Carla Cristina da Silva

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.

QTL Mapping of Growth-Related Traits in a Full-Sib Family of Rubber Tree ( Hevea brasiliensis ) Evaluated in a Sub-Tropical Climate

The rubber tree (Hevea spp.), cultivated in equatorial and tropical countries, is the primary plant used in natural rubber production. Due to genetic and physiological constraints, inbred lines of this species are not available. Therefore, alternative approaches are required for the characterization of this species, such as the genetic mapping of full-sib crosses derived from outbred parents. In the present study, an integrated genetic map was obtained for a full-sib cross family with simple sequence repeats (SSRs) and expressed sequence tag (EST-SSR) markers, which can display different segregation patterns. To study the genetic architecture of the traits related to growth in two different conditions (winter and summer), quantitative trait loci (QTL) mapping was also performed using the integrated map. Traits evaluated were height and girth growth, and the statistical model was based in an extension of composite interval mapping. The obtained molecular genetic map has 284 markers distributed among 23 linkage groups with a total length of 2688.8 cM. A total of 18 QTLs for growth traits during the summer and winter seasons were detected. A comparison between the different seasons was also conducted. For height, QTLs detected during the summer season were different from the ones detected during winter season. This type of difference was also observed for girth. Integrated maps are important for genetics studies in outbred species because they represent more accurately the polymorphisms observed in the genitors. QTL mapping revealed several interesting findings, such as a dominance effect and unique segregation patterns that each QTL could exhibit, which were independent of the flanking markers. The QTLs identified in this study, especially those related to phenotypic variation associated with winter could help studies of marker-assisted selection that are particularly important when the objective of a breeding program is to obtain phenotypes that are adapted to sub-optimal regions.

Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

Genetic Diversity Strategy for the Management and Use of Rubber Genetic Resources: More than 1,000 Wild and Cultivated Accessions in a 100-Genotype Core Collection.

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.

Assisted-selection of naturally caffeine-free coffee cultivars—characterization of SNPs from a methyltransferase gene

Breeding of caffeine-free coffee cultivars require tools for an early selection of progenies bearing this later trait. Genes from caffeine synthesis and degradation represent major targets for the development of molecular markers for assisted selection. In this study, we characterized SNPs identified on the caffeine synthase gene from AC1 mutant, a naturally caffeine-free arabica coffee plant. Molecular analysis of normal and mutant sequences indicates the occurrence of SNPs in protein domains, potentially associated with caffeine synthesis in coffee. Progenies F2, F1BC1 and BC from crosses of AC mutants and elite cultivars were evaluated regarding caffeine content in grains and genomic segregation profile of selected SNPs. Genotyping analysis allowed the discrimination between homozygous and heterozygous plants. Quantification of caffeine content indicated a significant variability among progenies and a low frequency of caffeine-free plants. Statistical analyses of genotyping and phenotyping results showed significant association between presence of selected SNPs and reduced caffeine content. Moreover, this association occurs through all evaluated genetic backgrounds and generations, indicating an inheritance stability of both trait and markers. The molecular markers described here represent a successful case of assisted-selection in coffee, indicating their potential use for breeding of caffeine-free cultivars.

Transcriptome Analysis of Distinct Cold Tolerance Strategies in the Rubber Tree (Hevea brasiliensis)

Natural rubber is an indispensable commodity used in approximately 40,000 products and is fundamental to the tire industry. Among the species that produce latex, the rubber tree [Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg.], a species native to the Amazon rainforest, is the major producer of latex used worldwide. The Amazon Basin presents optimal conditions for rubber tree growth, but the occurrence of South American leaf blight, which is caused by the fungus Microcyclus ulei (P. Henn) v. Arx, limits rubber tree production. Currently, rubber tree plantations are located in scape regions that exhibit suboptimal conditions such as high winds and cold temperatures. Rubber tree breeding programs aim to identify clones that are adapted to these stress conditions. However, rubber tree breeding is time-consuming, taking more than 20 years to develop a new variety. It is also expensive and requires large field areas. Thus, genetic studies could optimize field evaluations, thereby reducing the time and area required for these experiments. Transcriptome sequencing using next-generation sequencing (RNA-seq) is a powerful tool to identify a full set of transcripts and for evaluating gene expression in model and non-model species. In this study, we constructed a comprehensive transcriptome to evaluate the cold response strategies of the RRIM600 (cold-resistant) and GT1 (cold-tolerant) genotypes. Furthermore, we identified putative microsatellite (SSR) and single-nucleotide polymorphism (SNP) markers. Alternative splicing, which is an important mechanism for plant adaptation under abiotic stress, was further identified, providing an important database for further studies of cold tolerance.

Environment-driven genomic and trait divergence in a neotropical mangrove highlight differential sensitivities to climate change

Integrating genomic and ecological data is instrumental for understanding the mechanisms of adaptive processes in natural ecosystems. In non-model species, such studies can be particularly challenging but often yield results with implications for conservation. Here, we integrate molecular and ecophysiological approaches to assess the role of selection in the north-south organisation of genetic variation in the mangrove species Avicennia schaueriana, a new-world tree found in tropical to temperate coastal forests along the Atlantic coast of the Americas. We found substantial divergences between populations occurring north and south of the north-eastern extremity of South America, possibly reflecting the roles of contrasting environmental forces in shaping the genetic structure of the species. In a common garden experiment, individuals from equatorial and subtropical forests were found to be divergent in traits involved in water balance and carbon acquisition, suggesting a genetic basis of the observed differences. RNA-sequencing highlighted the molecular effects of different light, temperature and air humidity regimes on individuals under field conditions at contrasting latitudes. Additionally, genome-wide polymorphisms in trees sampled along most of the species range showed signatures of selection in sequences associated with the biogenesis of the photosynthetic apparatus, anthocyanin biosynthesis and osmotic and hypoxia stress responses. The observed functional divergence might differentially affect sensitivities of populations to our changing climate. We emphasize the necessity of independent conservation management for the long-term persistence of the species diversity. Moreover, we demonstrate the power of using a multidisciplinary approach in adaptation studies of non-model species.Integrating genomic and ecological data are instrumental for understanding the mechanisms of adaptive processes in natural ecosystems. In non-model species, such studies can be particularly challenging, but often brings to light results with implications for conservation. Here, we integrate molecular and ecophysiological approaches to assess the role of selection in the north-south organization of the genetic variation in the mangrove species Avicennia schaueriana, a new-world tree found from tropical to temperate coastal forests in the Atlantic coast of the Americas. In a common garden experiment, individuals from equatorial and subtropical forests diverged in traits involved in water balance and carbon acquisition, suggesting a genetic basis of the observed differences. RNA-sequencing highlighted the molecular effects of different light, temperature and air humidity in individuals under field conditions at contrasting latitudes. Additionally, genetic polymorphisms in trees sampled along most of the species range showed signatures of selection in sequences associated with the biogenesis of the photosynthetic apparatus, anthocyanin biosynthesis and osmotic and hypoxia stress responses. We found substantial divergences between populations occurring north and south of the north-eastern extremity of South America, underpinning roles of contrasting environmental forces in shaping the genetic structure of the species. The observed functional divergence might differentially affect sensitivities of populations to our changing climate, as discussed here. We indicate the necessity of independent conservation management for the long-term persistence of the species diversity. Moreover, we demonstrate the power of using a multidisciplinary approach in adaptation studies of non-model species.Local adaptation is often a product of environmental variations in the geographical space and has implications for biodiversity conservation. We investigated the role of latitudinal heterogeneity in climate on the organization of genetic and phenotypic variation in the dominant coastal tree, Avicennia schaueriana. In a common garden experiment, samples from an equatorial region, marked by rainy/dry seasons, accumulated less biomass, showed lower stomatal conductance and transpiration, narrower xylem vessels, smaller leaves and higher reflectance of long wavelengths (red light) on the stem epidermis, than samples from a subtropical region, marked by warm/cold seasons. Transcriptome differences identified between trees sampled under field conditions at equatorial and subtropical sites, were enriched in functional categories as responses to temperature, solar radiation, water deficit, photosynthesis and cell wall biosynthesis. The diversity based on thousands of SNP loci revealed a north-south genetic structure. Remarkably, signatures of selection were identified in loci associated with photosynthesis, anthocyanin accumulation and the responses to osmotic and hypoxia stresses. Our results suggest the existence of divergence in key resource-use characteristics, likely driven by climate seasonality, based on water-deficit and solar radiation. These findings provide a basis for conservation plans and for predictions for coastal plant responses to climate change.Summary Local adaptation is often a product of environmental variation in the geographical space and has important implications for species’ response to climate change. Ongoing changes in climate are predicted to particularly affect coastal ecosystems, which frequently span broad latitudinal ranges and remarkable environmental variation. We investigated the local adaptation to contrasting environments over the distribution of a coastal habitat-forming tree, based on analyses of key resource-use characteristics. We integrated results from comparative ecophysiology under a common garden experiment, with transcriptome sequencing and genome-wide scans for selection in one of the most widely distributed trees in wetlands of the Atlantic coast of South America, Avicennia schaueriana. We observed differences related to water and carbon balance between plants from contrasting latitudes, supported by gene expression under field conditions and morphophysiological traits under common garden. Additionally, we found signatures of selection in putative genes associated with photosynthesis, anthocyanin accumulation and the response to osmotic and hypoxia stresses. Our results strongly suggest the existence of a north-south divergence in key resource-use characteristics, likely driven by seasonality in water-deficit and light availability. These findings have implications for conservation and provide the basis for more realistic predictions on coastal plants responses to a rapidly changing climate.

Local adaptation insights from genomics and ecophysiology of a neotropical mangrove

Integrating genomic and ecological data is instrumental for understanding the mechanisms of adaptive processes in natural ecosystems. In non-model species, such studies can be particularly challenging but often yield results with implications for conservation. Here, we integrate molecular and ecophysiological approaches to assess the role of selection in the north-south organisation of genetic variation in the mangrove species Avicennia schaueriana, a new-world tree found in tropical to temperate coastal forests along the Atlantic coast of the Americas. We found substantial divergences between populations occurring north and south of the north-eastern extremity of South America, possibly reflecting the roles of contrasting environmental forces in shaping the genetic structure of the species. In a common garden experiment, individuals from equatorial and subtropical forests were found to be divergent in traits involved in water balance and carbon acquisition, suggesting a genetic basis of the observed differences. RNA-sequencing highlighted the molecular effects of different light, temperature and air humidity regimes on individuals under field conditions at contrasting latitudes. Additionally, genome-wide polymorphisms in trees sampled along most of the species range showed signatures of selection in sequences associated with the biogenesis of the photosynthetic apparatus, anthocyanin biosynthesis and osmotic and hypoxia stress responses. The observed functional divergence might differentially affect sensitivities of populations to our changing climate. We emphasize the necessity of independent conservation management for the long-term persistence of the species diversity. Moreover, we demonstrate the power of using a multidisciplinary approach in adaptation studies of non-model species.Integrating genomic and ecological data are instrumental for understanding the mechanisms of adaptive processes in natural ecosystems. In non-model species, such studies can be particularly challenging, but often brings to light results with implications for conservation. Here, we integrate molecular and ecophysiological approaches to assess the role of selection in the north-south organization of the genetic variation in the mangrove species Avicennia schaueriana, a new-world tree found from tropical to temperate coastal forests in the Atlantic coast of the Americas. In a common garden experiment, individuals from equatorial and subtropical forests diverged in traits involved in water balance and carbon acquisition, suggesting a genetic basis of the observed differences. RNA-sequencing highlighted the molecular effects of different light, temperature and air humidity in individuals under field conditions at contrasting latitudes. Additionally, genetic polymorphisms in trees sampled along most of the species range showed signatures of selection in sequences associated with the biogenesis of the photosynthetic apparatus, anthocyanin biosynthesis and osmotic and hypoxia stress responses. We found substantial divergences between populations occurring north and south of the north-eastern extremity of South America, underpinning roles of contrasting environmental forces in shaping the genetic structure of the species. The observed functional divergence might differentially affect sensitivities of populations to our changing climate, as discussed here. We indicate the necessity of independent conservation management for the long-term persistence of the species diversity. Moreover, we demonstrate the power of using a multidisciplinary approach in adaptation studies of non-model species.Local adaptation is often a product of environmental variations in the geographical space and has implications for biodiversity conservation. We investigated the role of latitudinal heterogeneity in climate on the organization of genetic and phenotypic variation in the dominant coastal tree, Avicennia schaueriana. In a common garden experiment, samples from an equatorial region, marked by rainy/dry seasons, accumulated less biomass, showed lower stomatal conductance and transpiration, narrower xylem vessels, smaller leaves and higher reflectance of long wavelengths (red light) on the stem epidermis, than samples from a subtropical region, marked by warm/cold seasons. Transcriptome differences identified between trees sampled under field conditions at equatorial and subtropical sites, were enriched in functional categories as responses to temperature, solar radiation, water deficit, photosynthesis and cell wall biosynthesis. The diversity based on thousands of SNP loci revealed a north-south genetic structure. Remarkably, signatures of selection were identified in loci associated with photosynthesis, anthocyanin accumulation and the responses to osmotic and hypoxia stresses. Our results suggest the existence of divergence in key resource-use characteristics, likely driven by climate seasonality, based on water-deficit and solar radiation. These findings provide a basis for conservation plans and for predictions for coastal plant responses to climate change.Summary Local adaptation is often a product of environmental variation in the geographical space and has important implications for species’ response to climate change. Ongoing changes in climate are predicted to particularly affect coastal ecosystems, which frequently span broad latitudinal ranges and remarkable environmental variation. We investigated the local adaptation to contrasting environments over the distribution of a coastal habitat-forming tree, based on analyses of key resource-use characteristics. We integrated results from comparative ecophysiology under a common garden experiment, with transcriptome sequencing and genome-wide scans for selection in one of the most widely distributed trees in wetlands of the Atlantic coast of South America, Avicennia schaueriana. We observed differences related to water and carbon balance between plants from contrasting latitudes, supported by gene expression under field conditions and morphophysiological traits under common garden. Additionally, we found signatures of selection in putative genes associated with photosynthesis, anthocyanin accumulation and the response to osmotic and hypoxia stresses. Our results strongly suggest the existence of a north-south divergence in key resource-use characteristics, likely driven by seasonality in water-deficit and light availability. These findings have implications for conservation and provide the basis for more realistic predictions on coastal plants responses to a rapidly changing climate.

Local adaptation to water-deficit and light limitation suggested from genomic and ecophysiological approaches in a coastal habitat-forming tree

Integrating genomic and ecological data is instrumental for understanding the mechanisms of adaptive processes in natural ecosystems. In non-model species, such studies can be particularly challenging but often yield results with implications for conservation. Here, we integrate molecular and ecophysiological approaches to assess the role of selection in the north-south organisation of genetic variation in the mangrove species Avicennia schaueriana, a new-world tree found in tropical to temperate coastal forests along the Atlantic coast of the Americas. We found substantial divergences between populations occurring north and south of the north-eastern extremity of South America, possibly reflecting the roles of contrasting environmental forces in shaping the genetic structure of the species. In a common garden experiment, individuals from equatorial and subtropical forests were found to be divergent in traits involved in water balance and carbon acquisition, suggesting a genetic basis of the observed differences. RNA-sequencing highlighted the molecular effects of different light, temperature and air humidity regimes on individuals under field conditions at contrasting latitudes. Additionally, genome-wide polymorphisms in trees sampled along most of the species range showed signatures of selection in sequences associated with the biogenesis of the photosynthetic apparatus, anthocyanin biosynthesis and osmotic and hypoxia stress responses. The observed functional divergence might differentially affect sensitivities of populations to our changing climate. We emphasize the necessity of independent conservation management for the long-term persistence of the species diversity. Moreover, we demonstrate the power of using a multidisciplinary approach in adaptation studies of non-model species.Integrating genomic and ecological data are instrumental for understanding the mechanisms of adaptive processes in natural ecosystems. In non-model species, such studies can be particularly challenging, but often brings to light results with implications for conservation. Here, we integrate molecular and ecophysiological approaches to assess the role of selection in the north-south organization of the genetic variation in the mangrove species Avicennia schaueriana, a new-world tree found from tropical to temperate coastal forests in the Atlantic coast of the Americas. In a common garden experiment, individuals from equatorial and subtropical forests diverged in traits involved in water balance and carbon acquisition, suggesting a genetic basis of the observed differences. RNA-sequencing highlighted the molecular effects of different light, temperature and air humidity in individuals under field conditions at contrasting latitudes. Additionally, genetic polymorphisms in trees sampled along most of the species range showed signatures of selection in sequences associated with the biogenesis of the photosynthetic apparatus, anthocyanin biosynthesis and osmotic and hypoxia stress responses. We found substantial divergences between populations occurring north and south of the north-eastern extremity of South America, underpinning roles of contrasting environmental forces in shaping the genetic structure of the species. The observed functional divergence might differentially affect sensitivities of populations to our changing climate, as discussed here. We indicate the necessity of independent conservation management for the long-term persistence of the species diversity. Moreover, we demonstrate the power of using a multidisciplinary approach in adaptation studies of non-model species.Local adaptation is often a product of environmental variations in the geographical space and has implications for biodiversity conservation. We investigated the role of latitudinal heterogeneity in climate on the organization of genetic and phenotypic variation in the dominant coastal tree, Avicennia schaueriana. In a common garden experiment, samples from an equatorial region, marked by rainy/dry seasons, accumulated less biomass, showed lower stomatal conductance and transpiration, narrower xylem vessels, smaller leaves and higher reflectance of long wavelengths (red light) on the stem epidermis, than samples from a subtropical region, marked by warm/cold seasons. Transcriptome differences identified between trees sampled under field conditions at equatorial and subtropical sites, were enriched in functional categories as responses to temperature, solar radiation, water deficit, photosynthesis and cell wall biosynthesis. The diversity based on thousands of SNP loci revealed a north-south genetic structure. Remarkably, signatures of selection were identified in loci associated with photosynthesis, anthocyanin accumulation and the responses to osmotic and hypoxia stresses. Our results suggest the existence of divergence in key resource-use characteristics, likely driven by climate seasonality, based on water-deficit and solar radiation. These findings provide a basis for conservation plans and for predictions for coastal plant responses to climate change.Summary Local adaptation is often a product of environmental variation in the geographical space and has important implications for species’ response to climate change. Ongoing changes in climate are predicted to particularly affect coastal ecosystems, which frequently span broad latitudinal ranges and remarkable environmental variation. We investigated the local adaptation to contrasting environments over the distribution of a coastal habitat-forming tree, based on analyses of key resource-use characteristics. We integrated results from comparative ecophysiology under a common garden experiment, with transcriptome sequencing and genome-wide scans for selection in one of the most widely distributed trees in wetlands of the Atlantic coast of South America, Avicennia schaueriana. We observed differences related to water and carbon balance between plants from contrasting latitudes, supported by gene expression under field conditions and morphophysiological traits under common garden. Additionally, we found signatures of selection in putative genes associated with photosynthesis, anthocyanin accumulation and the response to osmotic and hypoxia stresses. Our results strongly suggest the existence of a north-south divergence in key resource-use characteristics, likely driven by seasonality in water-deficit and light availability. These findings have implications for conservation and provide the basis for more realistic predictions on coastal plants responses to a rapidly changing climate.

Apomixis-related genes identified from a coexpression network in Paspalum notatum, a Neotropical grass

Apomixis is a highly desirable trait in modern agriculture; however, incorporating it into breeding programs requires a deeper comprehension of apomictic regulatory mechanisms. Paspalum notatum is considered a good model for such studies because it exhibits both sexual and apomictic cytotypes. A transcriptomic approach to identifying differentially expressed genes between these cytotypes would constitute an important tool for the identification of genes in the apomictic pathway. We generated leaf and inflorescence transcriptomes of apomictic tetraploids and sexual diploids/tetraploids of P. notatum accessions. We de novo assembled and annotated 114,306 unigenes. Coexpression network and ontological annotation were applied to recover the interactions and biological processes of exclusive and differentially expressed genes from the apomictic libraries. Additionally, genes potentially linked to the apomixis-controlling region and several genes reported in the literature were identified. The reference transcriptome obtained in this study represents a robust set of expression data for P. notatum. Our approach to sequencing florets and leaves from different cytotypes enabled us to isolate a set of candidate genes. Using a gene expression network, we recovered transcriptionally coordinated genes related to mechanisms regulating apomixis. These findings provide an important contribution to our knowledge of apomixis and are valuable for Paspalum breeding programs.