Identification, molecular characterization and differential expression studies of genes activated during Coffea arabica L. - Hemileia vastatrix interactions Berk. & Broome
Data
2017-02-20
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Editor
Universidade Federal de Viçosa
Resumo
O café é uma das culturas de maior valor econômico mundial, além de proporcionar e qualidade de vida para milhões de pessoas em países em desenvolvimento. Embora existam vários programas de melhoramento para essa cultura e cultivares comerciais disponíveis que apresentam fatores de resistência a estresse biótico, verifica-se ainda prejuízos significativos devidos à ferrugem do cafeeiro causada por Hemileia vastatrix. A compreensão dos mecanismos moleculares da resistência à ferrugem desempenha papel importante na eficiência do desenvolvimento de novas cultivares resistentes. O objetivo do presente trabalho é identificar, caracterizar e compreender os padrões de expressão de genes de resistência do cafeeiro que são ativados após inoculação com H. vastatrix. Foi utilizada a metodologia de Hibridação Subtrativa de Supressão (HSS) para identificar os genes diferencialmente expressos (upregulated e dowregulated) às 12 e 24 horas após inoculação (h.a.i.) de Coffea arabica com o patógeno, em interações compatíveis e incompatíveis. A partir de 433 clones obtidos e sequenciados, 352 foram anotados e categorizados. Observou-se proporção relativamente menor de genes expressos em interação compatível. A análise RT-qPCR de sete genes de sinalização de resistência mostrou padrões de expressão semelhantes para a maioria dos genes em ambas as interações, indicando que esses genes estão envolvidos na resistência (não específica) durante a qual as reações imunes são semelhantes. Na segunda etapa do trabalho, resistance gene analogs (RGAs), que conferem resistência à ferrugem do café, foram identificados, sequenciados e caracterizados a partir de uma biblioteca BAC do cafeeiro. Cinco RGAs foram anotados e mapeados no cromossomo 0 (zero) de C. canephora. Destes, quatro RGAs são ativados na interação incompatível entre C. arabica e H. vastatrix. Os resultados obtidos no trabalho sugerem que um desses genes RGA sequenciado (gene 11) é um novo gene S H (S H 10) ainda não identificado biologicamente. Com base nesses dados, foi verificado pela primeira vez o novo gene S H (S H 10) no clone diferenciador 644/18 H. Kawisari. Foi realizado a análise comparativa entre os cincos RGAs e verificado alta similaridade entre dois destes, os quais são pertencentes à família de genes CC-NBS-LRR. Foi verificado intensa seleção diversificada promovida pela substituição não sinônima e pela recombinação genética. Foi realizada a análise filogenética de genes ortólogos para as espécies de café, tomate e uva e observou-se alta variabilidade intraespecífica destes dois genes CC-NBS-LRR para as espécies, exceto para o café. Estes genes sequenciados são as maiores e mais completas sequências disponíveis para o C. arabica. Estes resultados são de extrema importância para o melhoramento genético molecular visando a resistência à ferrugem do cafeeiro. De modo geral, a compreensão dos padrões de expressão de genes de resistência e a caracterização molecular de novos RGAs são resultados valiosos e estabelece nova base para estudos futuros.
Coffee is one of the most valued cash crops making the economies of many developing countries and sustaining the livelihoods of millions around the world. Despite many decades of breeding efforts with great achievements in incorporating resistance components into elite cultivars, coffee leaf rust (caused by Hemileia vastatrix) is increasingly damaging coffee production. Understanding the molecular mechanisms of rust resistance is believed to play a vital part in enhancing resistant cultivar development. The objective of the present work is to understand the expression patterns of resistance genes activated following pathogen inoculation and characterize some major resistance genes. Suppression subtractive hybridization (SSH) was used to identify genes differentially over expressed and repressed at 12 and 24 hours after pathogen inoculation during incompatible and compatible interactions between C. arabica and H. vastatrix. From 433 clones of expressed sequence tags (ESTs) sequenced, 352 were annotated and categorized of which the proportion of genes expressed during compatible interaction were relatively smaller. RT-qPCR analysis of seven resistance-signaling genes showed similar expression patterns for most of the genes in both interactions, indicating these genes are involved in basal (non-specific) defense during which immune reactions are similar. In another experiment, resistance gene analogs (RGAs) conferring coffee rust resistance were identified from a BAC library, sequenced and characterized. Five RGAs were annotated and mapped to chromosome 0 of C. canephora. Four of the RGAs are actively expressed during C. arabica-H. vastatrix incompatible interaction. The result obtained in this work suggests that one of the RGAs sequenced (gene 11) is a new S H gene (S H 10) not yet identified biologically. We also report an S H gene (S H 10) in differential host clone 644/18 H. Kawisari for the first time. Moreover, comparative analysis of two RGAs belonging to the CC-NBS-LRR gene family showed intense diversifying selection due to nonsynonymous substitution and genetic recombination. Phylogenetic analysis of orthologous genes showed high interaspecies variability among the two genes in related species than in coffee. Overall, differential gene expression analysis provided a compiled expression profile of genes upregulated and downregulated at 12 and 24 h. a. i. during incompatible and compatible interactions. Likewise, the NBS- LRR genes sequenced in this work are the largest and most complete gene reported in Arabica coffee to date, which makes the work extremely important for molecular breeding of coffee rust resistance.
Coffee is one of the most valued cash crops making the economies of many developing countries and sustaining the livelihoods of millions around the world. Despite many decades of breeding efforts with great achievements in incorporating resistance components into elite cultivars, coffee leaf rust (caused by Hemileia vastatrix) is increasingly damaging coffee production. Understanding the molecular mechanisms of rust resistance is believed to play a vital part in enhancing resistant cultivar development. The objective of the present work is to understand the expression patterns of resistance genes activated following pathogen inoculation and characterize some major resistance genes. Suppression subtractive hybridization (SSH) was used to identify genes differentially over expressed and repressed at 12 and 24 hours after pathogen inoculation during incompatible and compatible interactions between C. arabica and H. vastatrix. From 433 clones of expressed sequence tags (ESTs) sequenced, 352 were annotated and categorized of which the proportion of genes expressed during compatible interaction were relatively smaller. RT-qPCR analysis of seven resistance-signaling genes showed similar expression patterns for most of the genes in both interactions, indicating these genes are involved in basal (non-specific) defense during which immune reactions are similar. In another experiment, resistance gene analogs (RGAs) conferring coffee rust resistance were identified from a BAC library, sequenced and characterized. Five RGAs were annotated and mapped to chromosome 0 of C. canephora. Four of the RGAs are actively expressed during C. arabica-H. vastatrix incompatible interaction. The result obtained in this work suggests that one of the RGAs sequenced (gene 11) is a new S H gene (S H 10) not yet identified biologically. We also report an S H gene (S H 10) in differential host clone 644/18 H. Kawisari for the first time. Moreover, comparative analysis of two RGAs belonging to the CC-NBS-LRR gene family showed intense diversifying selection due to nonsynonymous substitution and genetic recombination. Phylogenetic analysis of orthologous genes showed high interaspecies variability among the two genes in related species than in coffee. Overall, differential gene expression analysis provided a compiled expression profile of genes upregulated and downregulated at 12 and 24 h. a. i. during incompatible and compatible interactions. Likewise, the NBS- LRR genes sequenced in this work are the largest and most complete gene reported in Arabica coffee to date, which makes the work extremely important for molecular breeding of coffee rust resistance.
Descrição
Tese de Doutorado defendida na Universidade Federal de Viçosa.
Palavras-chave
Coffea arabica, Hemileia vastatrix, Doenças e pragas, Melhoramento genético
Citação
BARKA, G. D. Identification, molecular characterization and differential expression studies of genes activated during Coffea arabica L. - Hemileia vastatrix interactions Berk. & Broome. 2017. 149 f. Tese (Doutorado em Genética e melhoramento) - Universidade Federal de Viçosa, Viçosa-MG. 2017.