A Detection Method for Tropical Race 4 of the Banana Pathogen Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Fusarium wilt, the devastating disease that ruined the ‘Gros Michel’ (AAA)-based banana production in the first half of the 20th century. The occurrence of a new variant in Southeast Asia that overcomes the resistance in Cavendish clones such as ‘Grand Naine’ (AAA) is a major concern to current banana production worldwide. The threat posed by this new variant, called tropical race 4 (TR4), may be overcome by the introduction of resistant cultivars. However, the identification of new resistant sources or breeding for resistance is a long-term effort. Currently, the only option to control the disease is to avoid or reduce the spread of the pathogen by eradication of infected plants and isolation of infested plantations. This requires sensitive and highly specific diagnostics that enable early detection of the pathogen. A two-locus database of DNA sequences, from over 800 different isolates from multiple formae speciales of F. oxysporum, was used to develop a molecular diagnostic tool that specifically detects isolates from the vegetative compatibility group (VCG) 01213, which encompasses the Foc TR4 genotype. This diagnostic tool was able to detect all Foc TR4 isolates tested, while none of the Foc isolates from 19 VCGs other than 01213 showed any reaction. In addition, the developed diagnostic tool was able to detect Foc TR4 when using DNA samples from different tissues of ‘Grand Naine’ plants inoculated with TR4 isolate

Identification and Validation of EST-Derived Molecular Markers, TRAP and VNTRs, for Banana Research

The advent of high-throughput sequencing technology has generated abundant information on DNA sequences for the genomes of many plant species. Expressed Sequence Tags (ESTs), which are unique DNA sequences derived from a cDNA library and therefore representing genes transcribed in specific tissues or at some stage of development, are one type of DNA sequences highly available today for many important crop species. Molecular markers are used for bridging DNA sequence information with particular phenotypes and are useful tools for genotyping germplasm collections and also for tagging genes involved in desirable agronomic traits. In this sense, there is always a strong demand for suitable marker techniques to better utilise existing sequence information. A transcriptome database from banana (Musa spp.), DATAMusa, containing 42,724 ESTs from 11 different cDNA libraries and encompassing approximately 24 Mb of DNA sequence, was used in this study for the design of primers to PCR-amplify two types of EST-derived molecular markers, Variable Nucleotide Tandem Repeat (VNTR) and Target Region Amplification Polymorphism (TRAP). These primers were then validated against a panel of 14 diploid Musa genotypes and produced 32 (VNTR) and 119 (TRAP) alleles. Used separately or together, both types of markers were able to discriminate Musa genotypes from different genome background (A or B genomes). The TRAP alleles identified were derived from only one EST, while the VNTR alleles were derived from 12 unigenes. Based on the results of this study, EST-derived markers can be an important source of polymorphism to be used in genetic diversity and gene discovery studies in banan

Analysis of CcDREB1D promoter region from drought-tolerant and susceptible clones of Coffea canephora by homologous genetic transformation of Coffea arabica

In several plant species, the DREB genes play a key role in responses to abiotic stress. Since the development of molecular markers is one of the major goals for accelerating breeding programs, a study was done to evaluate the sequence variability of the DREBID gene in several Coffee genotypes. The promoter and coding regions of DREBID gene were cloned and sequenced from 16 coffee plants (10 from C. arabica and 4 from C. canephora), most of them characterized by different phenotypes (tolerance vs. susceptibility) regarding to drought. This showed a high conservation of DREB1 D proteins among the homologous sequences due to the low level of diversity and the high number of synonymous mutations and neutral changes which represents the majority of sequence variations. However, several nucleic polymorphisms ("single nucleotide polymorphism" and insertion/deletion [InDels]) were found in the coffee DREBID promoters. A comparison of predicted cis-acting elements for all the promoter sequences signaled the loss of some regulatory DNA elements. The sequence variation and the loss of some regulatory DNA elements could explain the differences of DREBID gene expression previously observed in leaves of drought tolerant (clone 14) and susceptible (clone 22) clones of C. canephora. In fact, both clones 14 and 22, have one same CcDREBID allelic sequence (hp15), and diverge at a second allele. Thus, the CcDREBID allele in the tolerant 14 (hp16) was considered to be the favorable/tolerant allele and the allele in 22 (hp17) was inferior/sensitive. The capacity of CcDREBID promoter to control the expression of the uidA reporter gene is under evaluation in transgenic plants of Coffee arabica cv. caturra stably transformed by Agrobacterium tumefaciens mediated gene transfer procedure. Caturra transgenic embryos were placed on a clean bench and subjected to dehydration tests. Preliminary results of bioassays checking GUS (/3-glucuronidase) activities indicate that the observed sequence variations have a direct role in the regulation of CcDREBID expression. The proximal promoter of CcDREBID for the three alleles tested (hp15, hp16 and hp17) equally induced the uidA gene expression, however, expression of uidA under control of the complete CcDREBID promoter was significantly induced in the tolerant allele (hp16) in response to the osmotic stress, whereas, it was not significantly upregulated for the common (hp15) and sensitive alleles (hp17). These results also evidence that the sequence variation present at the first -700 by of CcDREBID promoter do not interfere the regulation activity of the promoter, probably due to the non-overlapping of SNPs and cis-regulatory elements. Though, the higher sequence variation and co-occurrence of SNPs and cis-regulatory elements observed between -700 and -1500 by seems to affect the regulation of CcDREBID promoter in response to drought stress.Support: CAPES COFECUB, INCT-Café, CNPq and ConsOrcio Pesquisa Café. (Texte intégral

Alternativa para Promocao do Crescimento In Vitro de Microenxertos de Citros.

A tecnica da microenxertia possibilita a limpeza da planta em rela-cao a viroses altamente prejudiciais a exploracao desta cultura, como: tristeza, xiloporose, sorose e exocorte. O rapido crescimento destes materiais para que possam ser brevemente submetidos aos testes de indexacao e de grande interesse. E descrito, neste trabalho, metodo empregado para obtencao de crescimento rapido, em casa de vegetacao, de materiais citricos submetidos ao pro-cesso de microenxertia in vitro, na qual as hastes caulinares contendo os micro-enxertos sao enxertadas por garfagem lateral em caules de plantas previamente estabelecidas em vasos e mantidas sob condicoes de casa de vegetacao. Considerando os resultados obtidos com melhor crescimento e consequente reducao do tempo necessario para a obtencao de borbulhas, e a aplicabilidade do meto-do, sugere-se sua utilizacao em trabalhos envolvendo o uso da tecnica de microenxertia, visando a limpeza de materiais citricos.Made available in DSpace on 2011-04-09T12:13:16Z (GMT). No. of bitstreams: 1 pab13set93.pdf: 785121 bytes, checksum: c08b38e29590ff12e2c20f11899f8663 (MD5) Previous issue date: 2001-08-16199