Vânia C. R. Azevedo

Analysis of the leaf transcriptome of Musa acuminata during interaction with Mycosphaerella musicola: gene assembly, annotation and marker development.

BackgroundAlthough banana (Musa sp.) is an important edible crop, contributing towards poverty alleviation and food security, limited transcriptome datasets are available for use in accelerated molecular-based breeding in this genus. 454 GS-FLX Titanium technology was employed to determine the sequence of gene transcripts in genotypes of Musa acuminata ssp. burmannicoides Calcutta 4 and M. acuminata subgroup Cavendish cv. Grande Naine, contrasting in resistance to the fungal pathogen Mycosphaerella musicola, causal organism of Sigatoka leaf spot disease. To enrich for transcripts under biotic stress responses, full length-enriched cDNA libraries were prepared from whole plant leaf materials, both uninfected and artificially challenged with pathogen conidiospores.ResultsThe study generated 846,762 high quality sequence reads, with an average length of 334 bp and totalling 283 Mbp. De novo assembly generated 36,384 and 35,269 unigene sequences for M. acuminata Calcutta 4 and Cavendish Grande Naine, respectively. A total of 64.4% of the unigenes were annotated through Basic Local Alignment Search Tool (BLAST) similarity analyses against public databases.Assembled sequences were functionally mapped to Gene Ontology (GO) terms, with unigene functions covering a diverse range of molecular functions, biological processes and cellular components. Genes from a number of defense-related pathways were observed in transcripts from each cDNA library. Over 99% of contig unigenes mapped to exon regions in the reference M. acuminata DH Pahang whole genome sequence. A total of 4068 genic-SSR loci were identified in Calcutta 4 and 4095 in Cavendish Grande Naine. A subset of 95 potential defense-related gene-derived simple sequence repeat (SSR) loci were validated for specific amplification and polymorphism across M. acuminata accessions. Fourteen loci were polymorphic, with alleles per polymorphic locus ranging from 3 to 8 and polymorphism information content ranging from 0.34 to 0.82.ConclusionsA large set of unigenes were characterized in this study for both M. acuminata Calcutta 4 and Cavendish Grande Naine, increasing the number of public domain Musa ESTs. This transcriptome is an invaluable resource for furthering our understanding of biological processes elicited during biotic stresses in Musa. Gene-based markers will facilitate molecular breeding strategies, forming the basis of genetic linkage mapping and analysis of quantitative trait loci.

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.

Isolation and characterization of microsatellite loci for Hymenaea courbaril and transferability to Hymenaea stigonocarpa, two tropical timber species

Hymenaea courbaril is a tropical timber species, intensely exploited and found in the Amazon, Atlantic Forest and Brazilian Cerrado biome. Nine highly polymorphic microsatellite loci were developed from a genomic library enriched for AG/TC repeats. In a total of 41 individuals, from two natural populations, seven to 13 alleles per locus were detected and expected heterozygosity ranged from 0.75 to 0.90. Seven loci were effectively transferred to Hymenaea stigonocarpa. High levels of polymorphism make the present primers useful for population genetic studies and are a powerful tool to investigate mating system, gene flow and spatial genetic structure.

Genetic structure of Bertholletia excelsa populations from the Amazon at different spatial scales

Population genetic structure and genetic diversity levels are important issues to understand population dynamics and to guide forest management plans. The Brazil nut tree (Bertholletia excelsa Bonpl.) is an endemic species, widely distributed through Amazonian upland forests and also an important species for the local extractive economy. Our aim was to analyze the genetic structure of Brazil nut trees at both fine and large scales throughout the Amazon Basin, contributing to the knowledge base on this species and to generate information to support plans for its conservation. We genotyped individuals from nine sites distributed in five regions of the Brazilian Amazon using 11 microsatellite loci. We found an excess of heterozygotes in most populations, with significant negative inbreeding coefficients (f) for five of them and the fine-scale structure, when present, was very small. These results, as a consequence of self-incompatibility, indicate that conservation plans for B. excelsa must include the maintenance of genetic diversity within populations to ensure viable amounts of seeds for both economic purposes and for the local persistence of the species.

Development of expressed sequence tag and expressed sequence tag-simple sequence repeat marker resources for Musa acuminata.

Many varieties of banana (Musa acuminata) lack resistance to biotic stresses. An EST collection was developed, including transcripts expressed in banana-Mycosphaerella fijiensis interactions. Developed polymorphic gene-derived SSR markers are applicable for genetic mapping, diversity characterization and marker assisted breeding.

Diversidade genética e tamanho efetivo de duas populações de Myracrodruon urundeuva Fr. All., sob conservação ex situ

O objetivo deste trabalho foi investigar a diversidade, a estrutura genetica e o tamanho efetivo, retido em um banco de germoplasma, de duas populacoes de Myracrodruon urundeuva Fr. All. (aroeira), procedentes de Aramina-SP e Selviria-MS. As populacoes foram avaliadas a partir da amostragem de 25 progenies de polinizacao aberta de cada populacao. De cada progenie, 17 a 20 individuos foram amostrados e genotipados para oito locos microssatelites. Os maiores valores para o numero total de alelos (At), numero medio de alelos por loco (A), numero efetivo de alelos (Ae), heterozigosidade observada (H0) e esperada (He) foram detectados na populacao Selviria (At = 105, A = 13,13, Ae = 3,98, H0 = 0,669 e He = 0,749), enquanto Aramina teve At = 94, A = 13,75, Ae = 3,10, H 0 = 0,535 e He = 0,678. A diferenciacao nas frequencias alelicas das duas populacoes, com relacao ao polen cruzado (0,159) e do ovulo (0,235), indica que cerca de 84% e 77%, respectivamente, da diversidade genetica esta dentro das populacoes. O coeficiente medio de coancestria foi maior (Selviria Θ = 0,165, Aramina Θ = 0,169) e o tamanho efetivo medio das progenies (Selviria Ne(v) = 3,04, Aramina Ne(v) = 2,69) foi menor do que o esperado em progenies de populacoes panmiticas (Θ = 0,125, Ne(v) = 4). O tamanho efetivo total retido no banco ex situ foi estimado em 67,5 na populacao Selviria e 71,1 na populacao Aramina, valores menores do que o requerido (Ne = 150) para a conservacao de populacoes em curto prazo. Entretanto, as duas populacoes apresentaram alta diversidade genetica, o que as qualifica para serem utilizadas em programas de conservacao e melhoramento genetico da especie, desde que seja aumentado o tamanho efetivo populacional conservado ex situ.

Mating system variation among populations, individuals and within and among fruits in Bertholletia excelsa

Abstract The aim of this study was to investigate variation in mating system among three Brazilian Amazon populations of the tree Bertholletia excelsa with different levels of anthropogenic interventions. We collected open-pollinated seeds from one natural population, remnant trees dispersed in a pasture, and trees from a plantation. Outcrossing rate not varied among the populations and indicates that all seeds were originated from outcrossing (tm=1.0). Mating among relatives was significant higher in the plantation than forest and pasture populations, probably due the fact that many trees are related in the plantation. Correlated mating was significantly higher in pasture (rp=0.47) and plantation (rp=0.51) than in the natural population (rp=0.22), suggesting that trees in natural population are pollinated by a higher number of pollen donors. The paternity correlation was significantly higher within (rp(w)=0.41) than among fruits (rp(a)=0.18), showing a higher probability to find full-sibs within than among fruits. The fixation index was generally lower in seed trees than in their seedlings, suggesting selection for heterozygous individuals from seedling to adult stages. Progeny arrays collected from the natural population had a lower proportion of pairwise full-sibs than in pasture and plantation and higher variance effective size (2.75) than trees in pasture (2.15) and plantations (2.22). Results highlight that seed collections for conservation, breeding and reforestation programs preferentially should be carried out in natural populations due low proportion highest variance effective size within progeny.

Shotgun sequencing for microsatellite identification in Ilex paraguariensis (Aquifoliaceae).

Premise of the study: Ilex paraguariensis is a native tree species from Brazil, Argentina, and Paraguay that is used in the production of beverages, medicines, and cosmetics. Primers flanking microsatellites were developed to investigate genetic parameters in the species. Methods and Results: Using microsatellites cloned from an I. paraguariensis shotgun genomic library, 25 pairs of primers were designed and synthesized. Levels of polymorphism were evaluated in 24 individuals from two populations. Twenty loci were polymorphic, and an average of 4.8 and 4.5 alleles per locus were detected in the two populations, respectively. The mean observed heterozygosity was lower than the expected heterozygosity (0.54 vs. 0.60), indicating a departure from Hardy—Weinberg equilibrium and suggesting endogamy in both populations. Conclusions: The reported set of markers is highly informative and constitutes a powerful tool for the development of genetic characterization studies in I. paraguariensis.

Gene flow between vicariant tree species: insights into savanna-forest evolutionary relationships

Studying the genetic structure of vicariant species (i.e., closely related species that occupy ecologically distinct yet adjacent habitats) can shed light on the evolution and divergence of species with different ecological requirements. A previous phylogeographic study identified chloroplast DNA haplotype sharing between two vicariant tree species, one from forest (Hymenaea courbaril) and one from savanna (H. stigonocarpa) habitats. These species co-occur in the Brazilian Cerrado, a biome that encompasses forest patches and riverine forests within a savanna matrix. In order to investigate the evolutionary processes involved in the genetic divergence of these trees, we used nuclear microsatellite markers, statistical methods including approximate Bayesian computation (ABC), and leaf morphology to analyze neighboring and distant populations. Bayesian analysis revealed admixture between the species. ABC analysis supported the scenarios with the occurrence of gene flow between species during the Last Glacial Maximum or from the Holocene to the present, when compared to alternative scenarios of no gene flow or constant gene flow since divergence. However, putative hybrids did not exhibit intermediate leaflet morphology, which could be related to distinct selective pressures maintaining species integrity even in the face of gene flow. Our results suggest that despite morphological differences between savanna and forest species, interspecific barriers to gene flow might not be fully developed between vicariant tree species and that interspecific hybridization in trees from Cerrado biome may be an underdiagnosed process.

Expression of stress-related genes in zebrawood (Astronium fraxinifolium, Anacardiaceae) seedlings following germination in microgravity

Seeds of a tropical tree species from Brazil, Astronium fraxinifolium, or zebrawood, were germinated, for the first time in microgravity, aboard the International Space Station for nine days. Following three days of subsequent growth under normal terrestrial gravitational conditions, greater root length and numbers of secondary roots was observed in the microgravity-treated seedlings compared to terrestrially germinated controls. Suppression subtractive hybridization of cDNA and EST analysis were used to detect differential gene expression in the microgravity-treated seedlings in comparison to those initially grown in normal gravity (forward subtraction). Despite their return to, and growth in normal gravity, the subtracted library derived from microgravity-treated seedlings was enriched in known microgravity stress-related ESTs, corresponding to large and small heat shock proteins, 14-3-3-like protein, polyubiquitin, and proteins involved in glutathione metabolism. In contrast, the reverse-subtracted library contained a comparatively greater variety of general metabolism-related ESTs, but was also enriched for peroxidase, possibly indicating the suppression of this protein in the microgravity-treated seedlings. Following continued growth for 30 days, higher concentrations of total chlorophyll were detected in the microgravity-exposed seedlings.