Desenvolvimento e aplicações de DArT (Diversity Arrays Technology) e genotipagem por sequenciamento (Genotyping-by-Sequencing) para análise genética em eucalyptus

Tese (doutorado)—Universidade de Brasília, Departamento de Biologia Celular, 2012.Espécies de Eucalyptus tem sido utilizadas com sucesso para plantios florestais devido ao seu rápido crescimento, sua capacidade de adaptação às diversas condições edafo-climáticas e pelo seu potencial econômico na produção de energia, fibra e madeira sólida, reduzindo assim a pressão sobre as florestas tropicais e a biodiversidade associada. Marcadores moleculares tais como RAPD, AFLP, microssatélites, e mais recentemente SFP e SNPs têm contribuído para a caracterização e conservação dos recursos genéticos do gênero Eucalyptus, auxiliando também na compreensão da evolução do gênero, além de permitir a construção de mapas de ligação e identificação de QTLs (Quantitative trait loci). Entretanto, estas técnicas são lentas, laboriosas, apresentam limitações de cobertura genômica e envolvem custos elevados para a análise de muitos indivíduos. Diversity Arrays Technology (DArT) é um método baseado em hibridização que permite genotipar centenas a milhares de marcadores num simples ensaio. Esta tecnologia gera um perfil genômico com um alto rendimento e um grande poder de transferibilidade entre espécies. Neste trabalho é apresentado o desenvolvimento da primeira plataforma de genotipagem de alto desempenho de marcadores DArTs em microarranjo para o gênero Eucalyptus, e demonstrada sua eficiência em estudos de diversidade genética, filogenia e mapeamento genético. Foram desenvolvidas 18 bibliotecas genômicas de complexidade reduzida a partir de 64 espécies diferentes do gênero. Um total de 23.808 fragmentos de DNA foram avaliados para revelação de polimorfismos DArT, e 13.300 (56%) destes declarados polimórficos entre um painel de triagem composto por 284 indivíduos. Destes, 7.680 marcadores foram selecionados para a construção de um microarranjo de genotipagem para uso em rotina. Em um estudo de diversidade intra-específica, 4.752 marcadores foram polimórficos e 5.013 mostraram segregação mendeliana em seis populações segregantes não relacionadas, com uma média de 2.211 marcadores polimórficos por população. Na etapa seguinte do trabalho, foi otimizada a tecnologia de genotipagem por sequenciamento DArT-seq para a construção de um mapa genético de alta densidade para uma população segregante do cruzamento entre árvores elite de E. grandis (BRASUZ1 x M4D31). A população foi genotipada com o microarranjo DArT e com a técnica DArTseq. Enquanto o microarranjo DArT forneceu 1.088 marcadores, a genotipagem DArT-seq forneceu 2.449. No total, um mapa de ligação integrado por 564 marcadores DArT, 1.930 marcadores DArTseq e 29 microssatélites foi construído. Além destes marcadores, mais de 1.500 SNPs derivados da metodologia DArT-seq foram obtidos proporcionando uma vantagem adicional pela inclusão de marcadores co-dominantes no mapa. O desenvolvimento de metodologias de genotipagem por sequenciamento (GbS) via enzimas de restrição como DArT-seq ou captura com sondas, representa uma aplicação adicional das tecnologias de "next generation sequencing" além do sequenciamento, 2 potencializando a análise genética com marcadores moleculares. A combinação do elevado número de marcadores, baixo custo, metodologia relativamente accessível e uso de reagentes universais, aponta para um uso crescente de GbS nos próximos anos nas mais diversas aplicações em estudos de genética de populações, investigações evolutivas e em apoio ao melhoramento acelerando e aumentando a precisão da seleção direcional de características multifatoriais complexas. _______________________________________________________________________________________ ABSTRACTSpecies of Eucalyptus have been successfully used for forest plantations due to its rapid growth, its ability to adapt to various soil and climatic conditions and its economic use in energy, fiber and solid wood, reducing pressure on tropical forests and associated biodiversity. Molecular markers such as RAPD, AFLP, microsatellites, and more recently SFP and SNPs have contributed to the characterization and conservation of genetic resources of the genus, to the understanding of the evolution of the genus, and allowing the construction of linkage and QTLs maps. However, these techniques are slow, laborious, provide limited genome coverage and are costly for the analysis of large sample sizes. Diversity Arrays Technology (DArT) is a hybridization-based method that allows genotyping hundreds to thousands of markers in a single assay. This technology generates a genomic profile with high throughput and transferability between species. This study presents the development of the first high throughput genotyping platform for species of Eucalyptus based on a DArT microarray and demonstrates its use for diversity, phylogeny and mapping studies. A total of 18 reduced representation genomic libraries from 64 different species of the genus were developed. A total of 23,808 DNA fragments were screened for polymorphism and 13,300 (56%) of them declared polymorphic in a panel of 284 individuals. Out of these, 7,680 markers were selected to populate a routine DArT genotyping microarray. In an inter-specific diversity study, 4,752 were deemed polymorphic while 5,013 showed Mendelian segregation when assessed in six inter-specific mapping pedigrees, with an average of 2,211 polymorphic markers per pedigree. The subsequent step of the study, involved the optimization of the genotyping-by-sequencing technology called DArT-seq to construct a high density genetic map for a segregating population derived from the E. grandis elite trees (BRASUZ1 x M4D31). The population was genotyped both with the DArT microarray and with DArT-seq. While the DArT microarray yielded 1,088 markers, the DArT-seq method supplied 2,449 markers. In total, an integrated linkage map with 564 DArT markers, 1,930 DArT-NGS markers and 29 microsatellites was built. Besides these mapped markers, an additional set of over 1,500 SNPs derived from DArT-seq were scored providing an additional advantage by the inclusion of co-dominant markers on the map. The development of genotyping by sequencing (GBS) 3 methods via restriction enzymes as DART-seq or capture probes, represents a further application of the technologies of "next generation sequencing" beyond sequencing, empowering the genetic analysis with molecular markers. The combination of the large number of markers, low cost, relatively accessible methodology and use of universal reagents, points to an increased use of GBS in the coming years in several applications in studies of population genetics, evolutionary investigations and in support to plant breeding accelerating and increasing accuracy of directional selection for complex multi-factorial traits

Labelling Selective Sweeps Used in Durum Wheat Breeding from a Diverse and Structured Panel of Landraces and Cultivars

A panel of 387 durum wheat genotypes including Mediterranean landraces and modern cultivars was characterized with 46,161 diversity arrays technology (DArTseq) markers. Analysis of population structure uncovered the existence of five subpopulations (SP) related to the pattern of migration of durum wheat from the domestication area to the west of the Mediterranean basin (SPs 1, 2, and 3) and further improved germplasm (SPs 4 and 5). The total genetic diversity (HT) was 0.40 with a genetic differentiation (GST) of 0.08 and a mean gene flow among SPs of 6.02. The lowest gene flow was detected between SP 1 (presumably the ancient genetic pool of the panel) and SPs 4 and 5. However, gene flow from SP 2 to modern cultivars was much higher. The highest gene flow was detected between SP 3 (western Mediterranean germplasm) and SP 5 (North American and European cultivars). A genome wide association study (GWAS) approach using the top ten eigenvectors as phenotypic data revealed the presence of 89 selective sweeps, represented as quantitative trait loci (QTL) hotspots, widely distributed across the durum wheat genome. A principal component analysis (PCoA) using 147 markers with −log10p > 5 identified three regions located on chromosomes 2A, 2B and 3A as the main drivers for differentiation of Mediterranean landraces. Gene flow between SPs offers clues regarding the putative use of Mediterranean old durum germplasm by the breeding programs represented in the structure analysis. EigenGWAS identified selective sweeps among landraces and modern cultivars. The analysis of the corresponding genomic regions in the ‘Zavitan’, ‘Svevo’ and ‘Chinese Spring’ genomes discovered the presence of important functional genes including Ppd, Vrn, Rht, and gene models involved in important biological processes including LRR-RLK, MADS-box, NAC, and F-box.info:eu-repo/semantics/publishedVersio

Multi-environment QTL analysis using an updated genetic map of a widely distributed Seri × Babax spring wheat population

Seri/Babax spring wheat RIL population was developed to minimize the confounding effect of phenology in the genetic dissection of abiotic stress traits. An existing linkage map (< 500 markers) was updated with 6470 polymorphic Illumina iSelect 90K array and DArTseq SNPs to a genetic map of 5576.5 cM with 1748 non-redundant markers (1165 90K SNPs, 207 DArTseq SNPs, 183 AFLP, 111 DArT array, and 82 SSR) assigned to 31 linkage groups. We conducted QTL mapping for yield and related traits phenotyped in several major wheat growing areas in Egypt, Sudan, Iran, India, and Mexico (nine environments: heat, drought, heat plus drought, and yield potential). QTL analysis identified 39 (LOD 2.5–23.6; PVE 4.8–21.3%), 36 (LOD 2.5–15.4; PVE 2.9–21.4%), 30 (LOD 2.5–13.1; PVE 3.6–26.8%), 39 (LOD 2.7–14.4; PVE 2.6–15.9%), and 22 (LOD 2.8–4.8; PVE 6.8–12.9%) QTLs for grain yield, thousand-grain weight, grain number, days to heading, and plant height, respectively. The present study confirmed QTLs from previous studies and identified novel QTLs. QTL analysis based on high-yielding and low-yielding environmental clusters identified 11 QTLs (LOD 2.6–14.9; PVE 2.7–19.7%). The updated map thereby provides a better genome coverage (3.5-fold) especially on the D genome (4-fold), higher density (1.1-fold), and a good collinearity with the IWGSC RefSeq v1.0 genome, and increased the number of detected QTLs (5-fold) compared with the earlier map. This map serves as a useful genomic resource for genetic analyses of important traits on this wheat population that was widely distributed around the world.info:eu-repo/semantics/acceptedVersio

Comparison of Genomic Prediction Methods for Yellow, Stem, and Leaf Rust Resistance in Wheat Landraces from Afghanistan

Wheat rust diseases, including yellow rust (Yr; also known as stripe rust) caused by Puccinia striiformis Westend. f. sp. tritici, leaf rust (Lr) caused by Puccinia triticina Eriks. and stem rust (Sr) caused by Puccinia graminis Pres f. sp. tritici are major threats to wheat production all around the globe. Durable resistance to wheat rust diseases can be achieved through genomic-assisted prediction of resistant accessions to increase genetic gain per unit time. Genomic prediction (GP) is a promising technology that uses genomic markers to estimate genomic-assisted breeding values (GBEVs) for selecting resistant plant genotypes and accumulating favorable alleles for adult plant resistance (APR) to wheat rust diseases. To evaluate GP we compared the predictive ability of nine different parametric, semi-parametric and Bayesian models including Genomic Unbiased Linear Prediction (GBLUP), Ridge Regression (RR), Least Absolute Shrinkage and Selection Operator (LASSO), Elastic Net (EN), Bayesian Ridge Regression (BRR), Bayesian A (BA), Bayesian B (BB), Bayesian C (BC) and Reproducing Kernel Hilbert Spacing model (RKHS) to estimate GEBV’s for APR to yellow, leaf and stem rust of wheat in a panel of 363 bread wheat landraces of Afghanistan origin. Based on five-fold cross validation the mean predictive abilities were 0.33, 0.30, 0.38, and 0.33 for Yr (2016), Yr (2017), Lr, and Sr, respectively. No single model outperformed the rest of the models for all traits. LASSO and EN showed the lowest predictive ability in four of the five traits. GBLUP and RR gave similar predictive abilities, whereas Bayesian models were not significantly different from each other as well. We also investigated the effect of the number of genotypes and the markers used in the analysis on the predictive ability of the GP model. The predictive ability was highest with 1000 markers and there was a linear trend in the predictive ability and the size of the training population. The results of the study are encouraging, confirming the feasibility of GP to be effectively applied in breeding programs for resistance to all three wheat rust diseases.info:eu-repo/semantics/publishedVersio

Genetic Diversity and Population Structure Analysis of Triticum aestivum L. Landrace Panel from Afghanistan

Landraces are a potential source of genetic diversity and provide useful genetic resources to cope with the current and future challenges in crop breeding. Afghanistan is located close to the centre of origin of hexaploid wheat. Therefore, understanding the population structure and genetic diversity of Afghan wheat landraces is of enormous importance in breeding programmes for the development of high-yielding cultivars as well as broadening the genetic base of bread wheat. Here, a panel of 363 bread wheat landraces collected from seven north and north-eastern provinces of Afghanistan were evaluated for population structure and genetic diversity using single nucleotide polymorphic markers (SNPs). The genotyping-by-sequencing of studied landraces after quality control provided 4897 high-quality SNPs distributed across the genomes A (33.75%), B (38.73%), and D (27.50%). The population structure analysis was carried out by two methods using model-based STRUCTURE analysis and cluster-based discriminant analysis of principal components (DAPC). The analysis of molecular variance showed a higher proportion of variation within the sub-populations compared with the variation observed as a whole between sub-populations. STRUCTURE and DAPC analysis grouped the majority of the landraces from Badakhshan and Takhar together in one cluster and the landraces from Baghlan and Kunduz in a second cluster, which is in accordance with the micro-climatic conditions prevalent within the north-eastern agro-ecological zone. Genetic distance analysis was also studied to identify differences among the Afghan regions; the strongest correlation was observed for the Badakhshan and Takhar (0.003), whereas Samangan and Konarha (0.399) showed the highest genetic distance. The population structure and genetic diversity analysis highlighted the complex genetic variation present in the landraces which were highly correlated to the geographic origin and micro-climatic conditions within the agro-climatic zones of the landraces. The higher proportions of admixture could be attributed to historical unsupervised exchanges of seeds between the farmers of the central and north-eastern provinces of Afghanistan. The results of this study will provide useful information for genetic improvement in wheat and is essential for association mapping and genomic prediction studies to identify novel sources for resistance to abiotic and biotic stresses.info:eu-repo/semantics/publishedVersio

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

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

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

<p>Abstract</p> <p>Background</p> <p>A number of molecular marker technologies have allowed important advances in the understanding of the genetics and evolution of <it>Eucalyptus</it>, 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 <it>Eucalyptus</it>.</p> <p>Findings</p> <p>After testing several genome complexity reduction methods we identified the <it>Pst</it>I/<it>Taq</it>I method as the most effective for <it>Eucalyptus </it>and developed 18 genomic libraries from <it>Pst</it>I/<it>Taq</it>I representations of 64 different <it>Eucalyptus </it>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 <it>E. grandis </it>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.</p> <p>Conclusions</p> <p>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 <it>Eucalyptus</it>. The transferability of DArT across species and pedigrees is particularly valuable for a large genus such as <it>Eucalyptus </it>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 <it>Eucalyptus </it>reference genome, which will soon be completed.</p