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 valuation, 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. brasil-iensis 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

Microsatellite In Aeschynomene Falcata (leguminosae): Diversity, Cross-amplification, And Chromosome Localization.

Aeschynomene falcata is an important forage species; however, because of low seed production, it is underutilized as forage species. Aeschynomene is a polyphyletic genus with a challenging taxonomic position. Two subgenera have been proposed, and it is suggested that Aeschynomene can be split in 2 genera. Thus, new markers, such as microsatellite sequences, are desirable for improving breeding programs for A. falcata. Based on transferability and in situ localization, these microsatellite sequences can be applied as chromosome markers in the genus Aeschynomene and closely related genera. Here, we report the first microsatellite library developed for this genus; 11 microsatellites were characterized, with observed and expected heterozygosities ranging from 0.0000 to 0.7143 and from 0.1287 to 0.8360, respectively. Polymorphic information content varied from 0.1167 to 0.7786. The departure from Hardy-Weinberg equilibrium may have resulted from frequent autogamy, which is characteristic of A. falcata. Of the 11 microsatellites, 9 loci were cross-amplified in A. brevipes and A. paniculata and 7 in Dalbergia nigra and Machaerium vestitum. Five of these 7 cross-amplified microsatellites were applied as probes during the in situ hybridization assay and 2 showed clear signals on A. falcata chromosomes, ensuring their viability as chromosome markers.1310390-

Microsatellite in Aeschynomene falcata (Leguminosae): Diversity, cross-amplification, and chromosome localization

Aeschynomene falcata is an important forage species; however, because of low seed production, it is underutilized as forage species. Aeschynomene is a polyphyletic genus with a challenging taxonomic position. Two subgenera have been proposed, and it is suggested that Aeschynomene can be split in 2 genera. Thus, new markers, such as microsatellite sequences, are desirable for improving breeding programs for A. falcata. Based on transferability and in situ localization, these microsatellite sequences can be applied as chromosome markers in the genus Aeschynomene and closely related genera. Here, we report the first microsatellite library developed for this genus; 11 microsatellites were characterized, with observed and expected heterozygosities ranging from 0.0000 to 0.7143 and from 0.1287 to 0.8360, respectively. Polymorphic information content varied from 0.1167 to 0.7786. The departure from Hardy-Weinberg equilibrium may have resulted from frequent autogamy, which is characteristic of A. falcata. Of the 11 microsatellites, 9 loci were cross-amplified in A. brevipes and A. paniculata and 7 in Dalbergia nigra and Machaerium vestitum. Five of these 7 cross-amplified microsatellites were applied as probes during the in situ hybridization assay and 2 showed clear signals on A. falcata chromosomes, ensuring their viability as chromosome markers1341039010397CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP471708/2010-02011/22215-3; 2010/19132-

Microsatellite Markers For Studies With The Carnivorous Plant Philcoxia Minensis (plantaginaceae).

Microsatellite markers were developed for the critically endangered carnivorous species Philcoxia minensis (Plantaginaceae) for further population genetic studies aiming at its conservation. We identified 29 clones containing 40 microsatellites from a genomic enriched library. A total of 27 primer pairs were developed and evaluated in 30 individuals of a natural P. minensis population. Seventeen markers successfully presented amplification products within the expected size range, of which 12 were polymorphic. The expected and observed heterozygosities ranged from 0.03 to 0.65 and from 0.00 to 0.77, respectively. Positive transferability with the related species P. bahiensis was observed for the same 17 markers. The 12 polymorphic microsatellite markers are suitable for studies in genetic diversity and structure, mating system, and gene flow in P. minensis and also may be useful for similar issues regarding the related species P. bahiensis.

Microsatellite markers for studies with the carnivorous plant Philcoxia minensis (Plantaginaceae)

Premise of the study: Microsatellite markers were developed for the critically endangered carnivorous species Philcoxia minensis (Plantaginaceae) for further population genetic studies aiming at its conservation. Methods and Results: We identifi ed 29 clones containing 40 microsatellites from a genomic enriched library. A total of 27 primer pairs were developed and evaluated in 30 individuals of a natural P. minensis population. Seventeen markers successfully presented amplifi cation products within the expected size range, of which 12 were polymorphic. The expected and observed heterozygosities ranged from 0.03 to 0.65 and from 0.00 to 0.77, respectively. Positive transferability with the related species P. bahiensis was observed for the same 17 markers. Conclusions: The 12 polymorphic microsatellite markers are suitable for studies in genetic diversity and structure, mating system, and gene fl ow in P. minensis and also may be useful for similar issues regarding the related species P. bahiensis38CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informaçãoSem informação2012/12927-

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

Abstract: 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