene content annotation to identify genes related to the production of specialized metabolites. Conclusions: This genome assembly is the first well-curated resource for a Neotropical forest tree and the first one for a member of the Bignoniaceae family, opening exceptional opportunities to empower molecular, phytochemical, and breeding studies. This work should inspire the development of similar genomic resources for the largely neglected forest trees of the mega-diverse tropical biomes. [ABSTRACT FROM AUTHOR]

Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a human pathogen that causes diverse human diseases including streptococcal toxic shock syndrome (STSS). A GAS outbreak occurred in Brasilia, Brazil, during the second half of the year 2011, causing 26 deaths. Whole genome sequencing was performed using Illumina platform. The sequences were assembled and genes were predicted for comparative analysis with emm type 1 strains: MGAS5005 and M1 GAS. Genomics comparison revealed one of the invasive strains that differ from others isolates and from emm 1 reference genomes. Also, the new invasive strain showed differences in the content of virulence factors compared to other isolated in the same outbreak. The evolution of contemporary GAS strains is strongly associated with horizontal gene transfer. This is the first genomic study of a Streptococcal emm 1 outbreak in Brazil, and revealed the rapid bacterial evolution leading to new clones. The emergence of new invasive strains can be a consequence of the injudicious use of antibiotics in Brazil during the past decades. [ABSTRACT FROM AUTHOR]

Background: The advent of high-throughput genotyping technologies coupled to genomic prediction methods established a new paradigm to integrate genomics and breeding. We carried out whole-genome prediction and contrasted it to a genome-wide association study (GWAS) for growth traits in breeding populations of Eucalyptus benthamii (n =505) and Eucalyptus pellita (n =732). Both species are of increasing commercial interest for the development of germplasm adapted to environmental stresses. Results: Predictive ability reached 0.16 in E. benthamii and 0.44 in E. pellita for diameter growth. Predictive abilities using either Genomic BLUP or different Bayesian methods were similar, suggesting that growth adequately fits the infinitesimal model. Genomic prediction models using ~5000-10,000 SNPs provided predictive abilities equivalent to using all 13,787 and 19,506 SNPs genotyped in the E. benthamii and E. pellita populations, respectively. No difference was detected in predictive ability when different sets of SNPs were utilized, based on position (equidistantly genome-wide, inside genes, linkage disequilibrium pruned or on single chromosomes), as long as the total number of SNPs used was above ~5000. Predictive abilities obtained by removing relatedness between training and validation sets fell near zero for E. benthamii and were halved for E. pellita. These results corroborate the current view that relatedness is the main driver of genomic prediction, although some short-range historical linkage disequilibrium (LD) was likely captured for E. pellita. A GWAS identified only one significant association for volume growth in E. pellita, illustrating the fact that while genome-wide regression is able to account for large proportions of the heritability, very little or none of it is captured into significant associations using GWAS in breeding populations of the size evaluated in this study. Conclusions: This study provides further experimental data supporting positive prospects of using genome-wide data to capture large proportions of trait heritability and predict growth traits in trees with accuracies equal or better than those attainable by phenotypic selection. Additionally, our results document the superiority of the whole-genome regression approach in accounting for large proportions of the heritability of complex traits such as growth in contrast to the limited value of the local GWAS approach toward breeding applications in forest trees. [ABSTRACT FROM AUTHOR]

Background: Genomic prediction is a genomics assisted breeding methodology that can increase genetic gains by accelerating the breeding cycle and potentially improving the accuracy of breeding values. In this study, we use 41,304 informative SNPs genotyped in a Eucalyptus breeding population involving 90 E.grandis and 78 E.urophylla parents and their 949 F1 hybrids to develop genomic prediction models for eight phenotypic traits - basic density and pulp yield, circumference at breast height and height and tree volume scored at age three and six years. We assessed the impact of different genomic prediction methods, the composition and size of the training and validation set and the number and genomic location of SNPs on the predictive ability (PA). Results: Heritabilities estimated using the realized genomic relationship matrix (GRM) were considerably higher than estimates based on the expected pedigree, mainly due to inconsistencies in the expected pedigree that were readily corrected by the GRM. Moreover, the GRM more precisely capture Mendelian sampling among related individuals, such that the genetic covariance was based on the true proportion of the genome shared between individuals. PA improved considerably when increasing the size of the training set and by enhancing relatedness to the validation set. Prediction models trained on pure species parents could not predict well in F1 hybrids, indicating that model training has to be carried out in hybrid populations if one is to predict in hybrid selection candidates. The different genomic prediction methods provided similar results for all traits, therefore either GBLUP or rrBLUP represents better compromises between computational time and prediction efficiency. Only slight improvement was observed in PA when more than 5000 SNPs were used for all traits. Using SNPs in intergenic regions provided slightly better PA than using SNPs sampled exclusively in genic regions. Conclusions: The size and composition of the training set and number of SNPs used are the two most important factors for model prediction, compared to the statistical methods and the genomic location of SNPs. Furthermore, training the prediction model based on pure parental species only provide limited ability to predict traits in interspecific hybrids. Our results provide additional promising perspectives for the implementation of genomic prediction in Eucalyptus breeding programs by the selection of interspecific hybrids. [ABSTRACT FROM AUTHOR]

• Although genome-wide association studies (GWAS) have provided valuable insights into the decoding of the relationships between sequence variation and complex phenotypes, they have explained little heritability. Regional heritability mapping (RHM) provides heritability estimates for genomic segments containing both common and rare allelic effects that individually contribute too little variance to be detected by GWAS. • We carried out GWAS and RHM for seven growth, wood and disease resistance traits in a breeding population of 768 Eucalyptus hybrid trees using EuCHIP60K. Total genomic heritabilities accounted for large proportions (64-89%) of pedigree-based trait heritabilities, providing additional evidence that complex traits in eucalypts are controlled by many sequence variants across the frequency spectrum, each with small contributions to the phenotypic variance. • RHM detected 26 quantitative trait loci (QTLs) encompassing 2191 single nucleotide polymorphisms (SNPs), whereas GWAS detected 13 single SNP-trait associations. RHM and GWAS QTLs individually explained 5-15% and 4-6% of the genomic heritability, respectively. RHM was superior to GWAS in capturing larger proportions of genomic heritability. Equated to previously mapped QTLs, our results highlighted genomic regions for further examination towards gene discovery. • RHM-QTLs bearing a combination of common and rare variants could be useful enhancements to incorporate prior knowledge of the underlying genetic architecture in genomic prediction models. [ABSTRACT FROM AUTHOR]

The genome-wide heterozygosity at 9590 genes, all heterozygous in a single Eucalyptus grandis parent tree, was examined in a group of 28 S1 offspring. Heterozygosity ranged from 52-79%, averaging 65.5%, much higher than the 50% expected under random segregation, supporting the occurrence of strong (47%) selection against homozygosity. The expected pattern of heterozygosity from theoretical calculations and simulations for recessive detrimentals (pseudo-overdominance) and intrinsic heterozygote advantage was examined and compared with that observed. The observed patterns are consistent with at least several detrimental loci with large effects on both parental chromosomes of the 11 pairs. It is likely that 100 or more genes, many with substantial effects on viability, are contributing to this inbreeding depression. Although our genome-wide analysis of nearly 10 000 genes strongly suggested that pseudo-overdominance was responsible for the observed high inbreeding depression, heterozygote advantage could not be excluded. Finding inconvertible evidence of the cause of inbreeding depression still presents a difficult challenge. This study is the first theoretical examination of the genomic effect of inbreeding in a forest tree and provides an approach to analyze these data to determine the extent and cause of inbreeding depression across other plant genomes. [ABSTRACT FROM AUTHOR]

Background: Micro RNAs are a class of small non coding RNAs of 20-24 nucleotides transcribed as single stranded precursors from MIR gene loci. Initially described as post-transcriptional regulators involved in development, two decades ago, miRNAs have been proven to regulate a wide range of processes in plants such as germination, morphology and responses to biotic and abiotic stress. Despite wide conservation in plants, a number of miRNAs are lineage specific. We describe the first genome wide survey of Eucalyptus miRNAs based on high throughput sequencing. Results: In addition to discovering small RNA sequences, MIR loci were mapped onto the reference genome and interspecific variability investigated. Sequencing was carried out for the two most world widely planted species, E. grandis and E. globulus. To maximize discovery, E. grandis samples were from BRASUZ1, the same tree whose genome provided the reference sequence. Interspecific analysis reinforces the variability in small RNA repertoire even between closely related species. Characterization of Eucalyptus small RNA sequences showed 95 orthologous to conserved miRNAs and 193 novel miRNAs. In silico target prediction confirmed 163 novel miRNAs and degradome sequencing experimentally confirmed several hundred targets. Experimental evidence based on the exclusive expression of a set of small RNAs across 16 species within Myrtaceae further highlighted variable patterns of conservation and diversity of these regulatory elements. Conclusions: The description of miRNAs in Eucalyptus contributes to scientific knowledge of this vast genre, which is the most widely planted hardwood crop in the tropical and subtropical world, adding another important element to the annotation of Eucalyptus grandis reference genome. [ABSTRACT FROM AUTHOR]

We used high-density single nucleotide polymorphism ( SNP) data and whole-genome pooled resequencing to examine the landscape of population recombination ( ρ) and nucleotide diversity ( ϴw), assess the extent of linkage disequilibrium ( r2) and build the highest density linkage maps for Eucalyptus., At the genome-wide level, linkage disequilibrium ( LD) decayed within c. 4-6 kb, slower than previously reported from candidate gene studies, but showing considerable variation from absence to complete LD up to 50 kb. A sharp decrease in the estimate of ρ was seen when going from short to genome-wide inter- SNP distances, highlighting the dependence of this parameter on the scale of observation adopted. Recombination was correlated with nucleotide diversity, gene density and distance from the centromere, with hotspots of recombination enriched for genes involved in chemical reactions and pathways of the normal metabolic processes., The high nucleotide diversity ( ϴw = 0.022) of E. grandis revealed that mutation is more important than recombination in shaping its genomic diversity ( ρ/ ϴw = 0.645). Chromosome-wide ancestral recombination graphs allowed us to date the split of E. grandis (1.7-4.8 million yr ago) and identify a scenario for the recent demographic history of the species., Our results have considerable practical importance to Genome Wide Association Studies ( GWAS), while indicating bright prospects for genomic prediction of complex phenotypes in eucalypt breeding. [ABSTRACT FROM AUTHOR]

Pedigree reconstruction using molecular markers enables efficient management of inbreeding in open-pollinated breeding strategies, replacing expensive and time-consuming controlled pollination. This is particularly useful in preferentially outcrossed, insect pollinated Eucalypts known to suffer considerable inbreeding depression from related matings. A single nucleotide polymorphism (SNP) marker panel consisting of 106 markers was selected for pedigree reconstruction from the recently developed high-density Eucalyptus Infinium SNP chip (EuCHIP60K). The performance of this SNP panel for pedigree reconstruction in open-pollinated progenies of two Eucalyptus nitens seed orchards was compared with that of two microsatellite panels with 13 and 16 markers respectively. The SNP marker panel out-performed one of the microsatellite panels in the resolution power to reconstruct pedigrees and out-performed both panels with respect to data quality. Parentage of all but one offspring in each clonal seed orchard was correctly matched to the expected seed parent using the SNP marker panel, whereas parentage assignment to less than a third of the expected seed parents were supported using the 13-microsatellite panel. The 16-microsatellite panel supported all but one of the recorded seed parents, one better than the SNP panel, although there was still a considerable level of missing and inconsistent data. SNP marker data was considerably superior to microsatellite data in accuracy, reproducibility and robustness. Although microsatellites and SNPs data provide equivalent resolution for pedigree reconstruction, microsatellite analysis requires more time and experience to deal with the uncertainties of allele calling and faces challenges for data transferability across labs and over time. While microsatellite analysis will continue to be useful for some breeding tasks due to the high information content, existing infrastructure and low operating costs, the multi-species SNP resource available with the EuCHIP60k, opens a whole new array of opportunities for high-throughput, genome-wide or targeted genotyping in species of Eucalyptus. [ABSTRACT FROM AUTHOR]

We used whole genome resequencing of pooled individuals to develop a high-density single-nucleotide polymorphism ( SNP) chip for Eucalyptus. Genomes of 240 trees of 12 species were sequenced at 3.5× each, and 46 997 586 raw SNP variants were subject to multivariable filtering metrics toward a multispecies, genome-wide distributed chip content., Of the 60 904 SNPs on the chip, 59 222 were genotyped and 51 204 were polymorphic across 14 Eucalyptus species, providing a 96% genome-wide coverage with 1 SNP/12-20 kb, and 47 069 SNPs at ≤ 10 kb from 30 444 of the 33 917 genes in the Eucalyptus genome., Given the EUChip60K multi-species genotyping flexibility, we show that both the sample size and taxonomic composition of cluster files impact heterozygous call specificity and sensitivity by benchmarking against 'gold standard' genotypes derived from deeply sequenced individual tree genomes. Thousands of SNPs were shared across species, likely representing ancient variants arisen before the split of these taxa, hinting to a recent eucalypt radiation. We show that the variable SNP filtering constraints allowed coverage of the entire site frequency spectrum, mitigating SNP ascertainment bias., The EUChip60K represents an outstanding tool with which to address population genomics questions in Eucalyptus and to empower genomic selection, GWAS and the broader study of complex trait variation in eucalypts. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mass controlled pollination (MCP), involving large-scale application of pollen on physically isolated female reproductive organs, has been a lower cost alternative to controlled pollination for the commercial production of genetically improved seeds. Nevertheless, rare are the studies that examined the efficacy of operational MCP and no such assessment has been done in loblolly pine to date. The success of MCP was assessed by a microsatellite-based investigation of the realized versus expected parentage of a set of 300 Pinus taeda offspring in 19 families generated in two subsequent rounds of MCP in 2005 and 2006 in a clonal seed orchard in Brazil. Multi-locus combined probability of parentage exclusion both theoretical and realized from actual testing was >99 % for single parent and parent pair testing when using nine or ten markers. Parentage assignments carried out under a maximum likelihood framework revealed a significantly higher success rate of MCP in 2006 (84 %) following technical improvements adopted to minimize pollen contamination and maximize male reproductive success, although significant variability in the correct maternity and full parentage was seen among individual families. The observed patterns of unexpected parentage indicated that this variability likely resulted from mislabeling of clonal ramets of the parents used in the crosses which impacted both maternity and paternity. Preventing pollen contamination will not be sufficient for successful MCP if inaccuracies exist in the identity of the clonal plants that ultimately provide pollen and female strobili, showing that DNA marker auditing and correction of identity of all ramets in a clonal seed orchard should be a standard practice in the operational implementation of MCP. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The promise of association genetics to identify genes or genomic regions controlling complex traits has generated a flurry of interest. Such phenotype-genotype associations could be useful to accelerate tree breeding cycles, increase precision and selection intensity for late expressing, low heritability traits. However, the prospects of association genetics in highly heterozygous undomesticated forest trees can be severely impacted by the presence of cryptic population and pedigree structure. To investigate how to better account for this, we compared the GLM and five combinations of the Unified Mixed Model (UMM) on data of a low-density genome-wide association study for growth and wood property traits carried out in a Eucalyptus globulus population (n = 303) with 7,680 Diversity Array Technology (DArT) markers. Model comparisons were based on the degree of deviation from the uniform distribution and estimates of the mean square differences between the observed and expected p-values of all significant marker-trait associations detected. Our analysis revealed the presence of population and family structure. There was not a single best model for all traits. Striking differences in detection power and accuracy were observed among the different models especially when population structure was not accounted for. The UMM method was the best and produced superior results when compared to GLM for all traits. Following stringent correction for false discoveries, 18 marker-trait associations were detected, 16 for tree diameter growth and two for lignin monomer composition (S∶G ratio), a key wood property trait. The two DArT markers associated with S∶G ratio on chromosome 10, physically map within 1 Mbp of the ferulate 5-hydroxylase (F5H) gene, providing a putative independent validation of this marker-trait association. This study details the merit of collectively integrate population structure and relatedness in association analyses in undomesticated, highly heterozygous forest trees, and provides additional insights into the nature of complex quantitative traits in Eucalyptus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The status of genomics and genetics research in the Myrtaceae, a large family of dicotyledonous woody plants, is reviewed with Eucalyptus as the focal genus. The family contains over 5,650 species in 130 to 150 genera, predominantly of neo-tropical and Southern Hemisphere distribution. Several genera are well known for their economic importance worldwide. Myrtaceae are typically diploids with small to intermediate genome size. Microsatellites have been developed for several genera while higher throughput marker systems such as diversity arrays technology and single nucleotide polymorphism are available for Eucalyptus. Molecular data have been fundamental to current perspectives on the phylogeny, phylogeography and taxonomy of the Myrtaceae, while numerous studies of genetic diversity have been carried out particularly as it relates to endangered, rare, fragmented, overharvested or economically important species. Large expressed sequence tag collections for species of Eucalyptus have recently become public to support the annotation of the Eucalyptus grandis genome. Transcriptomics in Eucalyptus has advanced by microarrays and next-generation sequencing focusing on wood development. Linkage maps for Eucalyptus display high synteny across species and have been extensively used to map quantitative trait loci for a number of traits including growth, wood quality, disease and insect resistance. Candidate gene-based association genetics have successfully found marker-trait associations for wood and fiber traits. Genomic selection experiments have demonstrated clear potential to improve the efficiency of breeding programs while freeze-tolerant transgenic Eucalyptus trials have recently been initiated. The recently released E. grandis genome, sequenced to an average coverage of 8×, will open up exceptional opportunities to advance Myrtaceae genetics and genomics research. [ABSTRACT FROM AUTHOR]

Summary [ABSTRACT FROM AUTHOR]

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; ), 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. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Genomic selection (GS) involves selection decisions based on genomic breeding values estimated as the sum of the effects of genome-wide markers capturing most quantitative trait loci (QTL) for the target trait(s). GS is revolutionizing breeding practice in domestic animals. The same approach and concepts can be readily applied to forest tree breeding where long generation times and late expressing complex traits are also a challenge. GS in forest trees would have additional advantages: large training populations can be easily assembled and accurately phenotyped for several traits, and the extent of linkage disequilibrium (LD) can be high in elite populations with small effective population size ( N) frequently used in advanced forest tree breeding programs. Deterministic equations were used to assess the impact of LD (modeled by N and intermarker distance), the size of the training set, trait heritability, and the number of QTL on the predicted accuracy of GS. Results indicate that GS has the potential to radically improve the efficiency of tree breeding. The benchmark accuracy of conventional BLUP selection is reached by GS even at a marker density ~2 markers/cM when N ≤ 30, while up to 20 markers/cM are necessary for larger N. Shortening the breeding cycle by 50% with GS provides an increase ≥100% in selection efficiency. With the rapid technological advances and declining costs of genotyping, our cautiously optimistic outlook is that GS has great potential to accelerate tree breeding. However, further simulation studies and proof-of-concept experiments of GS are needed before recommending it for operational implementation. [ABSTRACT FROM AUTHOR]

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 Hind III and BstY I, 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 FROM AUTHOR]

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]