Mapeamento de QTLs e eQTLs relacionados à resistência à Phytophthora parasitica (agente causador da gomose dos citros) em citrandarins
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Data
2016-10-21
Autores
Lima, Rômulo Pedro Macêdo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Phytophthora nicotianae Breda de Haan (Phytophthora parasitica Dastur) e Phytophthora citrophthora (Smith & Smith), agentes causadores da gomose e podridão radicular, têm trazido graves danos em viveiros e pomares de citros no mundo inteiro. O Centro de Citricultura Sylvio Moreira/IAC vem realizando um amplo programa de melhoramento genético de citros via cruzamentos dirigidos com relação ao patossistema Phytophthora-citros, em que já foram verificadas diferenças no nível de resistência na progênie do cruzamento entre tangerina Sunki (Citrus sunki) e trifoliata Rubidoux (Poncirus trifoliata), a partir da detecção de genes diferencialmente expressos utilizando microarranjos, identificando transcritos envolvidos na resistência à Phytophthora, e do mapeamento genético. Este trabalho teve como objetivo principal mapear nos grupos de ligação dos mapas de P. trifoliata e C. sunki as regiões genômicas associadas à resistência à Phytophthora parasitica por meio de análise fenotípica (QTLs) e de expressão (eQTLs). Uma população de 110 híbridos, seus genitores e dois porta-enxertos de referência para a citricultura (limão Cravo e citrumelo Swingle), foi enxertada em limão Cravo e estabelecida em casa de vegetação. Cada híbrido foi conduzido com uma única haste e a inoculação foi realizada pelo método do disco a partir do meio de cultura contendo o micélio de P. parasitica, a 10 cm e 15 cm acima da região da enxertia, totalizando duas inoculações por genótipo. As plantas foram mantidas em ambiente com iluminação artificial e fotoperíodo de 16 horas, temperatura de 25°C e umidade relativa (UR) de 85%, com irrigações diárias. As lesões decorrentes foram avaliadas após 60 dias da inoculação, medindo-se o comprimento da área lesionada (CL) após a remoção da casca do ramo. O delineamento experimental foi o inteiramente casualizado com duas repetições do mesmo genótipo e uma planta por parcela. Constataram-se diferenças entre os híbridos, com os CL variando de 1,15 a 11,13 mm. A herdabilidade do caráter foi de 65%. Para o mapeamento de QTLs e eQTLs (QTLs de expressão), mapas de ligação foram previamente construídos de Poncirus trifoliata (L.) Raf. cv. Rubidoux e Citrus sunki Hort. ex Tan. com o uso de marcadores DArT-seqTM e utilizando a estratégia pseudo-testcross. Foram detectados três QTLs associados à resistência à Phytophthora presentes nos grupos de ligação 2, 7 e 10 do mapa de P. trifoliata, e apenas um QTL no grupo de ligação 2 de C. sunki. A proporção da variação fenotípica, explicada pelo QTL, variou de 1,77% a 7,41% para o genitor trifoliata Rubidoux, enquanto que o QTL detectado no mapa da tangerina Sunki, explicou 16,8% do fenótipo. Esses resultados sugerem que há grande possibilidade de sucesso na seleção de híbridos resistentes dentro do experimento. Além disso, 19 genes possivelmente envolvidos em processo de respostas de defesa da planta à infecção do patógeno foram avaliados e validados na população de 51 híbridos selecionados e genitores. As análises dos dados de expressão gênica, bem como as correlações fenotípicas e genotípicas, demonstram uma enorme variação intra e intergrupos quanto à ativação das vias dos fitormônios relacionados à resistência em plantas, sugerindo que os citrandarins possuem diversas estratégias para controlar a infecção do oomiceto. Essas informações possibilitaram localizar regiões genômicas associadas à resistência pela análise dos dados de expressão gênica. Para identificação dos eQTLs, os genes selecionados foram quantificados por meio da técnica de PCR quantitativo em tempo real (RT-qPCR). Foram detectados 74 eQTLs associados à resistência à Phytophthora no mapa de P. trifoliata, e 90 eQTLs no de C. sunki. A proporção da variação genotípica, explicada pelo eQTL, variou de 0 a 13,66% para o genitor trifoliata Rubidoux, enquanto que no mapa da tangerina Sunki, variou de 0 a 18,87%. Os resultados do mapeamento de QTLs e eQTLs sugerem que a resistência de alguns citrandarins frente à inoculação de P. parasitica seja devido a uma combinação favorável de QTLs e eQTLs transmitidos pelos dois genitores, com a tangerina Sunki parecendo ter uma contribuição maior para passar alelos de resistência à progênie.
Phytophthora nicotianae Breda de Haan (Phytophthora parasitica Dastur) e Phytophthora citrophthora (Smith & Smith), causative agents of gummosis and root rot, have brought serious damage in nurseries and citrus groves worldwide. The Citrus Center Sylvio Moreira/IAC has been conducting an extensive citrus breeding program by directing crossings regarding pathosystem Phytophthora - citrus, where differences have been observed in the level of resistance in the progeny of the cross between Sunki mandarin (Citrus sunki) and trifoliate Rubidoux (Poncirus trifoliata), from the detection of differentially expressed genes using microarray identifying transcripts involved in resistance to Phytophthora and genetic mapping. This work aimed to map the binding groups of P. trifoliata and C. sunki maps the genomic regions related to the resistance of Phytophthora parasitica by phenotypic analysis (QTLs) and expression (eQTLs). A population of 110 hybrids, their genitors and two reference rootstocks for citrus (Rangpur lime and Swingle citrumelo) was grafted at Rangpur lime and established in the greenhouse. Each hybrid was conducted with a single rod and the inoculation was performed by the disk method from the culture medium containing the mycelium of P. Parasitica, 10 cm and 15 cm above the grafted area, totaling two inoculations per genotype. The plants were kept in a room with artificial light and a 16 hour photoperiod, 25°C and relative humidity (RH) of 85%, with daily irrigations. The resulting lesions were evaluated 60 days after inoculation, by measuring the length of the damaged area (CL) after the branch bark removal. The experimental design was completely randomized with two repetitions of the same genotype and one plant per plot. We observed differences between hybrids, with CL ranging from 1.15 to 11.13 mm. The character’s heritability was 65%. For the QTLs and eQTLs (QTL expression) mapping, linkage maps were previously constructed from Poncirus trifoliata (L.) Raf. cv. Rubidoux e Citrus sunki Hort. ex Tan. by DArT-seqTM markers and using the pseudo-testcross strategy. Three QTLs were identified associated with resistance to Phytophthora in the linking groups 2, 7 and 10 of P. trifoliata map, and only one QTL on linkage group 2 of C. sunki. The proportion of the phenotypic variation explained by QTL ranged from 1.77% to 7.41 % for the genitor trifoliate Rubidoux while detected at the Sunki mandarin map explain 16.8% of the phenotype. These results suggest that there is great possibility of success in the selection of resistant hybrids within the experiment. Furthermore, 19 genes possibly involved in the process of plant defense responses to pathogen infection were assessed and validated in the population of 51 selected hybrids and genitors. The analysis of gene expression data and the genotypic and phenotypic correlations show a huge variation within and between groups as the activation of plant hormone pathways related to resistance in plants, suggesting that citrandarins have several strategies to control the oomycete infection. These informations made it possible to locate genomic regions associated with resistance by analysis of gene expression data. To identify eQTLs, selected genes were quantified by quantitative real time PCR (RT- qPCR). Seventy-four eQTLs were detected associated with resistance to Phytophthora in P. trifoliata map, and 90 eQTLs in C. sunki. The proportion of genotype variation explained by eQTL ranged from 0 to 13.66% for the genitor trifoliate Rubidoux while at the Sunki mandarin map ranged from 0 to 18.87 %. QTLs results and eQTLs suggest that the resistance of some citrandarins front of the inoculation of P. parasitica is due to a favorable combination of QTLs and eQTLs transmitted by both genitors, with Sunki mandarin seeming to have a greater contribution to pass alleles resistance to progeny.
Phytophthora nicotianae Breda de Haan (Phytophthora parasitica Dastur) e Phytophthora citrophthora (Smith & Smith), causative agents of gummosis and root rot, have brought serious damage in nurseries and citrus groves worldwide. The Citrus Center Sylvio Moreira/IAC has been conducting an extensive citrus breeding program by directing crossings regarding pathosystem Phytophthora - citrus, where differences have been observed in the level of resistance in the progeny of the cross between Sunki mandarin (Citrus sunki) and trifoliate Rubidoux (Poncirus trifoliata), from the detection of differentially expressed genes using microarray identifying transcripts involved in resistance to Phytophthora and genetic mapping. This work aimed to map the binding groups of P. trifoliata and C. sunki maps the genomic regions related to the resistance of Phytophthora parasitica by phenotypic analysis (QTLs) and expression (eQTLs). A population of 110 hybrids, their genitors and two reference rootstocks for citrus (Rangpur lime and Swingle citrumelo) was grafted at Rangpur lime and established in the greenhouse. Each hybrid was conducted with a single rod and the inoculation was performed by the disk method from the culture medium containing the mycelium of P. Parasitica, 10 cm and 15 cm above the grafted area, totaling two inoculations per genotype. The plants were kept in a room with artificial light and a 16 hour photoperiod, 25°C and relative humidity (RH) of 85%, with daily irrigations. The resulting lesions were evaluated 60 days after inoculation, by measuring the length of the damaged area (CL) after the branch bark removal. The experimental design was completely randomized with two repetitions of the same genotype and one plant per plot. We observed differences between hybrids, with CL ranging from 1.15 to 11.13 mm. The character’s heritability was 65%. For the QTLs and eQTLs (QTL expression) mapping, linkage maps were previously constructed from Poncirus trifoliata (L.) Raf. cv. Rubidoux e Citrus sunki Hort. ex Tan. by DArT-seqTM markers and using the pseudo-testcross strategy. Three QTLs were identified associated with resistance to Phytophthora in the linking groups 2, 7 and 10 of P. trifoliata map, and only one QTL on linkage group 2 of C. sunki. The proportion of the phenotypic variation explained by QTL ranged from 1.77% to 7.41 % for the genitor trifoliate Rubidoux while detected at the Sunki mandarin map explain 16.8% of the phenotype. These results suggest that there is great possibility of success in the selection of resistant hybrids within the experiment. Furthermore, 19 genes possibly involved in the process of plant defense responses to pathogen infection were assessed and validated in the population of 51 selected hybrids and genitors. The analysis of gene expression data and the genotypic and phenotypic correlations show a huge variation within and between groups as the activation of plant hormone pathways related to resistance in plants, suggesting that citrandarins have several strategies to control the oomycete infection. These informations made it possible to locate genomic regions associated with resistance by analysis of gene expression data. To identify eQTLs, selected genes were quantified by quantitative real time PCR (RT- qPCR). Seventy-four eQTLs were detected associated with resistance to Phytophthora in P. trifoliata map, and 90 eQTLs in C. sunki. The proportion of genotype variation explained by eQTL ranged from 0 to 13.66% for the genitor trifoliate Rubidoux while at the Sunki mandarin map ranged from 0 to 18.87 %. QTLs results and eQTLs suggest that the resistance of some citrandarins front of the inoculation of P. parasitica is due to a favorable combination of QTLs and eQTLs transmitted by both genitors, with Sunki mandarin seeming to have a greater contribution to pass alleles resistance to progeny.
Descrição
Palavras-chave
Citros, Gomose, Expressão gênica, Marcadores DArT-seq, Mapas genéticos