Genetic linkage mapping in Hevea brasiliensis

Orientadores: Anete Pereira de Souza, Antonio Augusto Franco GarciaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Aproximadamente 2.500 espécies são conhecidas por produzirem borracha natural e a seringueira, [Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg.], espécie nativa da Amazônia e pertencente ao gênero Hevea, é a maior fonte de borracha natural do mundo. A borracha natural é matéria prima para fabricação de mais de 40.000 produtos tendo importância fundamental na indústria de pneus. Apesar de a região Amazônica oferecer condições climáticas adequadas para seu crescimento e desenvolvimento, esta área também possui condições favoráveis à ocorrência do mal-das-folhas (Microcyclus ulei P. Henn v. Arx), doença também conhecida como mal sulamericano das folhas (South American Leaf Bligth ¿ SALB). Dessa maneira, a heveicultura se expandiu para áreas de escape que propiciam novas condições de estresse, limitando o seu crescimento e a produção de látex. O melhoramento genético vem buscando cultivares adaptados a estas regiões de escape, porém o ciclo de melhoramento da seringueira é longo e não permite o rápido desenvolvimento de novos cultivares. O desenvolvimento de ferramentas na biologia molecular permite o melhor entendimento da espécie e pode diminuir o tempo gasto nos ensaios de campo. O presente trabalho desenvolveu novos marcadores microssatélites (SSRs) a partir de bibliotecas enriquecidas em microssatélites. A caracterização destes marcadores mostrou a alta variabilidade alélica dentro de H. brasiliensis e o teste de transferibilidade dos SSRs em outras seis espécies do gênero Hevea mostrou alelos exclusivos para as mesmas e taxas de amplificação superior a 80%. Com o objetivo desenvolver novos marcadores SSRs e single nucleotide polymorphisms (SNPs) em larga escala, foi sequenciado na plataforma Illumina GAIIx o transcriptoma de painel de dois cultivares importantes para a heveicultura (GT1 e PR255). A montagem e a caracterização do transcriptoma permitiu o melhor entendimento da dinâmica do transcriptoma em H. brasiliensis e identificou novos transcritos para a espécie. As sequências do transcriptoma foram submetidas à busca de SSRs e SNPs. No transcriptoma, foram identificados 1.709 novas sequências contendo SSRs para seringueira, a uma frequência de um SSR a cada 2,8 kb. Já a busca de SNPs identificou 404.114 SNPs com frequência de um SNP a cada 125 pb. Através da anotação no Kyoto Encyclopedia of Genes and Genomes (KEGG), foram identificadas sequências anotadas a todas as enzimas referentes às duas vias de síntese de látex (mevalonato - MVA e C-metileritritol 4-fosfato -MEP). Apesar de as vias MVA e MEP serem muitos estudadas, esta foi a primeira vez que SNPs foram identificados e validados. Os marcadores SSRs e SNPs foram então mapeados em uma população segregante F1. O mapa genético obtido contém 383 marcadores mapeados em 20 grupos de ligação. Neste trabalho foram desenvolvidos 52 SSRs e 51 SNPs do total de marcadores mapeados. Como o número de grupos de ligação esperado é 18 (2n=36), conclui-se que o mapa genético obtido mostra que ainda há uma cobertura incompleta do genoma. Devido à alta frequência de SNPs no genoma, o desenvolvimento de novos marcadores poderá saturar o mapa de forma homogênea, permitindo o agrupamento dos marcadores nos 18 grupos de ligação esperadosAbstract: Approximately 2.500 species are known to produce natural rubber. Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg. also known as rubber tree is a species native to the Amazon rainforest and is the largest source of natural rubber in the world. Natural rubber has been used in more than 40,000 products and has great importance in the tire industry. Although the Amazon rainforest offers optimal conditions for growth and rubber yields due to its warm and humid climate, this region also provides optimal conditions for the fungus Microcyclus ulei P. Henn v. Arx which causes the South American Leaf Blight (SALB) disease. Thus, rubber tree plantations have expanded to escape areas that provides new stress conditions limiting their growth and latex production. The rubber tree breeding is trying to create a new cultivar that is resistant to these new conditions but the rubber tree cycle breeding is long and does not allow a rapid cultivar development. Thus, molecular biology techniques could provide a greater knowledge of H. brasiliensis genetic and could optimize field evaluation and, thus, reduce the time and area required for experiments. The present work developed new microsatellites (SSRs) markers for rubber tree from genomic enriched libraries. The new microsatellites were characterized and demonstrated a high allelic variability within H. brasiliensis genotypes. The transferability rate in other six species of the genus Hevea was greater than 80%. To develop new SSRs and single nucleotide polymorphisms (SNPs) markers, the panel transcriptome from two important cultivars (GT1 and PR255) was sequenced in Illumina GAIIx platform. The transcriptome obtained allowed a better knowledge about H. brasiliensis transcriptome and identified new transcripts for rubber tree public database. The sequences were submitted to a SSR and SNP search. The SSR frequency was one SSR each 2.8 kb and it was identified 1.709 new sequences with new SSRs for rubber tree database. A total of 404.114 putative SNPs were detected with a frequency of one SNP every 125 bp. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, it was identified contigs corresponding to all the enzymes of mevalonate (MVA) and -C-methyl-D-erythritol 4-phosphate (MEP). Despite MVA and MEP pathways are being very well studied, since they are directly involved to rubber biosynthesis, this is the first time that molecular markers have been developed for such important pathways. The SSRs and SNPs developed were mapped in a full-sib population. The genetic linkage map has 383 molecular markers distributed in 20 linkage groups. This project contributed with 52 SSRs and 51 SNPs of the total mapped markers. Although the expected number of linkage groups are 18 (2n=36), the new genetic linkage map still has an incomplete coverage of the genome. Due to the high frequency of the SNPs in the genome, the development of new markers can saturate this map homogeneouslyDoutoradoGenetica Vegetal e MelhoramentoDoutora em Genética e Biologia Molecula

Deep expression analysis reveals distinct cold-response strategies in rubber tree (hevea brasiliensis)

Natural rubber, an indispensable commodity used in approximately 40,000 products, is fundamental to the tire industry. The rubber tree species Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg., which is native the Amazon rainforest, is the major producer of latex worldwide. Rubber tree breeding is time consuming, expensive and requires large field areas. Thus, genetic studies could optimize field evaluations, thereby reducing the time and area required for these experiments. In this work, transcriptome sequencing was used to identify a full set of transcripts and to evaluate the gene expression involved in the different cold-response strategies of the RRIM600 (cold-resistant) and GT1 (cold-tolerant) genotypes.ResultsWe built a comprehensive transcriptome using multiple database sources, which resulted in 104,738 transcripts clustered in 49,304 genes. The RNA-seq data from the leaf tissues sampled at four different times for each genotype were used to perform a gene-level expression analysis. Differentially expressed genes (DEGs) were identified through pairwise comparisons between the two genotypes for each time series of cold treatments.DEG annotation revealed that RRIM600 and GT1 exhibit different chilling tolerance strategies. To cope with cold stress, the RRIM600 clone upregulates genes promoting stomata closure, photosynthesis inhibition and a more efficient reactive oxygen species (ROS) scavenging system. The transcriptome was also searched for putative molecular markers (single nucleotide polymorphisms (SNPs) and microsatellites) in each genotype. and a total of 27,111 microsatellites and 202,949 (GT1) and 156,395 (RRIM600) SNPs were identified in GT1 and RRIM600. Furthermore, a search for alternative splicing (AS) events identified a total of 20,279 events.ConclusionsThe elucidation of genes involved in different chilling tolerance strategies associated with molecular markers and information regarding AS events provides a powerful tool for further genetic and genomic analyses of rubber tree breeding20CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP478701/2012–8; 402954/2012Sem informação2007/50392–1; 2012/50491–8; 2014/18755–0; 2015/24346–

De Novo Assembly And Transcriptome Analysis Of The Rubber Tree (hevea Brasiliensis) And Snp Markers Development For Rubber Biosynthesis Pathways.

Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.9e10266

Genetic analysis of wheat sensitivity to the ToxB fungal effector from Pyrenophora tritici-repentis, the causal agent of tan spot.

Genetic mapping of sensitivity to the Pyrenophora tritici-repentis effector ToxB allowed development of a diagnostic genetic marker, and investigation of wheat pedigrees allowed transmission of sensitive alleles to be tracked. Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis, is a major disease of wheat (Triticum aestivum). Secretion of the P. tritici-repentis effector ToxB is thought to play a part in mediating infection, causing chlorosis of plant tissue. Here, genetic analysis using an association mapping panel (n = 480) and a multiparent advanced generation intercross (MAGIC) population (n founders = 8, n progeny = 643) genotyped with a 90,000 feature single nucleotide polymorphism (SNP) array found ToxB sensitivity to be highly heritable (h2 ≥ 0.9), controlled predominantly by the Tsc2 locus on chromosome 2B. Genetic mapping of Tsc2 delineated a 1921-kb interval containing 104 genes in the reference genome of ToxB-insensitive variety 'Chinese Spring'. This allowed development of a co-dominant genetic marker for Tsc2 allelic state, diagnostic for ToxB sensitivity in the association mapping panel. Phenotypic and genotypic analysis in a panel of wheat varieties post-dated the association mapping panel further supported the diagnostic nature of the marker. Combining ToxB phenotype and genotypic data with wheat pedigree datasets allowed historic sources of ToxB sensitivity to be tracked, finding the variety 'Maris Dove' to likely be the historic source of sensitive Tsc2 alleles in the wheat germplasm surveyed. Exploration of the Tsc2 region gene space in the ToxB-sensitive line 'Synthetic W7984' identified candidate genes for future investigation. Additionally, a minor ToxB sensitivity QTL was identified on chromosome 2A. The resources presented here will be of immediate use for marker-assisted selection for ToxB insensitivity and the development of germplasm with additional genetic recombination within the Tsc2 region

Genetic analysis of wheat sensitivity to the ToxB fungal effector from Pyrenophora tritici-repentis, the causal agent of tan spot

Abstract: Key message: Genetic mapping of sensitivity to the Pyrenophora tritici-repentis effector ToxB allowed development of a diagnostic genetic marker, and investigation of wheat pedigrees allowed transmission of sensitive alleles to be tracked. Abstract: Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis, is a major disease of wheat (Triticum aestivum). Secretion of the P. tritici-repentis effector ToxB is thought to play a part in mediating infection, causing chlorosis of plant tissue. Here, genetic analysis using an association mapping panel (n = 480) and a multiparent advanced generation intercross (MAGIC) population (n founders = 8, n progeny = 643) genotyped with a 90,000 feature single nucleotide polymorphism (SNP) array found ToxB sensitivity to be highly heritable (h2 ≥ 0.9), controlled predominantly by the Tsc2 locus on chromosome 2B. Genetic mapping of Tsc2 delineated a 1921-kb interval containing 104 genes in the reference genome of ToxB-insensitive variety ‘Chinese Spring’. This allowed development of a co-dominant genetic marker for Tsc2 allelic state, diagnostic for ToxB sensitivity in the association mapping panel. Phenotypic and genotypic analysis in a panel of wheat varieties post-dated the association mapping panel further supported the diagnostic nature of the marker. Combining ToxB phenotype and genotypic data with wheat pedigree datasets allowed historic sources of ToxB sensitivity to be tracked, finding the variety ‘Maris Dove’ to likely be the historic source of sensitive Tsc2 alleles in the wheat germplasm surveyed. Exploration of the Tsc2 region gene space in the ToxB-sensitive line ‘Synthetic W7984’ identified candidate genes for future investigation. Additionally, a minor ToxB sensitivity QTL was identified on chromosome 2A. The resources presented here will be of immediate use for marker-assisted selection for ToxB insensitivity and the development of germplasm with additional genetic recombination within the Tsc2 region

Leaf-, Panel- And Latex-expressed Sequenced Tags From The Rubber Tree (hevea Brasiliensis) Under Cold-stressed And Suboptimal Growing Conditions: The Development Of Gene-targeted Functional Markers For Stress Response.

Hevea brasiliensis is a native species of the Amazon Basin of South America and the primary source of natural rubber worldwide. Due to the occurrence of South American Leaf Blight disease in this area, rubber plantations have been extended to suboptimal regions. Rubber tree breeding is time-consuming and expensive, but molecular markers can serve as a tool for early evaluation, thus reducing time and costs. In this work, we constructed six different cDNA libraries with the aim of developing gene-targeted molecular markers for the rubber tree. A total of 8,263 reads were assembled, generating 5,025 unigenes that were analyzed; 912 expressed sequence tags (ESTs) represented new transcripts, and two sequences were highly up-regulated by cold stress. These unigenes were scanned for microsatellite (SSR) regions and single nucleotide polymorphisms (SNPs). In total, 169 novel EST-SSR markers were developed; 138 loci were polymorphic in the rubber tree, and 98 % presented transferability to six other Hevea species. Locus duplication was observed in H. brasiliensis and other species. Additionally, 43 SNP markers in 13 sequences that showed similarity to proteins involved in stress response, latex biosynthesis and developmental processes were characterized. cDNA libraries are a rich source of SSR and SNP markers and enable the identification of new transcripts. The new markers developed here will be a valuable resource for linkage mapping, QTL identification and other studies in the rubber tree and can also be used to evaluate the genetic variability of other Hevea species, which are valuable assets in rubber tree breeding.341035-105

Wheat genetic loci conferring resistance to stripe rust in the face of genetically diverse races of the fungus Puccinia striiformis f. sp. tritici.

Funder: Roger Harrison TrustAnalysis of a wheat multi-founder population identified 14 yellow rust resistance QTL. For three of the four most significant QTL, haplotype analysis indicated resistance alleles were rare in European wheat. Stripe rust, or yellow rust (YR), is a major fungal disease of wheat (Triticum aestivum) caused by Puccinia striiformis Westend f. sp. tritici (Pst). Since 2011, the historically clonal European Pst races have been superseded by the rapid incursion of genetically diverse lineages, reducing the resistance of varieties previously showing durable resistance. Identification of sources of genetic resistance to such races is a high priority for wheat breeding. Here we use a wheat eight-founder multi-parent population genotyped with a 90,000 feature single nucleotide polymorphism array to genetically map YR resistance to such new Pst races. Genetic analysis of five field trials at three UK sites identified 14 quantitative trait loci (QTL) conferring resistance. Of these, four highly significant loci were consistently identified across all test environments, located on chromosomes 1A (QYr.niab-1A.1), 2A (QYr.niab-2A.1), 2B (QYr.niab-2B.1) and 2D (QYr.niab-2D.1), together explaining ~ 50% of the phenotypic variation. Analysis of these four QTL in two-way and three-way combinations showed combinations conferred greater resistance than single QTL, and genetic markers were developed that distinguished resistant and susceptible alleles. Haplotype analysis in a collection of wheat varieties found that the haplotypes associated with YR resistance at three of these four major loci were rare (≤ 7%) in European wheat, highlighting their potential utility for future targeted improvement of disease resistance. Notably, the physical interval for QTL QYr.niab-2B.1 contained five nucleotide-binding leucine-rich repeat candidate genes with integrated BED domains, of which two corresponded to the cloned resistance genes Yr7 and Yr5/YrSp