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.Citation: Mantello CC, Cardoso-Silva CB, da Silva CC, de Souza LM, Scaloppi Junior EJ, et al. (2014) De Novo Assembly and Transcriptome Analysis of the Rubber Tree (Hevea brasiliensis) and SNP Markers Development for Rubber Biosynthesis Pathways. PLoS ONE 9(7): e102665.

Several genomic prediction models combining genotype × environment (G×E) interactions have recently been developed and used for genomic selection (GS) in plant breeding programs. G×E interactions reduce selection accuracy and limit genetic gains in plant breeding. Two data sets were used to compare the prediction abilities of multienvironment G×E genomic models and two kernel methods. Specifically, a linear kernel, or GB (genomic best linear unbiased predictor [GBLUP]), and a nonlinear kernel, or Gaussian kernel (GK), were used to compare the prediction accuracies (PAs) of four genomic prediction models: 1) a single-environment, main genotypic effect model (SM); 2) a multienvironment, main genotypic effect model (MM); 3) a multienvironment, single-variance G×E deviation model (MDs); and 4) a multienvironment, environment-specific variance G×E deviation model (MDe). We evaluated the utility of genomic selection (GS) for 435 individual rubber trees at two sites and genotyped the individuals via genotyping-by-sequencing (GBS) of single-nucleotide polymorphisms (SNPs). Prediction models were used to estimate stem circumference (SC) during the first 4 years of tree development in conjunction with a broad-sense heritability (H 2) of 0.60. Applying the model (SM, MM, MDs, and MDe) and kernel method (GB and GK) combinations to the rubber tree data revealed that the multienvironment models were superior to the single-environment genomic models, regardless of the kernel (GB or GK) used, suggesting that introducing interactions between markers and environmental conditions increases the proportion of variance explained by the model and, more importantly, the PA. Compared with the classic breeding method (CBM), methods in which GS is incorporated resulted in a 5-fold increase in response to selection for SC with multienvironment GS (MM, MDe, or MDs). Furthermore, GS resulted in a more balanced selection response for SC and contributed to a reduction in selection time when used in conjunction with traditional genetic breeding programs. Given the rapid advances in genotyping methods and their declining costs and given the overall costs of large-scale progeny testing and shortened breeding cycles, we expect GS to be implemented in rubber tree breeding programs.

Rubber tree (Hevea brasiliensis) cultivation is the main source of natural rubber worldwide and has been extended to areas with suboptimal climates and lengthy drought periods; this transition affects growth and latex production. High-density genetic maps with reliable markers support precise mapping of quantitative trait loci (QTL), which can help reveal the complex genome of the species, provide tools to enhance molecular breeding, and shorten the breeding cycle. In this study, QTL mapping of the stem diameter, tree height, and number of whorls was performed for a full-sibling population derived from a GT1 and RRIM701 cross. A total of 225 simple sequence repeats (SSRs) and 186 single-nucleotide polymorphism (SNP) markers were used to construct a base map with 18 linkage groups and to anchor 671 SNPs from genotyping by sequencing (GBS) to produce a very dense linkage map with small intervals between loci. The final map was composed of 1,079 markers, spanned 3,779.7 cM with an average marker density of 3.5 cM, and showed collinearity between markers from previous studies. Significant variation in phenotypic characteristics was found over a 59-month evaluation period with a total of 38 QTLs being identified through a composite interval mapping method. Linkage group 4 showed the greatest number of QTLs (7), with phenotypic explained values varying from 7.67 to 14.07%. Additionally, we estimated segregation patterns, dominance, and additive effects for each QTL. A total of 53 significant effects for stem diameter were observed, and these effects were mostly related to additivity in the GT1 clone. Associating accurate genome assemblies and genetic maps represents a promising strategy for identifying the genetic basis of phenotypic traits in rubber trees. Then, further research can benefit from the QTLs identified herein, providing a better understanding of the key determinant genes associated with growth of Hevea brasiliensis under limiting water conditions.

The rubber tree [Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell. Arg.] is the only plant species worldwide that is cultivated for the commercial production of natural rubber. This study describes the genetic diversity of the Hevea spp. complex that is available in the main ex situ collections of South America, including Amazonian populations that have never been previously described. Genetic data were analyzed to determine the genetic structure of the wild populations, quantify the allelic diversity and suggest the composition of a core collection to capture the maximum genetic diversity within a minimal sample size. A total of 1,117 accessions were genotyped with 13 microsatellite markers. We identified a total of 408 alleles, 319 of which were shared between groups and 89 that were private in different groups of accessions. In a population structure and principal component analysis, the level of clustering reflected a primary division into the following two subgroups: cluster 1, which consisted of varieties from the advanced breeding germplasm that originated from the Wickham and Mato Grosso accessions; and cluster 2, which consisted of the wild germplasm from the Acre, Amazonas, Pará and Rondônia populations and Hevea spp. The analyses revealed a high frequency of gene flow between the groups, with the genetic differentiation coefficient (GST) estimated to be 0.018. Additionally, no distinct separation among the H. brasiliensis accessions and the other species from Amazonas was observed. A core collection of 99 accessions was identified that captured the maximum genetic diversity. Rubber tree breeders can effectively utilize this core collection for cultivar improvement. Furthermore, such a core collection could provide resources for forming an association panel to evaluate traits with agronomic and commercial importance. Our study generated a molecular database that should facilitate the management of the Hevea germplasm and its use for subsequent genetic and genomic breeding.

Hevea brasiliensis (rubber tree) is a large tree species of the Euphorbiaceae family with inestimable economic importance. Rubber tree breeding programs currently aim to improve growth and production, and the use of early genotype selection technologies can accelerate such processes, mainly with the incorporation of genomic tools, such as marker-assisted selection (MAS). However, few quantitative trait loci (QTLs) have been used successfully in MAS for complex characteristics. Recent research shows the efficiency of genome-wide association studies (GWAS) for locating QTL regions in different populations. In this way, the integration of GWAS, RNA-sequencing (RNA-Seq) methodologies, coexpression networks and enzyme networks can provide a better understanding of the molecular relationships involved in the definition of the phenotypes of interest, supplying research support for the development of appropriate genomic based strategies for breeding. In this context, this work presents the potential of using combined multiomics to decipher the mechanisms of genotype and phenotype associations involved in the growth of rubber trees. Using GWAS from a genotyping-by-sequencing (GBS) Hevea population, we were able to identify molecular markers in QTL regions with a main effect on rubber tree plant growth under constant water stress. The underlying genes were evaluated and incorporated into a gene coexpression network modelled with an assembled RNA-Seq-based transcriptome of the species, where novel gene relationships were estimated and evaluated through in silico methodologies, including an estimated enzymatic network. From all these analyses, we were able to estimate not only the main genes involved in defining the phenotype but also the interactions between a core of genes related to rubber tree growth at the transcriptional and translational levels. This work was the first to integrate multiomics analysis into the in-depth investigation of rubber tree plant growth, producing useful data for future genetic studies in the species and enhancing the efficiency of the species improvement programs.

Background 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. Results We 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. Conclusions The 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 breeding. Electronic supplementary material The online version of this article (10.1186/s12864-019-5852-5) contains supplementary material, which is available to authorized users.

A produção de mudas de caramboleira é um dos fatores limitantes à expansão comercial da cultura, devido ao tempo que estas levam para serem formadas e iniciarem a produção. Uma boa alternativa pode estar na otimização dos métodos de propagação vegetativa, através de estudos relativos aos processos e fatores envolvidos no enraizamento de estacas. Este trabalho foi desenvolvido com o objetivo de estudar o enraizamento de estacas apicais e basais de caramboleira, tratadas com ácido indolbutírico (IBA), em condições de nebulização intermitente. Estacas apicais e basais de caramboleira foram coletadas de ramos de plantas-matrizes da cultivar B-10 e submetidas à aplicação de cinco concentrações de IBA (0; 1.000; 3.000; 5.000 e 7000 mg.L-1), em imersão por 10 segundos, para avaliar a capacidade de sobrevivência, enraizamento e número médio de raízes/estaca. Posteriormente, as estacas foram colocadas em caixas de madeira contendo vermiculita média como substrato e mantidas em casa de vegetação, sob nebulização intermitente, durante 70 dias. O delineamento experimental utilizado foi em esquema fatorial 2 x 5, com 4 repetições e 10 estacas/parcela. As estacas apicais obtiveram melhores resultados para porcentagem de sobrevivência (49,26%), enraizamento (34,84%) e número médio de raízes (20,51), mostrando-se superiores às basais. O uso de IBA não influenciou em nenhuma das variáveis analisadas.

Basic knowledge of genetic characteristics of populations is necessary to conduct effective breeding and selection. The objective of this paper is describing the genetic variation of rubber yield and the correlation with other traits, and estimating the genetic parameters for girth growth and total number of latex vessels. Sixty seven clones of Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell.-Arg. were tested at five sites during 10 years. Characters girth growth at panel opening and rubber yield, showed broad sense heritability on plot mean level, from 0.32 to 0.66 and 0.59 to 0.92, respectively. Predicted genetic gains equal to 0.73 cm and 0.79 g increase respectively on girth and yield in the opening panel and mature phases seems realistic, even with moderate selection intensities. Genetic correlations with rubber yield, bark thickness and total number of latex vessels were very large, and almost no genotype-environment interaction was present for girth growth. High genotype-environment interaction was present for rubber yield with genetic and phenotypic correlations across the sites, ranging from 0.64 to 0.92 (genetic) and 0.63 to 0.89 (phenotypic). Total number of latex vessels rings had a high heritability, ranging from 20.0% to 64.0% in the sites E and B, respectively. Key words: rubber tree, heritability, genetic correlations, genotype-environment interaction VARIABILIDADE GENÉTICA PARA CRESCIMENTO DO CAULE E PRODUÇÃO DE BORRACHA EM Hevea brasiliensis RESUMO: O conhecimento básico das características presentes nas populações de plantas é essencial para a condução dos trabalhos de seleção e melhoramento. Neste trabalho, objetivou-se descrever a variação genética da produção de borracha e correlacioná-las com outras características, assim como estimar os parâmetros genéticos para perímetro do caule e o número de anéis de vasos laticíferos. Um total de 67 clones de seringueira [Hevea brasiliensis (Willd. ex Adr. Juss.) Muell.-Arg.] foram avaliados em cinco locais durante 10 anos. Os caracteres perímetro do caule na abertura do painel e produção de borracha mostraram herdabilidades no sentido amplo ao nível de média de parcela entre 0,32 a 0,66 e 0,59 a 0,92, respectivamente. Ganhos genéticos preditos de 0,73 cm e 0,79g de incremento de perímetro na fase juvenil parecem viáveis mesmo com uma intensidade de seleção moderada. Correlações genéticas entre produção de borracha, espessura de casca e número total de vasos laticíferos foram muito altas, e praticamente não houve interação genótipo-ambiente para perímetro do caule. Alta interação genótipo-ambiente foi detectada para produção de borracha, com correlações genéticas e fenotípicas por locais variando entre 0,64 e 0,92 (genéticas) e 0,63 e 0,89 (fenotípicas). O caráter total de anéis de vasos laticíferos apresentou alta herdabilidade, variando de 20% até 63% nos locais E e B, respectivamente.