Marcadores moleculares para distúrbio fisiológico em eucalipto
Data
2023-02-28
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Universidade Federal de Viçosa
Resumo
O distúrbio fisiológico em Eucaliptus grandis, se apresenta como uma síndrome abiótica que afeta diretamente a qualidade da madeira, interferindo de forma significativa na economia brasileira, uma vez que o país é o principal produtor de madeira e celulose. Os principais sintomas relatados para esse distúrbio são a perda da dominância apical, seca do ponteiro, necrose do tronco principal e amarelecimento das folhas, sendo que alguns desses sintomas são também resultantes de outros estresses abióticos, como seca e estresse nutricional. Consequentemente, investigar a expressão diferencial de genes que respondem ao distúrbio fisiológico e desenvolver marcadores moleculares constituem ferramentas em potencial para melhor entendimento do distúrbio, gerando subsídios para o diagnóstico molecular, ainda na fase de muda. Para definir marcadores moleculares associados ao distúrbio fisiológico em eucalipto e analisar a presença de SNPs, o DNA dos caules de clones resistentes (1572, 1644, 1407 e 1404) e suscetíveis (520, 1296, 5204, 2361 e 5341) foi extraído e foram amplificados fragmentos sobrepostos de promotores dos genes selecionados (KASIII, KCSI, TIL1, MSH2, RPT2 e Galactose Oxidase), cuja expressão é indicativo do estado de distúrbio fisiológico. O sequenciamento de aproximadamente 2000 pb de regiões 5’ flanqueadoras dos respectivos genes permitiu o alinhamento dos promotores sequenciados, possibilitando a identificação de polimorfismos (SNPs e Indels) nos diferentes genótipos, devido à variação natural. Entre os diversos SNPs e Indels identificadas, apenas dois SNPs nas posições -1366 e -1373,-1374 no promotor do gene RPT2 puderam ser associados ao caráter resistência e susceptibilidade. Experimentos estão em progresso para avaliar a capacidade de marcação genética desses SNPs com relação ao distúrbio fisiológico. Também foi identificado o transcriptoma induzido pelo distúrbio em folhas de eucalipto, sendo extraído RNA total de clones 520 sintomáticos, 520 assintomático e 1404 resistente. A qualidade do RNA extraído foi avaliada pelo Bioanalyser e submetido ao RNA-seq, pelo método Illummina. O contraste 520-SS vs 520-AS revelou uma predominância de genes regulados positivamente pelo distúrbio fisiológico relacionados com estresse abiótico. Consistente com os resultados previamente obtidos para o transcriptoma induzido pelo distúrbio fisiológico em caules de clones suscetíveis, a síndrome do distúrbio fisiológico em folhas promoveu a repressão de genes relacionados com a síntese de ácidos graxos de cadeia longa, que são essenciais para proteção e desenvolvimentos das plantas. Estes resultados confirmam que os genes relacionados com o metabolismo de ácidos graxos podem ser utilizados como marcadores do distúrbio fisiológico. A análise dos genes diferencialmente regulados pelo contraste 1404-R vs 520-AS confirmou a repressão de genes do sistema imune da planta no clone resistente 1404, submetido às mesmas condições indutoras do distúrbio do clone suscetível 520. Esses últimos resultados corroboram o argumento de que os estímulos bióticos não são o gatilho do distúrbio fisiológico, sendo há muito tempo erroneamente considerado. Palavras-chave: Distúrbio fisiológico. Eucalipto. Marcador molecular. RNA-seq
The physiological disorder in Eucaliptus grandis, presents itself as an abiotic syndrome that directly affects the quality of the wood, interfering significantly in the Brazilian economy, since the country is the leading producer of wood and cellulose. The main symptoms reported for this disorder are the loss of apical dominance, point wilting, necrosis of the main trunk and yellowing of the leaves. Some of these symptoms also result from other abiotic stresses, such as drought and nutritional stress. Consequently, investigating differential expression of genes that respond to the physiological disorder and developing molecular markers are potential tools to better understanding the disorder, generating subsidies for molecular and more accurate diagnosis, still at the molting stage. To define molecular markers associated with the physiological disorder in eucalyptus and analyze the presence of SNPs, DNA from the stems of resistant clones (1572, 1644, 1407, and 1404) and susceptible clones (520, 1296, 5204, 2361, and 5341) was extracted. Overlapping fragments of the selected gene promoters (KASIII, KCSI, TIL1, MSH2, RPT2, and Galactose Oxidase), whose expression is indicative of the state of physiological disturbance, were amplified. The sequencing of approximately 2000-bp 5' flanking regions of the respective genes allowed the alignment of the sequenced promoters and the identification of polymorphisms (SNPs and Indels) in the different genotypes due to natural variation. Among the various SNPs and Indels identified, only two SNPs at positions -1366 and -1373, -1374 in the RPT2 gene promoter could be associated with resistance and susceptibility traits. Experiments are in progress to assess the genetic marker capacity of these SNPs concerning a physiological disturbance. The disturbance-induced transcriptome in eucalyptus leaves was also identified. For this, total RNA was extracted from symptomatic 520, asymptomatic 520, and 1404 resistant leaves. The RNA quality was evaluated by the Bioanalyser and submitted to RNA-seq by the Illummina method. The contrast 520-SS vs 520- AS revealed a predominance of abiotic-related genes upregulated by the physiological disturbance. Consistent with the results of a previously identified transcriptome induced by the physiological disturbance in stems of the susceptible 520 clone, the state of physiological disturbance in leaves promoted the repression of genes related to the synthesis of long-chain fatty acids, which are essential for protection and development of plants. These results confirm the disorder marker nature of genes related to fatty acid metabolism. The analysis of the genes differentially regulated by the contrast 1404-R vs 520-AS confirmed repression of genes of the plant's immune system in the resistant clone 1404, submitted to the same disorder-inducing conditions as the 520 susceptible clone. These latter results substantiate the argument that biotic stimuli are not the trigger of the physiological disorder, which has been long mistakenly considered. Keywords: Physiological disorder. Eucalyptus. Molecular marker. RNA-seq
The physiological disorder in Eucaliptus grandis, presents itself as an abiotic syndrome that directly affects the quality of the wood, interfering significantly in the Brazilian economy, since the country is the leading producer of wood and cellulose. The main symptoms reported for this disorder are the loss of apical dominance, point wilting, necrosis of the main trunk and yellowing of the leaves. Some of these symptoms also result from other abiotic stresses, such as drought and nutritional stress. Consequently, investigating differential expression of genes that respond to the physiological disorder and developing molecular markers are potential tools to better understanding the disorder, generating subsidies for molecular and more accurate diagnosis, still at the molting stage. To define molecular markers associated with the physiological disorder in eucalyptus and analyze the presence of SNPs, DNA from the stems of resistant clones (1572, 1644, 1407, and 1404) and susceptible clones (520, 1296, 5204, 2361, and 5341) was extracted. Overlapping fragments of the selected gene promoters (KASIII, KCSI, TIL1, MSH2, RPT2, and Galactose Oxidase), whose expression is indicative of the state of physiological disturbance, were amplified. The sequencing of approximately 2000-bp 5' flanking regions of the respective genes allowed the alignment of the sequenced promoters and the identification of polymorphisms (SNPs and Indels) in the different genotypes due to natural variation. Among the various SNPs and Indels identified, only two SNPs at positions -1366 and -1373, -1374 in the RPT2 gene promoter could be associated with resistance and susceptibility traits. Experiments are in progress to assess the genetic marker capacity of these SNPs concerning a physiological disturbance. The disturbance-induced transcriptome in eucalyptus leaves was also identified. For this, total RNA was extracted from symptomatic 520, asymptomatic 520, and 1404 resistant leaves. The RNA quality was evaluated by the Bioanalyser and submitted to RNA-seq by the Illummina method. The contrast 520-SS vs 520- AS revealed a predominance of abiotic-related genes upregulated by the physiological disturbance. Consistent with the results of a previously identified transcriptome induced by the physiological disturbance in stems of the susceptible 520 clone, the state of physiological disturbance in leaves promoted the repression of genes related to the synthesis of long-chain fatty acids, which are essential for protection and development of plants. These results confirm the disorder marker nature of genes related to fatty acid metabolism. The analysis of the genes differentially regulated by the contrast 1404-R vs 520-AS confirmed repression of genes of the plant's immune system in the resistant clone 1404, submitted to the same disorder-inducing conditions as the 520 susceptible clone. These latter results substantiate the argument that biotic stimuli are not the trigger of the physiological disorder, which has been long mistakenly considered. Keywords: Physiological disorder. Eucalyptus. Molecular marker. RNA-seq
Descrição
Palavras-chave
Marcadores genéticos, Eucalipto - Fisiologia, Distúrbios fisiológicos
Citação
ESTEVES, Márcia Helena Santos. Marcadores moleculares para distúrbio fisiológico em eucalipto. 2023. 141 f. Dissertação (Mestrado em Bioquímica Aplicada) - Universidade Federal de Viçosa, Viçosa. 2023.