Variabilidade genética de Xanthomonas axonopodis pv. passiflorae

A mancha bacteriana, causada por Xanthomonas axonopodis pv. passiflorae, é uma das mais importantes doenças do maracujazeiro, podendo limitar a produção dessa frutífera em algumas regiões do País. O uso de resistência genética e controle químico, juntamente com o emprego de medidas de exclusão, são as práticas de controle da doença mais recomendadas. Para o desenvolvimento de variedades resistentes é necessário conhecer tanto a variabilidade genética do hospedeiro quanto do patógeno. Nesse trabalho foi estudada a variabilidade de cinqüenta isolados patogênicos de Xanthomonas axonopodis pv. passiflorae, coletados em quatro diferentes locais no estado de São Paulo. No estudo da variabilidade genética foram usados dados de marcadores moleculares RAPD (Random Amplified Polymorphic DNA), os quais foram usados para o cálculo do coeficiente de similaridade de Dice entre os isolados, análise de variância molecular (AMOVA) entre e dentro das populações e agrupamento dos isolados pelo método UPGMA. Para análise da agressividade foram usados cinco isolados, mais divergentes, baseado no dendrograma. O coeficiente de similaridade variou entre 0,6887 e 0,9688. Na análise de agrupamento, os isolados foram separados em sete grupos e não houve relação evidente entre local de coleta com a composição dos grupos. Na análise da variância molecular (AMOVA) verificou-se que a maior parte da variabilidade genética está dentro das populações (89,4%) e apenas 10,6%, entre populações. Os resultados da análise de agrupamento e da AMOVA indicam que existe grande fluxo gênico entre isolados bacterianos nas regiões analisadas. No teste de patogenicidade verificou-se diferença significativa de agressividade entre os isolados. Os resultados demonstram a importância do conhecimento da variabilidade genética e da agressividade na seleção dos isolados para serem utilizados em testes de resistência genética no desenvolvimento de variedades resistentes.The bacterial spot, caused by Xanthomonas axonopodis pv. passiflorae , is one of the most important diseases of passion fruit, which can drastically limit the production in some regions of Brazil. The use of genetic resistance, chemical control and the adoption of exclusion measures are the more recommended practices for the control of this disease. To produce the resistant varieties knowledge about the variability of both host and pathogen is needed. In this study, we investigated the variability of 50 pathogenic isolates of X. axonopodis pv. passiflorae, collected from four production areas in São Paulo State. We used RAPD markers (Random Amplified Polymorphic DNA) for identification of the degree of genetic similarities through dice coefficient, analysis of molecular variance (AMOVA), among as well as within populations and clustering by the UPGMA method. Based on the clustering, the five more divergent isolates were selected for agressiveness trials. The similarity coefficient ranged from 0.6887 to 0.9688. Clustering analysis grouped the isolates in seven categories and neither apparent correlation was identified among their geographical origin no clustering pattern. The AMOVA data showed that the variability within and among populations were 89.4% and 10.6% respectively. The clustering analysis indicated genic flow among the different production areas. The results highlights the importance of taking into consideration the bacterial genetic variability of this bacterium as well as its agressiveness during the selection of resistant varieties

Optimization of PCR amplification of maize microsatellite loci

Maize (Zea mays L.) microsatellite loci are useful as genetic markers because they are numerous, occur in every chromosome, and have a high content of polymorphism information. Furthermore, they can be amplified by PCR and the resulting fragments resolved on agarose gels. A major problem, however, is that the primers used in their amplification often have different length and nucleic acid content, requiring optimization of PCR amplification programs for each locus. We developed "touchdown" PCR programs successfully used in the amplification of a set of 125-maize microsatellite loci, chosen as representative of all chromosomes. The programs varied both in relation to the annealing temperature (Tm) and primer concentration. Primers could be divided into four groups. A large group included primers that amplified well in a basic previously published amplification program but with different primer concentrations. A second group amplified in alternative programs in which the annealing temperatures were changed so as to include the Tm of either both primers or the highest Tm of the primer pair estimated based on their nucleotide composition. A third group included primers that amplified in programs with Tm higher than those estimated, and in a fourth group, with just two pairs of primers, the opposite situation prevailed

A homolog of the RPS2 disease resistance gene is constitutively expressed in Brassica oleracea

In this study, we identified disease resistance gene homologs in Brassica oleracea and assessed their expression in lines resistant and susceptible to Xanthomonas campestris pv. campestris (Xcc). Two DNA fragments of approximately 2.5 kb (BI-16/RPS2 and Lc201/RPS2) were amplified by PCR from two Brassica lines using primers based on an RPS2 homologous sequence previously described in the Brassica oleracea ecotype B117. The sequences of these fragments shared high similarity (95-98%) with RPS2 homologs from various Brassica species. The digestion of these fragments with restriction enzymes revealed polymorphisms at the Xba I restriction sites. The length polymorphisms were used as a co-dominant marker in an F2 population developed to segregate for resistance to Xcc, the causal agent of black rot. Linkage analysis showed no significant association between the marker and quantitative trait loci for black rot. RT-PCR with specific primers yielded an expected 453 bp fragment that corresponded to the RPS2 homologs in both resistant and susceptible lines inoculated with the pathogen, as well as in non-inoculated control plants. These results suggest that these homologs are constitutively expressed in B. oleracea

Effects of Phaseolus vulgaris QTL in controlling host-bacteria interactions under two levels of nitrogen fertilization

Molecular markers were used to estimate the effect of mineral nitrogen on the phenotypic expression of quantitative trait loci (QTL) controlling the number of Rhizobium nodules (NN) and resistance to Xanthomonas axonopodis pv. phaseoli in the common bean. Recombinant inbred lines derived from a BAT-93 x Jalo EEP558 cross were grown in a greenhouse in the absence or presence (5 mM NH4NO3) of nitrogen. Resistance to Xanthomonas was assessed as diseased leaf area (DLA) and the number of nodules was obtained by direct counting. Analyses of variance were used to detect significant associations between 85 marker loci from 12 linkage groups (LG) and quantitative traits. In the absence of nitrogen, 15 and 11 markers, distributed over 7 and 5 LG, showed a significant association with NN and DLA, respectively. The combined percentage of phenotypic variance explained by the marker-loci and QTL associations was 34% for NN and 42% for DLA. In the presence of nitrogen, there were only five significant associations for NN and eight for DLA, which explained 28 and 26% of the total phenotypic variance, respectively. The effects of some QTL were detected only at a certain level of nitrogen. The contribution of parental alleles at two NN QTL was dependent on the level of nitrogen. Four QTL were associated with both the number of Rhizobium nodules and resistance to Xanthomonas, suggesting a common genetic control of responses to bacterial infections in the common bean. Despite the dramatic environmental interactions noted with some QTL, in other cases the phenotypic effects were not affected by the amount of nitrogen. The stability of the latter QTL may be relevant when breeding cultivars adapted to variable soil fertility

Proportional odds model applied to mapping of disease resistance genes in plants

Molecular markers have been used extensively to map quantitative trait loci (QTL) controlling disease resistance in plants. Mapping is usually done by establishing a statistical association between molecular marker genotypes and quantitative variations in disease resistance. However, most statistical approaches require a continuous distribution of the response variable, a requirement not always met since evaluation of disease resistance is often done using visual ratings based on an ordinal scale of disease severity. This paper discusses the application of the proportional odds model to the mapping of disease resistance genes in plants amenable to expression as ordinal data. The model was used to map two resistance QTL of maize to Puccinia sorghi. The microsatellite markers bngl166 and bngl669, located on chromosomes 2 and 8, respectively, were used to genotype F2 individuals from a segregating population. Genotypes at each marker locus were then compared by assessing disease severity in F3 plants derived from the selfing of each genotyped F2 plant based on an ordinal scale severity. The residual deviance and the chi-square score statistic indicated a good fit of the model to the data and the odds had a constant proportionality at each threshold. Single-marker analyses detected significant differences among marker genotypes at both marker loci, indicating that these markers were linked to disease resistance QTL. The inclusion of the interaction term after single-marker analysis provided strong evidence of an epistatic interaction between the two QTL. These results indicate that the proportional odds model can be used as an alternative to traditional methods in cases where the response variable consists of an ordinal scale, thus eliminating the problems of heterocedasticity, non-linearity, and the non-normality of residuals often associated with this type of data.<br>Marcadores moleculares têm sido extensivamente usados para o mapeamento de loci de características quantitativas (QTL) que controlam a resistência às doenças em plantas. O mapeamento é usualmente feito estabelecendo uma associação estatística entre os genótipos dos marcadores moleculares e as variações quantitativas na resistência à doença. No entanto, a maioria dos métodos estatísticos requer uma distribuição contínua da variável resposta, um pressuposto nem sempre encontrado, já que a avaliação da resistência às doenças é freqüentemente feita visualmente através da atribuição de escores numa escala ordinal ao grau de severidade da doença. Este artigo apresenta a aplicação do modelo de chances proporcionais no mapeamento de genes de resistência à doença em plantas, adequado aos casos de dados ordinais. O modelo foi utilizado para mapear dois QTL de resistência a Puccinia sorghi em milho. Os marcadores moleculares bngl166 e bngl669, localizados nos cromossomos 2 e 8, respectivamente, foram usados para a genotipagem de indivíduos F2 de uma população segregante. Os genótipos em cada locus foram, então, comparados com relação ao grau de severidade da doença, avaliada em plantas F3 geradas através da auto-polinização das plantas F2, usando uma escala ordinal do grau de severidade. A "deviance" residual indicou uma boa qualidade de ajuste do modelo aos dados, assim como o teste escore confirmou a suposição de proporcionalidade constante das chances a cada ponto de corte. A análise individual dos marcadores detectou diferenças significativas entre os genótipos para ambos os loci, indicando que estes marcadores estão associados aos QTL de resistência à doença. Além disso, a inclusão do termo de interação indicou uma forte evidência de epistase entre os dois QTL. Os resultados indicaram que o modelo de chances proporcionais pode ser usado como uma alternativa aos métodos tradicionais, em casos onde a variável resposta segue uma escala ordinal, eliminando, assim, os problemas de falta de homogeneidade das variâncias, de linearidade e de normalidade dos resíduos, comuns neste tipo de dados