Mutagênese em Petunia x hybrida Vilm. e isolamento de um novo mutante morfológico

Traditionally, mutagenesis has been used to introduce novel genetic variability in ornamental crops. More recently, it has become a powerful tool in gene discovery and functional analyses in reverse genetics approaches. The present work aimed to compare the efficiency of physical and chemical agents in generating mutant populations of petunia. We have indirectly evaluated the genomic damage by analyzing developmental characteristics of the plantlets derived from treated seeds employing gamma radiation at 0, 20, 40, 60, 80 and 100 Gy and the alkylating agent ethyl-methanesulfonate (EMS) at 0, 0.05, 0.1, 0.15, 0.2 and 0.25% (v/v). Gamma rays and EMS caused developmental defects and decreased seedling viability in plants obtained from the mutagenized seeds. High mutagen doses reduced in approximately 44% the number of plants with primary leaves at 15 days after sowing (DAS) and decreased seedling survival rates to 55% (gamma) and 28% (EMS), in comparison to untreated controls. Seedling height decrease was proportional to increasing EMS dosage, whereas 40 and 60 Gy of gamma irradiation caused the most significant reduction in height. Moderate DNA damage allowing a high saturation of mutant alleles in the genome and the generation of viable plants for reverse genetics studies was correlated to the biological parameter LD50, the dose required to kill half of the tested population. It corresponded to 100 Gy for gamma radiation and 0.1% for EMS treatment. The optimized mutagen treatments were used to develop petunia mutant populations (M1 and M2) and novel morphological mutants were identified.A mutagênese tem sido tradicionalmente usada para gerar variabilidade genética em plantas ornamentais. Recentemente, tornou-se uma ferramenta poderosa na descoberta e análise da função gênica em genética reversa. Este trabalho objetivou comparar a eficiência da mutagênese física e química na geração de populações mutantes de petúnia. O dano genômico foi avaliado indiretamente por características de desenvolvimento de plântulas após o tratamento com doses de radiação gama de 0, 20, 40, 60, 80 e 100 Gy e do agente alquilante etil-metanossulfonato (EMS) de 0; 0,05; 0,1; 0,15; 0,2 e 0,25% (v/v). Radiação gama e EMS causaram danos ao desenvolvimento e reduziram a viabilidade das plântulas derivadas das sementes tratadas. As maiores doses de mutagênico diminuíram o número de plantas com folhas primárias aos 15 dias após a semeadura (DAS) em aproximadamente 44% e reduziram as taxas de sobrevivência a 55% (gama) e 28% (EMS) em relação aos controles. A redução na altura das plântulas foi proporcional ao aumento das dosagens de EMS, enquanto 40 e 60 Gy de radiação gama provocaram a redução mais significativa na altura de plantas. Abordagens de genética reversa requerem danos genômicos moderados, que permitam alta saturação de alelos mutantes com pequena redução no número de plantas viáveis, relacionados ao parâmetro biológico DL50, dosagem de mutagênico necessária para eliminar metade da população. Este valor correspondeu a 100 Gy de radiação gama e 0,1% de EMS. Os tratamentos foram empregados para a geração de populações mutantes de petúnia (M1 e M2) e novos mutantes morfológicos foram isolados.FAPES

Development and characterization of microsatellite markers for Piptadenia Gonoacantha (fabaceae)

Microsatellite primers were designed for Piptadenia gonoacantha (Fabaceae) and characterized to estimate genetic diversity parameters. The species is a native tree from the Atlantic Forest biome commonly used in forest restoration; it has medicinal potential and the wood is economically useful. Twenty-eight microsatellite loci were identified from an enriched genomic library. Fifteen loci resulted in successful amplifications and were characterized in a natural population of 94 individuals. Twelve loci were polymorphic, with allele numbers ranging from three to 15 per locus, and expected and observed heterozygosities ranging from 0.2142 to 0.8325 and 0.190 to 0.769, respectively. The developed markers will be used in further studies of population genetics of P. gonoacantha, aimed at conservation and management of the species in natural populations and in forest restoration projects32CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP140036/2011-3sem informaçãoBiota/FAPESP-11/50296-

Molecular Genetic Variability, Population Structure and Mating System in Tropical Forages

Microsatellite (SSR) markers were developed for the following tropical forage species, using accessions available from the plant genetic resources (PGR) collections held by EMBRAPA (Brazilian Agricultural Research Corporation): Brachiaria brizantha, B. humidicola, Panicum maximum, Paspalum spp., Stylosanthes capitata, S. guianensis, S. macrocephala, Calopogonium mucunoides and Centrosema spp. The markers were used to analyse population structure and genetic diversity, evolution and origin of the genetic variability in the centre of origin, mating systems and genetic resources in EMBRAPA’s germplasm bank. The results shed light on the amount of genetic variation within and between populations, revealed the need in some cases for further plant collection to adequately represent the species in PGR collections, allowed us to assemble core collections (subsets of the total collections) that should contain most of the available diversity and (in the case of the legumes) showed the need to avoid unwanted outcrossing when regenerating conserved material. The data will allow plant breeders to better select accessions for hybrid production, discriminate between genotypes and use marker-assisted selection in breeding programs. Our results will also underpin the construction of genetic maps, mapping of genes of agronomic interest and numerous other studies on genetic variability, population structure, gene flow and reproductive systems for the tropical forage species studied in this work

Genetic divergence of rubber tree estimated by multivariate techniques and microsatellite markers

Genetic diversity of 60 Hevea genotypes, consisting of Asiatic, Amazonian, African and IAC clones, and pertaining to the genetic breeding program of the Agronomic Institute (IAC), Brazil, was estimated. Analyses were based on phenotypic multivariate parameters and microsatellites. Five agronomic descriptors were employed in multivariate procedures, such as Standard Euclidian Distance, Tocher clustering and principal component analysis. Genetic variability among the genotypes was estimated with 68 selected polymorphic SSRs, by way of Modified Rogers Genetic Distance and UPGMA clustering. Structure software in a Bayesian approach was used in discriminating among groups. Genetic diversity was estimated through Nei's statistics. The genotypes were clustered into 12 groups according to the Tocher method, while the molecular analysis identified six groups. In the phenotypic and microsatellite analyses, the Amazonian and IAC genotypes were distributed in several groups, whereas the Asiatic were in only a few. Observed heterozygosity ranged from 0.05 to 0.96. Both high total diversity (HT' = 0.58) and high gene differentiation (G st' = 0.61) were observed, and indicated high genetic variation among the 60 genotypes, which may be useful for breeding programs. The analyzed agronomic parameters and SSRs markers were effective in assessing genetic diversity among Hevea genotypes, besides proving to be useful for characterizing genetic variability

Population structures of Brazilian tall coconut (Cocos nucifera L.) by microsatellite markers

Coconut palms of the Tall group were introduced to Brazil from the Cape Verde Islands in 1553. The present study sought to evaluate the genetic diversity among and within Brazilian Tall coconut populations. Samples were collected of 195 trees from 10 populations. Genetic diversity was accessed by investigating 13 simple sequence repeats (SSR) loci. This provided a total of 68 alleles, ranging from 2 to 13 alleles per locus, with an average of 5.23. The mean values of gene diversity (He ) and observed heterozygosity (Ho ) were 0.459 and 0.443, respectively. The genetic differentiation among populations was estimated at θ^P=0.1600and the estimated apparent outcrossing rate was ta = 0.92. Estimates of genetic distances between the populations varied from 0.034 to 0.390. Genetic distance and the corresponding clustering analysis indicate the formation of two groups. The first consists of the Baía Formosa, Georgino Avelino, and São José do Mipibu populations and the second consists of the Japoatã, Pacatuba, and Praia do Forte populations. The correlation matrix between genetic and geographic distances was positive and significant at a 1% probability. Taken together, our results suggest a spatial structuring of the genetic variability among the populations. Geographically closer populations exhibited greater similarities

High levels of genetic differentiation and selfing in the Brazilian cerrado fruit tree Dipteryx alata Vog. (Fabaceae)

Dipteryx alata is a native fruit tree species of the cerrado (Brazilian savanna) that has great economic potential because of its multiple uses. Knowledge of how the genetic variability of this species is organized within and among populations would be useful for genetic conservation and breeding programs. We used nine simple sequence repeat (SSR) primers developed for Dipteryx odorata to evaluate the genetic structure of three populations of D. alata located in central Brazil based on a leaf sample analysis from 101 adults. The outcrossing rate was evaluated using 300 open-pollinated offspring from 25 seed-trees. Pollen dispersal was measured by parentage analysis. We used spatial genetic structure (SGS) to test the minimal distance for harvesting seeds in conservation and breeding programs. Our data indicate that the populations studied had a high degree of genetic diversity and population structure, as suggested by the high level of divergence among populations . The estimated outcrossing rate suggested a mixed mating system, and the intrapopulation fixation index was influenced by SGS. We conclude that seed harvesting for genetic conservation and breeding programs requires a minimum distance between trees of 196 m to avoid collecting seeds from related seed-trees

Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data

INTRODUCTION: Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells. METHODS: Malignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays. RESULTS: MPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most highly overexpressed genes in this category, that encoding periostin, was analysed immunohistochemically on breast cancer tissue microarrays and its expression in neoplastic cells correlated with poor outcome in a cohort of poor prognosis estrogen receptor-positive tumours. CONCLUSION: Using highly enriched cell populations in combination with multiplatform gene expression profiling studies, a comprehensive analysis of molecular changes between the normal and malignant breast tissue was established. This study provides a basis for the identification of novel and potentially important targets for diagnosis, prognosis and therapy in breast cancer

Genetic diversity in cultivated carioca common beans based on molecular marker analysis

A wide array of molecular markers has been used to investigate the genetic diversity among common bean species. However, the best combination of markers for studying such diversity among common bean cultivars has yet to be determined. Few reports have examined the genetic diversity of the carioca bean, commercially one of the most important common beans in Brazil. In this study, we examined the usefulness of two molecular marker systems (simple sequence repeats – SSRs and amplified fragment length polymorphisms – AFLPs) for assessing the genetic diversity of carioca beans. The amount of information provided by Roger’s modified genetic distance was used to analyze SSR data and Jaccards similarity coefficient was used for AFLP data. Seventy SSRs were polymorphic and 20 AFLP primer combinations produced 635 polymorphic bands. Molecular analysis showed that carioca genotypes were quite diverse. AFLPs revealed greater genetic differentiation and variation within the carioca genotypes (Gst = 98% and Fst = 0.83, respectively) than SSRs and provided better resolution for clustering the carioca genotypes. SSRs and AFLPs were both suitable for assessing the genetic diversity of Brazilian carioca genotypes since the number of markers used in each system provided a low coefficient of variation. However, fingerprint profiles were generated faster with AFLPs, making them a better choice for assessing genetic diversity in the carioca germplasm