Genome sequencing of a Hevea brasiliensis for single nucleotide polymorphism discovery

The rubber tree (Hevea spp.), is the primary plant used in natural rubber production. Historically, the breeding of rubber trees has been based on techniques involving statistics and quantitative genetic approaches to determine the best genotypes to be used as new cultivars. The discovery of molecular genetic markers has provided new possibilities for characterizing genotypes for the purpose of identifying cultivars, analyzing genetic diversity, establishing relationships between agricultural traits and genetic factors (QTLs), and identifying genes of interest. The application of next generation sequencing technology has brought a new opportunity for high throughput single nucleotide polymorphism (SNP) discovery. Knowledge about SNPs markers is extremely important in the development of genotyping assays, allowing improvements in plant breeding through marker-assisted selection. In this project, we carried out genomic sequencing of two rubber tree cultivars. The DNA libraries were constructed for two cultivars of rubber tree (two from GT1 and two from RRIM701) and sequenced in Illumina plataform. The resulting short reads (72 bp) were submitted to quality filtering and then were de novo assembled using the CLC software. Next, Burrows-Wheeler Aligner (BWA) aligned short reads back to assembled contigs, and Freebayes was used to perform variant calling and snp detection. Genotype likelihoods were computed and variable positions in the aligned reads were compared to the reference contigs. Using the varFilter command of VCFutils script, SNPs were filtered only for positions with a minimal mapping quality (-Q) and coverage (-d) of 30 and 10 respectively. Unique and shared SNPs among the two cultivars were extracted with the VCFtools software. SNPs located in contigs containing open reading frames (ORFs) ?200 bp were also extracted using transdecoder script. A total of 10,993,648 reads were obtained. Only 10,071 contigs were retained after assembly and removal of singletons and repetitive regions. The contig length median was 3078 bp (N50), and GC content was 35.4%. After the step of clustering and homology search against H. brasiliensis draft genome via blastx, the remaining 6,995 contigs were used as reference for mapping and SNP calling. In total, Freebayes detected a total of 59,116 (39,455 transitions and 19,812 transversions, Ts/Tv=1,99) different heterozygous SNP position in sequences using the stringent parameters. Of these SNPs, 41,621 (70.4%) were found in contigs containing predicted open reading frames (ORFs). Regarding different genotypes, 19,118 putative SNPs were found in GT1 and 25,360 in RRIM701. These cultivars shared 14,568 SNPs positions. The results show that it is possible to combine next generation sequencing data combined with high-density SNP detection methods to discover large numbers of putative SNPs in Hevea brasiliensis, providing a framework for further population genomic studies to identify the molecular basis underlying phenotypic variation of relevant traits in a non-model species. Financial Support: FAPESP. (Résumé d'auteur

Population genetic structure based on microsatellite markers in rubber tree (Hevea brasiliensis) germplasm collection : [P483]

Hevea brasiliensis is native to the Amazonian rain forest and an important source of natural rubber. All high-yielding cultivars of rubber tree in the world originated from breeding programs initially developed in Southeast Asia with a very narrow genetic basis. Germplasm diversity is the mainstay for crop improvement and genetic dissection of complex traits. Understanding genetic diversity, population structure, and the level and distribution of linkage disequilibrium (LD) in target populations is of great importance and a prerequisite for association mapping. A part of germplasm collection is used here and 192 accessions from Agronomic Institute of Campinas (IAC) and Eduard Michelin Plantation were screened with 15 microsatellite markers (SSR). Genomic DNA was extracted from young leaf tissues and was amplified by PCR. The Mean number of alleles per locus, mean expected heterozygosity and polymorphic information content across all accessions were 16.58, 0.90, and 0.89, respectively. The STRUCTURE analyses identified three subgroups among this population panel. The consensus neighbor-joining tree resulted in three clusters that generally agreed with three subgroups from the structure analyses. Overall, the results suggest that these populations can be exploited for assess the extent and genome-wide distribution of linkage disequilibrium (LD). Additional molecular markers, including more SSRs and single nucleotide polymorphisms (SNPs), should be used to provide better coverage of the genome and more genotyping should be initiated with the germplasm collection. In addition, the germplasm collection can also be used in the selection of parents for future crosses, based on genetic distance of the accessions. (Résumé d'auteur

Functional annotation of genomic sequences of Hevea brasiliensis

The rubber tree (Hevea brasiliensis) is a recent domesticated endemic tree species from Amazonian rain forest. It is an important crop to rubber industries due the latex producing, which has better quality compared to the synthetic rubber. Difficulties for genetic improvement were attributed to its perennial nature and long juvenile period, so genomic resources should provide insights into the chromosome organization and help elucidate the regulatory architecture of loci involved in biological processes, including metabolism, growth, development and immunity mechanisms. In this context, functional annotation were developed from the genomic sequencing of two rubber tree cultivars (GT1 and RRIM 701) aiming the better understanding of the Hevea genome. DNA sequencing libraries were previously constructed from the cultivars of rubber tree using the Illumina GAIIx. A stringent filtering resulted in 6,995 contigs, which were screened for homolog sequences through Blastx search (e-value: ? 10-6) against NCBI's non-redundant protein database. Also, a bi-directional best hit blast was performed against Kyoto Encyclopedia of Genes and Genomes (KEGG) database using KEGG Automatic Annotation Server. After the step of clustering and homology search via blast, 2,369 ORFs were found, from which, 490 complete, 748 internal and 1,131 partial. Blastx search returned a total of 2,176 hits for a range of plant species. Among the various plants that have protein sequences in GenBank the top-hit species were attributed to Ricinus communis (1,146 hits), followed by Populus trichocarpa (350 hits) and Vitis vinifera (214 hits).A total of 1,523 ORFs were assigned to Gene Ontology (GO) categories, which were distributed to cellular components (33.53%), biological processes (40.40%) and molecular functions (26.07%). The most representatives GO terms among the biological processes were metabolic processes (11.05%) and cellular processes (9.94%). Both Hevea brasiliensis and Ricinus communis are classified in the Euphorbiaceae family, which may explain for the high similarities that were observed. Representative sequences were mapped to important pathways, such as glycolysis, carbon fixation, starch and sucrose metabolism, amino acid biosynthesis, plant hormone signal transduction, biosynthesis of secondary metabolites and plant-pathogen interactions, using the KEGG database. The possibility of utilize genome sequence information form a potential resource to study the biology, evolution, and future genomic-assisted crop improvements of the rubber tree. Financial support: FAPESP and CNPq. (Résumé d'auteur

Development of SNPs markers (single nucleotide polymorphisms) presents in regulatory regions and genotyping of mapping population Hevea brasiliensis

The rubber tree (Hevea spp.), primary plant used for natural rubber production, have a long breeding cycle. However, the advent of molecular genetic markers favored early assessment methods, allowing the characterization of genotypes, genetic diversity analysis, QTL analysis and identifying genes of interest. In this context, this work aimed at the development and genotyping of new SNP markers presents in regulatory regions in two populations of genetic mapping of rubber tree. The raw data for each pool of samples (GT1 and RRIM701 genotypes) were separately trimmed and de novo assembled in a unique file using the CLC Genomics Workbench software (Version 6.0.1, CLC Bio, Denmark). Next, BWA aligned short reads back to assembled contigs, and Freebayes was used to perform variant calling and SNP detection. Using the varFilter command of vcfutils script, SNPs were filtered only for positions with a minimal mapping quality and coverage of 30 and 10 respectively. Unique and shared SNPs among the two cultivars were extracted with the VCFtools software. The selected SNPs annotation was made in two databases of cis regions: PLACE and ALGEEN-PROMO. Identification of SNP allelic forms was performed using KASPAR chemistry, either with PCR final point analysis on rtPCR equipment (LC480) for testing the polymorphism of the parents, or with Fluidigm technology for progeny genotyping. We obtained an average of 76% success rate in genotyping. The results will be used for saturation of molecular genetic map of H. brasiliensis, the location of these cis regions mutated on the map. (Résumé d'auteur

Método cromatográfico para análise de Ácido salicílico nos frutos e folhas do morangueiro (Fragaria X Ananassa Duch.).

Nota Técnica

Technical performance of milk producers in the state of Goiás , Brazil, in the short and long terms

The objective of this study was to identify groups of technologically homogeneous milk producers in the State of Goiás and calculate the gains for inefficient producers, considering the ones of better performance (benchmarks) with the same technological level, i.e., in the short term, and the benchmarks in higher technological level, i.e., in the long term. Multivariate statistical analysis techniques were used for the formation of homogeneous producer groups and multi-stage model of data envelopment analysis (DEA) for the estimation of efficiency scores and identification of benchmarks. The results indicated the formation of four groups of producers and indicate that, on average, the dairy farming in Goiás presents mainly characteristics of traditional, less specialized production. Estimated average efficiency for producers as a whole, or efficiency of long-term, was 0.571. The short-term results vary according to the group analyzed, but were higher than the long-term values, indicating that the factor analysis promoted technological homogeneity between the producers of the same group. Results showed a trend in dairy farming of Goiás towards adequacy of the input/output ratio to the standards of less capitalintensive production systems

Beef quality traits of Nellore, F1 Simmental × Nellore and F1 Angus × Nellore steers fed at the maintenance level or ad libitum with two concentrate levels in the diet

This trial was conducted to evaluate some beef quality attributes of Nellore, F1 Simmental × Nellore and F1 Angus × Nellore steers finished on feedlot. The effects of feeding regime and genetic group on shear force, thawing losses, cooking (leak + evaporation) losses, total losses and muscle fiber type, as well as carcass pH and temperature during 24 h of chilling were evaluated. There was a genetic group effect on shear force, where the beef from F1 Simmental × Nellore and F1 Angus × Nellore animals had lower values than Nellore animals. Beef of the animals fed the diets with 1% and 2% of body weight on concentrated lost more liquid than the meat of the animals fed at maintenance during thawing and when considering total losses. During cooking there was a difference among the feeding regimes for drip losses which were greater on the animals fed the diet of 1% of body weight on concentrate, followed by the 2% diet and, finally, by the animals fed at maintenance. The muscle of the Nellore steers had larger proportion of intermediate fibers and lower proportion of oxidative fibers than the crossbred animals. The proportion of glycolytic fibers was not influenced by genetic group. The Nellore animals had larger proportion of fibers of fast contraction and smaller proportion of fibers of slow contraction when compared with the crossbred animals. Feeding regime did not influence the proportion of muscular fibers or shear force. Nellore cattle produce tougher beef than crossbred Simmental × Nellore or Angus × Nellore, although all of them have the potential to produce an acceptable beef when slaughtered at young age. Feed restriction up to 90 days is not enough to cause modification on muscle fiber frequencies, then not affecting beef quality