Claudia Fortes Ferreira

Genetic diversity and population structure of Musa accessions in ex situ conservation

BackgroundBanana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa.ResultsFrom the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis.ConclusionsThe data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping.

Genetic diversity of Phaeoisariopsis griseola in the State of Minas Gerais, Brazil

Due to the importance of common bean angular leaf spotin the state of Minas Gerais-Brazil and to the greatvariability of the pathogen, Phaeoisariopsisgriseola, monitoring races becomes an important toolfor breeding programs aiming at genetic resistance.The pathogenic variability of 30 isolates of the P. griseola, collected from various locations in thestate of Minas Gerais, was studied using the followingcommon bean differential series (Don Timóteo,Bolón Bayo, Montcalm, G 5686, Amendoin, G 11796,BAT 332, PAN 72, Cornell 49-242, México 54, Florde Mayo and G 2858). The first trifoliate leaf wasinoculated with a 2 × 104 conidia/mL. Plants weremaintained at 20–22 °C and 95% relativehumidity for 48 hours. Symptom evaluation wasperformed 15 days after inoculation. Thirteen raceswere identified demonstrating the wide geneticvariability of the pathogen in the state of MinasGerais. Race 63.63 was the most virulent, whereas race63.23 was the most frequent (10 of 30 isolates), beingwidely distributed among the regions studied. Thevirulence phenotype indicated that the races studiedbelonged to the Mesoamerican group, which wasconfirmed when the 30 isolates were compared to Andeanand Mesoamerican standards using RAPD markers.

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.

Agronomic and molecular characterization of gamma ray induced banana (Musa sp.) mutants using a multivariate statistical algorithm

Bananas are tropical fruits grown worldwide playing a key role in market trade and especially used as main food source for low income populations. In Brazil, bananas are mainly consumed in natura, occupying the second largest internal market. Nevertheless, this crop presents low availability of productive commercial varieties with good agronomic characteristics. A strategy undertaken to solve this problem is the development of new cultivars through conventional genetic breeding methods. However, this strategy presents some obstacles such as female sterility and low number of seeds. In order to overcome these shortcomings, use of mutation induction aiming the selection of mutants with desirable agronomic characteristics seems to have great potential for developing new cultivars. The objective of the present work was to evaluate the genetic variability in putative banana ‘Pacovan’ (AAB genome, subgroup Prata Type) mutants submitted to gamma ray irradiation, using a set of agronomical and molecular data (ISSR markers). The distance between the putative ‘Pacovan’ mutants varied from 0.26 to 0.64 with cophenetic correlation coefficient of 0.7669. Four mutants were selected based on best agronomical characteristics and height. This data also shows that there is variability that can be explored after the irradiation of ‘Pacovan’ banana mutants, which can be used in the genetic breeding program of banana aiming to develop short new varieties that also present good agronomic characteristics. This is the first attempt to use combined data in order to evaluate the genetic variability in putative banana mutants.

Análise de trilha de caracteres forrageiros do capim-elefante (Pennisetum purpureum Schum.)

The main goals of this work was to obtain estimates of phenotype, genotype and residual correlation coefficients and display genotype correlations in direct and indirect effects (path analysis) of height, diameter of stem at the base and number of tillers per meter (explanatory independent variables) on dry matter production (basic dependent variable) of elephantgrass clones in two harvest periods at conditions of northern Rio de Janeiro State, Brazil. Great differences among estimates in two harvest periods were observed, however it could be concluded that height of plants at cutting influenced dry matter production mainly in conditions of high tillering capable clones. Number of tillers per meter and diameter of stem explained better dry matter production potential, acting, respectively, in an direct and inverse way, alternating according to environmental conditions during growth.

Potential of SNP markers for the characterization of Brazilian cassava germplasm

Key messageHigh-throughput markers, such as SNPs, along with different methodologies were used to evaluate the applicability of the Bayesian approach and the multivariate analysis in structuring the genetic diversity in cassavas.AbstractThe objective of the present work was to evaluate the diversity and genetic structure of the largest cassava germplasm bank in Brazil. Complementary methodological approaches such as discriminant analysis of principal components (DAPC), Bayesian analysis and molecular analysis of variance (AMOVA) were used to understand the structure and diversity of 1,280 accessions genotyped using 402 single nucleotide polymorphism markers. The genetic diversity (0.327) and the average observed heterozygosity (0.322) were high considering the bi-allelic markers. In terms of population, the presence of a complex genetic structure was observed indicating the formation of 30 clusters by DAPC and 34 clusters by Bayesian analysis. Both methodologies presented difficulties and controversies in terms of the allocation of some accessions to specific clusters. However, the clusters suggested by the DAPC analysis seemed to be more consistent for presenting higher probability of allocation of the accessions within the clusters. Prior information related to breeding patterns and geographic origins of the accessions were not sufficient for providing clear differentiation between the clusters according to the AMOVA analysis. In contrast, the FST was maximized when considering the clusters suggested by the Bayesian and DAPC analyses. The high frequency of germplasm exchange between producers and the subsequent alteration of the name of the same material may be one of the causes of the low association between genetic diversity and geographic origin. The results of this study may benefit cassava germplasm conservation programs, and contribute to the maximization of genetic gains in breeding programs.

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.

Diferenciação molecular de cultivares elites de bananeira

The objective of this work was to characaterize elite banana genotypes, and those recommended, using RAPD and microsatellites. Forty-seven RAPD primers and 34 microsatellites primers were used. A contamination essay using the AGMI 24-25 primer was also carried out. Tropical variety was considered the standard sample and the Caipira and Prata Grauda, contaminants. Markers were able to separate the cultivars according to their origin and genomic constitution as well as defined molecular profiles for some of the cultivars evaluated. Garantida, Preciosa and Pacovan Ken cultivars presented genetic similarity with both markers. The AGMI 24-25 primer demonstrated high capacity to discriminate the genotypes in the contamination essay.

Melhoramento genético da bananeira: estratégias e tecnologias disponíveis

Bananas are cultivated in more than 107 countries in an area of 4.1 million hectares with a production of 95 million tons and considered the second most produced fruit worldwide. Bananas are attacked by viruses (CMV and BSV), fungi (Yellow and Black Sigatoka and Fusarium), bacteria (Moko), nematodes and insects (Weevils). However, through genetic breeding, it is possible to achieve resistance to most pests and diseases. The center of origin of most of Musa spp. germplasm is the Asian Continent, where diploid, triploid, Tetraploid with genome Musa acuminata and Musa balbisiana bananas are found. In banana breeding, especially for disease resistance, the following methods are employed: introduction and selection of clones; hybridization (crosses between diploids and diploids, triploids and diploids and diploids with tetraploids); chromosome duplication; mutation and transgenic. Methods involving hybridization, however, the method mostly used, is limited by pathernocarpy, sterility, variable number of ploidy levels and low seed production. All varieties produced by the program are then evaluated in banana producing regions in Brazil. Recently new breeding techniques based on genetic information on Musa ssp. are being incremented.

Development of a cassava core collection based on single nucleotide polymorphism markers

Single nucleotide polymorphism (SNP) markers were used in the largest cassava (Manihot esculenta Crantz) germplasm collection from Brazil to develop core collections based on the maximization strategy. Subsets with 61, 64, 84, 128, 256, and 384 cassava accessions were selected and named PoHEU, MST64, PoRAN, MST128, MST256, and MST384, respectively. All the 798 alleles identified by 402 SNP markers in the entire collection were captured in all core collections. Only small alterations in the diversity parameters were observed for the different core collections compared with the complete collection. Because of the optimal adjustment of the validation parameters representative of the complete collection, the absence of genotypes with high genetic similarity and the maximization of the genetic distances between accessions of the PoHEU core collection, which contained 4.7% of the accessions of the complete collection, maximized the genetic conservation of this important cassava collection. Furthermore, the development of this core collection will allow concentrated efforts toward future characterization and agronomic evaluation of accessions to maximize the diversity and genetic gains in cassava breeding programs.