Livia Moura Souza

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.

Microsatellite marker development for the rubber tree ( Hevea brasiliensis) : characterization and cross-amplification in wild Hevea species

BackgroundThe rubber tree (Hevea brasiliensis) is native to the Amazon region and it is the major source of natural rubber in the world. Rubber tree breeding is time-consuming and expensive. However, molecular markers such as microsatellites can reduce the time required for these programs. This study reports new genomic microsatellite markers developed and characterized in H. brasiliensis and the evaluation of their transferability to other Hevea species.FindingsWe constructed di- and trinucleotide-enriched libraries. From these two libraries, 153 primer pairs were designed and initially evaluated using 9 genotypes of H. brasiliensis. A total of 119 primer pairs had a good amplification product, 90 of which were polymorphic. We chose 46 of the polymorphic markers and characterized them in 36 genotypes of H. brasiliensis. The expected and observed heterozygosities ranged from 0.1387 to 0.8629 and 0.0909 to 0.9167, respectively. The polymorphism information content (PIC) values ranged from 0.097 to 0.8339, and the mean number of alleles was 6.4 (2–17). These 46 microsatellites were also tested in 6 other Hevea species. The percentage of transferability ranged from 82% to 87%. Locus duplication was found in H. brasiliensis and also in 5 of other species in which transferability was tested.ConclusionsThis study reports new microsatellite markers for H. brasiliensis that can be used for genetic linkage mapping, quantitative trait loci identification and marker- assisted selection. The high percentage of transferability may be useful in the evaluations of genetic variability and to monitor introgression of genetic variability from different Hevea species into breeding programs.

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.

Microsatellite Loci for Orthophytum ophiuroides (Bromelioideae, Bromeliaceae) Species Adapted to Neotropical Rock Outcrops

Premise of the study: Microsatellite primers were developed for Orthophytum ophiuroides, a rupicolous bromeliad species endemic to neotropical rocky fields. These microsatellite loci will be used to investigate population differentiation and species cohesion in such fragmented environments. The loci were tested for cross-amplification in related bromeliad species. Methods and Results: Eleven polymorphic microsatellite markers were isolated and characterized from an enriched library of O. ophiuroides. The loci were tested on 42 individuals from two populations of this species. The number of alleles per locus ranged from three to nine and the expected and observed heterozygosities ranged from 0.167 to 0.870 and from 0.369 to 0.958, respectively. Seven loci successfully amplified in other related bromeliad species. Conclusions: Our results suggest that the microsatellite loci developed here will be useful to assess genetic diversity and gene flow in O. ophiuroides for the investigation of population differentiation and species cohesion in neotropical mountainous habitats.

High-resolution genetic map and QTL analysis of growth-related traits of Hevea brasiliensis cultivated under suboptimal temperature and humidity conditions

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.

Linkage disequilibrium and population structure in wild and cultivated populations of rubber tree (Hevea brasiliensis).

Among rubber tree species, which belong to the Hevea genus of the Euphorbiaceae family, Hevea brasiliensis (Willd. ex Adr.de Juss.) Muell. Arg. is the main commercial source of natural rubber production worldwide. Knowledge of the population structure and linkage disequilibrium (LD) of this species is essential for the efficient organization and exploitation of genetic resources. Here, we obtained single-nucleotide polymorphisms (SNPs) using a genotyping-by-sequencing (GBS) approach and then employed the SNPs for the following objectives: (i) to identify the positions of SNPs on a genetic map of a segregating mapping population, (ii) to evaluate the population structure of a germplasm collection, and (iii) to detect patterns of LD decay among chromosomes for future genetic association studies in rubber tree. A total of 626 genotypes, including both germplasm accessions (368) and individuals from a genetic mapping population (254), were genotyped. A total of 77,660 and 21,283 SNPs were detected by GBS in the germplasm and mapping populations, respectively. The mapping population, which was previously mapped, was constructed with 1,062 markers, among which only 576 SNPs came from GBS, reducing the average interval between two adjacent markers to 4.4 cM. SNPs from GBS genotyping were used for the analysis of genetic structure and LD estimation in the germplasm accessions. Two groups, which largely corresponded to the cultivated and wild populations, were detected using STRUCTURE and via principal coordinate analysis. LD analysis, also using the mapped SNPs, revealed that non-random associations varied along chromosomes, with regions of high LD interspersed with regions of low LD. Considering the length of the genetic map (4,693 cM) and the mean LD (0.49 for cultivated and 0.02 for wild populations), a large number of evenly spaced SNPs would be needed to perform genome-wide association studies in rubber tree, and the wilder the genotypes used, the more difficult the mapping saturation.

QTL detection for growth and latex production in a full-sib rubber tree population cultivated under suboptimal climate conditions

BackgroundRubber tree is cultivated in mainly Southeast Asia and is by far the most significant source of natural rubber production worldwide. However, the genetic architecture underlying the primary agronomic traits of this crop has not been widely characterized. This study aimed to identify quantitative trait loci (QTLs) associated with growth and latex production using a biparental population established in suboptimal growth conditions in Brazil.ResultsA full-sib population composed of 251 individuals was developed from crossing two high-producing Asiatic rubber tree cultivars, PR 255 and PB 217. This mapping population was genotyped with microsatellite markers from enriched genomic libraries or transcriptome datasets and single-nucleotide polymorphism (SNP) markers, leading to construction of a saturated multipoint integrated genetic map containing 354 microsatellite and 151 SNP markers. Height and circumference measurements repeated over a six-year period and registration of cumulative latex production during six consecutive months on the same individuals allowed in-depth characterization of the genetic values of several growth traits and precocious latex production. Growth traits, circumference and height, were overall positively correlated, whereas latex production was not correlated or even negatively correlated with growth traits. A total of 86 distinct QTLs were identified, most of which were detected for only one trait. Among these QTLs, 15 were linked to more than one phenotypic trait (up to 4 traits simultaneously). Latex production and circumference increments during the last wintering period were associated with the highest numbers of identified QTLs (eleven and nine, respectively), jointly explaining the most significantly observed phenotypic variances (44.1% and 44.4%, respectively). The most important QTL for latex production, located on linkage group 16, had an additive effect of the male parent PB 217 and corresponded to a QTL at the same position detected in a previous study carried out in Thailand for the biparental population RRIM 600 x PB 217.ConclusionsOur results identified a set of significant QTLs for rubber tree, showing that the performance of modern Asiatic cultivars can still be improved and paving the way for further marker-assisted selection, which could accelerate breeding programs.

Development of microsatellite molecular markers and genetic diversity in Hevea Braziliensis

Background The rubber tree [Hevea Braziliensis (Willd. ex Adr. De Juss.) Muell-Arg.] is native from the Amazon region which has great economic importance for being the largest source of natural rubber in the world. Although Brazil is the center of origin and genetic diversity of this culture, the country is currently responsible for only 1% of the world production. Besides offering optimal weather conditions for crop development, the Amazon rainforest region is also favorable to the development of the fungus known as SALB (South American leaf blight), which causes the disease-of-leaves. Leaves fall sharply reducing the production of latex, limiting the production of rubber in this region. Thus breeding programs are seeking for clones which are resistant to this fungus and with high production in escape regions, which provide stress conditions such as low temperatures, high altitude, wind and other diseases [1]. The rubber tree is perennial and requires about 30 years to obtain an improved variety, starting from the controlled pollination of a clone to the final recommendation. Molecular markers such as microsatellites (Simple Sequence Repeats, SSRs) are an important tool for diversity studies and potentially to assist breeding programs. This study aimed to develop an enriched microsatellite library for H. Braziliensis, characterize these developed microsatellite markers and test the transferability of these markers to six other species of the genus Hevea. Material and methods For this study we used 36 accessions of H. Braziliensis donated by the Agronomy Institute of Campinas and one accession of each of six other species of genus Hevea (H. nitida, H.pauciflora (2), H.camargoana, H.guianensis, H. rigidifolia andH. benthamiana) provided by Embrapa. Genomic DNA samples were extracted from lyophilized leaf tissues using a modified CTAB method [2]. Trinucleotide and dinucleotide enriched genomic libraries for H. Braziliensis were constructed. The DNA samples were digested with AFAI and enriched using (CT)8 and (GT)8 biotinylated microsatellite probes for the dinucleotide library and (ATC)8 and (CCT)8 for the trinucleotide library. The clones obtained were sequenced and the sequences were evaluated with the Microsat program, which removes parts of the vector and the adapters and verifies the presence of restriction site within the sequence. After this step, the sequences were aligned and edited using the program SeqMan (DNAStar Inc.), which also allows analyzing the redundancy of the library. The identification of microsatellites was performed using a research tool SSRs SSRIT – “The Simple Sequence Repeat Identification Tool” avaiable at Gramene [http:// www.gramene.org]and primers complementary to sequences flaking the microsatellites were designed by Primer Select Program (DNAStar Inc) and Primer 3. Amplification tests were made from a temperature gradient to know the annealing temperature and the products were evaluated and resolved on 3% agarose gels stained with ethidium bromide and in denaturing 6% polyacrylamide and silver stained [3]. The loci were characterized on the number of alleles per locus, allele frequency and the Polymorph Information Content (PIC). It was also made analysis of ancestry for de accessions of H. Braziliensis using the program Structure v 2.3.3 [4]. * Correspondence: camila.mantello@gmail.com Molecular Genetic Analysis Laboratory Molecular Biology Center and Genetic Engineering (CBMEG), UNICAMP, Campinas, Sao Paulo, 13083-875, Brazil Full list of author information is available at the end of the article Mantello et al. BMC Proceedings 2011, 5(Suppl 7):P11 http://www.biomedcentral.com/1753-6561/5/S7/P11