Manoel Souza

Insights into the Musa genome: Syntenic relationships to rice and between Musa species

BackgroundMusa species (Zingiberaceae, Zingiberales) including bananas and plantains are collectively the fourth most important crop in developing countries. Knowledge concerning Musa genome structure and the origin of distinct cultivars has greatly increased over the last few years. Until now, however, no large-scale analyses of Musa genomic sequence have been conducted. This study compares genomic sequence in two Musa species with orthologous regions in the rice genome.ResultsWe produced 1.4 Mb of Musa sequence from 13 BAC clones, annotated and analyzed them along with 4 previously sequenced BACs. The 443 predicted genes revealed that Zingiberales genes share GC content and distribution characteristics with eudicot and Poaceae genomes. Comparison with rice revealed microsynteny regions that have persisted since the divergence of the Commelinid orders Poales and Zingiberales at least 117 Mya. The previously hypothesized large-scale duplication event in the common ancestor of major cereal lineages within the Poaceae was verified. The divergence time distributions for Musa-Zingiber (Zingiberaceae, Zingiberales) orthologs and paralogs provide strong evidence for a large-scale duplication event in the Musa lineage after its divergence from the Zingiberaceae approximately 61 Mya. Comparisons of genomic regions from M. acuminata and M. balbisiana revealed highly conserved genome structure, and indicated that these genomes diverged circa 4.6 Mya.ConclusionThese results point to the utility of comparative analyses between distantly-related monocot species such as rice and Musa for improving our understanding of monocot genome evolution. Sequencing the genome of M. acuminata would provide a strong foundation for comparative genomics in the monocots. In addition a genome sequence would aid genomic and genetic analyses of cultivated Musa polyploid genotypes in research aimed at localizing and cloning genes controlling important agronomic traits for breeding purposes.

Analysis of non-TIR NBS-LRR resistance gene analogs in Musa acuminata Colla: Isolation, RFLP marker development, and physical mapping

BackgroundMany commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.ResultsA computational strategy was developed for unbiased conserved motif discovery in NBS and LRR domains in R-genes and homologues in monocotyledonous plant species. Degenerate PCR primers targeting conserved motifs were tested on the wild cultivar Musa acuminata subsp. burmannicoides, var. Calcutta 4, which is resistant to a number of fungal pathogens and nematodes. One hundred and seventy four resistance gene analogs (RGAs) were amplified and assembled into 52 contiguous sequences. Motifs present were typical of the non-TIR NBS-LRR RGA subfamily. A phylogenetic analysis of deduced amino-acid sequences for 33 RGAs with contiguous open reading frames (ORFs), together with RGAs from Arabidopsis thaliana and Oryza sativa, grouped most Musa RGAs within monocotyledon-specific clades. RFLP-RGA markers were developed, with 12 displaying distinct polymorphisms in parentals and F1 progeny of a diploid M. acuminata mapping population. Eighty eight BAC clones were identified in M. acuminata Calcutta 4, M. acuminata Grande Naine, and M. balbisiana Pisang Klutuk Wulung BAC libraries when hybridized to two RGA probes. Multiple copy RGAs were common within BAC clones, potentially representing variation reservoirs for evolution of new R-gene specificities.ConclusionThis is the first large scale analysis of NBS-LRR RGAs in M. acuminata Calcutta 4. Contig sequences were deposited in GenBank and assigned numbers ER935972 – ER936023. RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the Musa A and B genome. The developed RFLP-RGA markers are applicable for genetic map development and marker assisted selection for defined traits such as pest and disease resistance.

Genomics of Banana and Plantain (Musa spp.), Major Staple Crops in the Tropics

This chapter on Musa (banana and plantain) genomics covers the latest information on activities and resources developed by the Global Musa Genomics Consortium. Section 4.1 describes the morphology of the plant, its socio-economical importance and usefulness as an experimental organism. Section 4.2 describes the complexity of Musa taxonomy and the importance of genetic diversity. Section 4.3 details the genetic maps which have recently been developed and those that are currently being developed. Section 4.4 presents the five BAC libraries which are now publicly available from the Musa Genome Resource Centre and can be distributed in various forms under a material transfer agreement. Section 4.5 gives an overview of cytogenetics and genome organization, showing that the genus Musa has a quite high proportion of repetitive DNA; the discovery of the first para-retrovirus integrated in the genome makes it unique. Section 4.6 explains the first attempts to sequence the genome by BAC end sequencing, whole BAC sequencing, and reduced representation sequencing. Section 4.7 addresses functional genomics with the description of cDNA libraries, gene validation using gene trapping, mutation induction and tilling techniques, as well as genetic transformation. Section 4.8 draws overall conclusions. This chapter demonstrates that by organizing the Global Musa Genomics Consortium (currently comprising 33 member institutions from 23 countries), duplication of effort can be minimized and the results of Musa genomics research are rapidly made accessible to taxonomists, breeders and the biotechnology community.

Sugar-mediated acclimation: the importance of sucrose metabolism in meristems.

We have designed an in vitro experimental setup to study the role of sucrose in sugar-mediated acclimation of banana meristems using established highly proliferating meristem cultures. It is a first step toward the systems biology of a meristem and the understanding of how it can survive severe abiotic stress. Using the 2D-DIGE proteomic approach and a meristem-specific EST library, we describe the long-term acclimation response of banana meristems (after 2, 4, 8, and 14 days) and analyze the role of sucrose in this acclimation by setting up a control, a sorbitol, and a sucrose acclimation treatment over time. Sucrose synthase is the dominant enzyme for sucrose breakdown in meristem tissue, which is most likely related to its lower energy consumption. Metabolizing sucrose is of paramount importance to survive, but the uptake of sugar and its metabolism also drive respiration, which may result in limited oxygen levels. According to our data, a successful acclimation is correlated to an initial efficient uptake of sucrose and subsequently a reduced breakdown of sucrose and an induction of fermentation likely by a lack of oxygen.

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.

Introduction of the anti-apoptotic baculovirus p35 gene in passion fruit induces herbicide tolerance, reduced bacterial lesions, but does not inhibits passion fruit woodiness disease progress induced by cowpea aphid-borne mosaic virus (CABMV)

The introduction of anti-apoptotic genes into plants leads to resistance to environmental stress and broad-spectrum disease resistance. The anti-apoptotic gene (p35) from a baculovirus was introduced into the genome of passion fruit plants by biobalistics. Eleven regenerated plants showed the presence of the p35 gene by PCR and/or dot blot hybridization. Transcriptional analysis of regenerated plants showed the presence of specific p35 transcripts in 9 of them. Regenerated plants containing the p35 gene were inoculated with the cowpea aphid-borne mosaic virus (CABMV), the bacterium Xanthomonas axonopodis pv passiflorae, and the herbicide, glufosinate, (Syngenta). None of the plants showed resistance to CABMV. Regenerated plants (p35+) showed less than half of local lesions showed by non-transgenic plants when inoculated with X. axonopodis and some p35+ plants showed increased tolerance to the glufosinate herbicide when compared to non-transgenic plants.

Combating a Global Threat to a Clonal Crop: Banana Black Sigatoka Pathogen Pseudocercospora fijiensis (Synonym Mycosphaerella fijiensis) Genomes Reveal Clues for Disease Control

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana 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.

Variable number of tandem repeat markers in the genome sequence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana (Musa spp)

We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence.

Dois novos sistemas de diagnose precoce da meleira do mamoeiro

Two new systems of early diagnosis of papaya sticky disease Papaya sticky disease was first reported affecting papaya (Carica papaya) in Brazil in the late 80s. Today this disease is found in the papaya production areas throughout Brazil, and in some of them it became the main limiting factor for the papaya industry. The primary disease symptom is an excessive exudation of highly fluid latex that becomes dark as result of oxidation and turns the fruit unmarketable. It is caused by a new virus that has an isometric particle (40-50 nm in diameter), and a unique 12 kb long dsRNA molecule. Since its diagnosis is done mainly by observation of the symptoms on the fruit, infected plants may be source of inoculum for several months before diagnosis. Another form of diagnosis is the detection of dsRNA from leaves and latex using CF11 columns. This is a laborious system not suited to large scale usage. The present work presents two cheap and fast diagnostic protocols. These protocols use latex obtained from fruits, leaves and stems, and are based on the extraction and visualization of nucleic acids. Presence of the virus is confirmed by the visualization of dsRNA on agarose (1%) gel in 1X TBE. Using these protocols it is possible to confirm the presence of the virus in young and assymptomatic plants.