Marcos Mota do Carmo Costa

Brazilian coffee genome project: an EST-based genomic resource

Coffee is one of the most valuable agricultural commodities and ranks second on international trade exchanges. The genus Coffea belongs to the Rubiaceae family which includes other important plants. The genus contains about 100 species but commercial production is based only on two species, Coffea arabica and Coffea canephora that represent about 70 % and 30 % of the total coffee market, respectively. The Brazilian Coffee Genome Project was designed with the objective of making modern genomics resources available to the coffee scientific community, working on different aspects of the coffee production chain. We have single-pass sequenced a total of 214,964 randomly picked clones from 37 cDNA libraries of C. arabica, C. canephora and C. racemosa, representing specific stages of cells and plant development that after trimming resulted in 130,792, 12,381 and 10,566 sequences for each species, respectively. The ESTs clustered into 17,982 clusters and 32,155 singletons. Blast analysis of these sequences revealed that 22 % had no significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function). The generated coffee EST database resulted in the identification of close to 33,000 different unigenes. Annotated sequencing results have been stored in an online database at http://www.lge.ibi.unicamp.br/cafe. Resources developed in this project provide genetic and genomic tools that may hold the key to the sustainability, competitiveness and future viability of the coffee industry in local and international markets.

Characterization of novel microsatellite markers in Musa acuminata subsp. burmannicoides , var. Calcutta 4

BackgroundBanana is a nutritionally important crop across tropical and sub-tropical countries in sub-Saharan Africa, Central and South America and Asia. Although cultivars have evolved from diploid, triploid and tetraploid wild Asian species of Musa acuminata (A genome) and Musa balbisiana (B genome), many of todays commercial cultivars are sterile triploids or diploids, with fruit developing via parthenocarpy. As a result of restricted genetic variation, improvement has been limited, resulting in a crop frequently lacking resistance to pests and disease. Considering the importance of molecular tools to facilitate development of disease resistant genotypes, the objectives of this study were to develop polymorphic microsatellite markers from BAC clone sequences for M. acuminata subsp. burmannicoides, var. Calcutta 4. This wild diploid species is used as a donor cultivar in breeding programs as a source of resistance to diverse biotic stresses.FindingsMicrosatellite sequences were identified from five Calcutta 4 BAC consensi datasets. Specific primers were designed for 41 loci. Isolated di-nucleotide repeat motifs were the most abundant, followed by tri-nucleotides. From 33 tested loci, 20 displayed polymorphism when screened across 21 diploid M. acuminata accessions, contrasting in resistance to Sigatoka diseases. The number of alleles per SSR locus ranged from two to four, with a total of 56. Six repeat classes were identified, with di-nucleotides the most abundant. Expected heterozygosity values for polymorphic markers ranged from 0.31 to 0.75.ConclusionsThis is the first report identifying polymorphic microsatellite markers from M. acuminata subsp. burmannicoides, var. Calcutta 4 across accessions contrasting in resistance to Sigatoka diseases. These BAC-derived polymorphic microsatellite markers are a useful resource for banana, applicable for genetic map development, germplasm characterization, evolutionary studies and marker assisted selection for traits.

Expressed sequence-tag analysis of ovaries of Brachiaria brizantha reveals genes associated with the early steps of embryo sac differentiation of apomictic plants

In apomixis, asexual mode of plant reproduction through seeds, an unreduced megagametophyte is formed due to circumvented or altered meiosis. The embryo develops autonomously from the unreduced egg cell, independently of fertilization. Brachiaria is a genus of tropical forage grasses that reproduces sexually or by apomixis. A limited number of studies have reported the sequencing of apomixis-related genes and a few Brachiaria sequences have been deposited at genebank databases. This work shows sequencing and expression analyses of expressed sequence-tags (ESTs) of Brachiaria genus and points to transcripts from ovaries with preferential expression at megasporogenesis in apomictic plants. From the 11 differentially expressed sequences from immature ovaries of sexual and apomictic Brachiaria brizantha obtained from macroarray analysis, 9 were preferentially detected in ovaries of apomicts, as confirmed by RT-qPCR. A putative involvement in early steps of Panicum-type embryo sac differentiation of four sequences from B. brizantha ovaries: BbrizHelic, BbrizRan, BbrizSec13 and BbrizSti1 is suggested. Two of these, BbrizSti1 and BbrizHelic, with similarity to a gene coding to stress induced protein and a helicase, respectively, are preferentially expressed in the early stages of apomictic ovaries development, especially in the nucellus, in a stage previous to the differentiation of aposporous initials, as verified by in situ hybridization.

Identification and expression profile of odorant-binding proteins in Halyomorpha halys (Hemiptera: Pentatomidae)

The brown marmorated stink bug, Halyomorpha halys, is a devastating invasive species in the USA. Similar to other insects, olfaction plays an important role in its survival and reproduction. As odorant‐binding proteins (OBPs) are involved in the initial semiochemical recognition steps, we used RNA‐Sequencing (RNA‐Seq) to identify OBPs in its antennae, and studied their expression pattern in different body parts under semiochemical stimulation by either aggregation or alarm pheromone or food odorants. Thirty full‐length putative HhalOBPs were identified, corresponding to 22 ‘classic’ OBPs and eight ‘Plus‐C’ OBPs. The similarity amongst them ranged from 4.95–70.92%, and with another 325 hemipteran OBPs similarity ranged from 1.94–91.51%, the highest levels being with other stink bug OBPs. Phylogenetic analysis confirmed the monophyly of seven groups of stink bug and other hemipteran OBPs. All 30 HhalOBPs were expressed and about 2/3 were expressed primarily in antennae. The expression of 21 HhalOBPs was higher in the antennae under alarm pheromone stimulus, indicating that multiple OBPs may be responding to this pheromone. Two were highest in antennae under aggregation pheromone stimulus. These findings should provide a basis for understanding the physiological functions of HhalOBPs and the chemosensory perception of this pest, which may help to uncover new control targets for behavioural interference.

SisGen: A CORBA---Based Data Management Program for DNA Sequencing Projects

Biological data deluge has challenged researchers over the last decade. Expressed sequence tag (EST) analyzes provide a rapid and economical means to identify candidate genes, gene expression profiles in different cell conditions, as well as functional annotation of putative gene products. Although EST analysis tools are publicly available there is still a lack of comprehensive data analysis and management programs. This work presents SisGen , an integrated software system capable of efficiently managing multi---user genomic projects. SisGen is a Java client---server application that uses CORBA as a middleware in a multi---layer architecture. The software integrates data management an annotation pipeline in a rich graphical visualization environment. The architectural design is presented and highlights the advantages in terms of portability, interconnectivity, modularity and user interface that can be achieved with this concept.

Transcriptome-Based Identification of Highly Similar Odorant-Binding Proteins among Neotropical Stink Bugs and Their Egg Parasitoid

Olfaction plays a fundamental role in insect survival through resource location and intra and interspecific communications. We used RNA-Seq to analyze transcriptomes for odorant-binding proteins (OBPs) from major stink bug pest species in Brazil, Euschistus heros, Chinavia ubica, and Dichelops melacanthus, and from their egg parasitoid, Telenomus podisi. We identified 23 OBPs in E. heros, 25 OBPs in C. ubica, 9 OBPs in D. melacanthus, and 7 OBPs in T. podisi. The deduced amino acid sequences of the full-length OBPs had low intraspecific similarity, but very high similarity between two pairs of OBPs from E. heros and C. ubica (76.4 and 84.0%) and between two pairs of OBPs from the parasitoid and its preferred host E. heros (82.4 and 88.5%), confirmed by a high similarity of their predicted tertiary structures. The similar pairs of OBPs from E. heros and C. ubica may suggest that they have derived from a common ancestor, and retain the same biological function to bind a ligand perceived or produced in both species. The T. podisi OBPs similar to E. heros were not orthologous to any known hymenopteran OBPs, and may have evolved independently and converged to the host OBPs, providing a possible basis for the host location of T. podisi using E. heros semiochemical cues.

The generation challenge programme platform: semantic standards and workbench for crop science.

The Generation Challenge programme (GCP) is a global crop research consortium directed toward crop improvement through the application of comparative biology and genetic resources characterization to plant breeding. A key consortium research activity is the development of a GCP crop bioinformatics platform to support GCP research. This platform includes the following: (i) shared, public platform-independent domain models, ontology, and data formats to enable interoperability of data and analysis flows within the platform; (ii) web service and registry technologies to identify, share, and integrate information across diverse, globally dispersed data sources, as well as to access high-performance computational (HPC) facilities for computationally intensive, high-throughput analyses of project data; (iii) platform-specific middleware reference implementations of the domain model integrating a suite of public (largely open-access/-source) databases and software tools into a workbench to facilitate biodiversity analysis, comparative analysis of crop genomic data, and plant breeding decision making.

First microsatellite markers developed from Cupuassu ESTs: Application in diversity analysis and cross-species transferability to cacao

The cupuassu tree (Theobroma grandiflorum) (Willd. ex Spreng.) Schum. is a fruitful species from the Amazon with great economical potential, due to the multiple uses of its fruit´s pulp and seeds in the food and cosmetic industries, including the production of cupulate, an alternative to chocolate. In order to support the cupuassu breeding program and to select plants presenting both pulp/seed quality and fungal disease resistance, SSRs from Next Generation Sequencing ESTs were obtained and used in diversity analysis. From 8,330 ESTs, 1,517 contained one or more SSRs (1,899 SSRs identified). The most abundant motifs identified in the EST-SSRs were hepta- and trinucleotides, and they were found with a minimum and maximum of 2 and 19 repeats, respectively. From the 1,517 ESTs containing SSRs, 70 ESTs were selected based on their functional annotation, focusing on pulp and seed quality, as well as resistance to pathogens. The 70 ESTs selected contained 77 SSRs, and among which, 11 were polymorphic in cupuassu genotypes. These EST-SSRs were able to discriminate the cupuassu genotype in relation to resistance/susceptibility to witches’ broom disease, as well as to pulp quality (SST/ATT values). Finally, we showed that these markers were transferable to cacao genotypes, and that genome availability might be used as a predictive tool for polymorphism detection and primer design useful for both Theobroma species. To our knowledge, this is the first report involving EST-SSRs from cupuassu and is also a pioneer in the analysis of marker transferability from cupuassu to cacao. Moreover, these markers might contribute to develop or saturate the cupuassu and cacao genetic maps, respectively.

SNP discovery in apple cultivars using next generation sequencing

Background Knowledge about single nucleotide polymorphism (SNP) markers is extremely important in the development of genotyping assays, allowing improvements in plant breeding through marker-assisted selection. With the emergence of next generation sequencing platforms, high-density SNP discovery in the genome of plant crops becomes more achievable. In this project, we carried out whole genome resequencing of two apple cultivars (M13/91 and Fred Hough, developed by Epagri [1,2]) using the recently released draft genome sequence of the domesticated apple genome (Malus x domestica Borkh.) [3] as a reference for SNP discovery.

p2pBIOFOCO: Proposing a Peer-to-Peer System for Distributed BLAST Execution

The peer-to-peer (P2P) model is currently being used for applications that demand great amount of time and space resources, taking advantage of idle and geographically distributed machines. Bioinformatics is a field that requires large amount of computational resources. In this context, we propose a P2P model that executes a particular bioinformatics application, named BLAST. This is a method for comparing two biological sequences at a time. But it requires a great amount of computation, since it compares a big file containing thousands of sequences that are being investigated and a very large database containing millions of biological sequences. We describe in detail the architecture of our framework, and present experiments using real biological data, that were executed on machines available in three institutions of Brazil - University of Brasilia, Catholic University of Brasilia and Embrapa/Genetic Resources and Biotechnology. The obtained results, when comparing the execution of BLAST in this P2P framework and its execution in the best machine of the three institutions, show that our P2P model can efficiently run other bioinformatics applications.