Caracterização da diversidade epigenética de cana-de-açúcar via MSAP (Methylation Sensitive Amplification Polymorphism)
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Data
2018-06-29
Autores
Martins, Alessandra Alves
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Universidade Estadual Paulista (Unesp)
Resumo
A busca por fontes de energia renováveis desperta o interesse em uma das principais culturas exploradas no Brasil, a cana-de-açúcar. As cultivares modernas derivaram de cruzamentos interespecíficos principalmente entre Saccharum officinarum e S. spontaneum nos quais poucos acessos destas espécies foram utilizados, proporcionando uma base genética estreita. Porém, existe uma vasta variabilidade genética e epigenética dentre os acessos que compõem o “complexo Saccharum” que ainda não foi explorada nos programas de melhoramento. A metilação do DNA, uma das principais modificações epigenéticas ocorrentes em plantas, pode ser quantificada pela técnica de MSAP (Methylation sensitive amplification polymorphism). Neste contexto, este trabalho teve como objetivo estimar a diversidade epigenética das espécies S. officinarum, S. spontaneum, S. robustum, S. barberi, Erianthus sp. e cultivares comerciais via marcadores MSAP. Para isso, duas amostras de cada acesso foram digeridas com as enzimas de restrição EcoRI-MspI e EcoRI-HpaII, ligadas aos respectivos adaptadores e submetidas as amplificações pré-seletivas e seletivas. Os padrões de metilação foram estimados, classificando os marcadores quanto ao número de loci suscetíveis (MSL) e não suscetíveis a metilação (NML). As relações de dissimilaridade entre os acessos assim como a distribuição da variabilidade epigenética dentre e entre os grupos das espécies foram analisadas. No total, foram obtidos 1341 loci, sendo que a diversidade foi significativamente maior nos MSL, resultando em uma maior variabilidade no epigenoma. A maioria dos polimorfismos identificados nos seis grupos analisados derivaram da metilação da citosina externa. A frequência de loci metilados variou significativamente entre os grupos de acordo com a combinação seletiva de primers utilizada. Os acessos foram divididos em duas subpopulações, com maior variabilidade epigenética dentro dos grupos investigados. Os marcadores MSAP avaliados indicaram uma diferença epigenética moderada entre as espécies. Os resultados obtidos sinalizaram que existem diferenças nos padrões de metilação entre as espécies que constituem o germoplasma básico da cana-de-açúcar e as cultivares comerciais, podendo esta modificação epigenética ser explorada nos programas de melhoramento da cultura.
The search for renewable energy sources has promoted the interest in one of the main crops explored in Brazil, the sugarcane. Modern sugarcane cultivars derived from interspecific crosses, mainly between Saccharum officinarum and S. spontaneum in which few assessions of these species were involved leading to a narrow genetic base. However, there is a huge genetic and epigenetic variability among the accessions of the “Saccharum complex” that has not yet been explored in the breeding programs. The DNA methylation, one of the main epigenetic modifications that occur in plants, can be quantified by using the MSAP (Methylation sensitive amplification polymorphism) technique. In this context, this study aimed to estimate the epigenetic diversity of S. officinarum, S. spontaneum, S. robustum, S. barberi, Erianthus sp. species and commercial cultivars by using MSAP derived markers. Two DNA samples of each accession were separetly digested with the restriction enzymes EcoRI-MspI and EcoRI-HpaII, ligated to adapters followed by pre-selective and selective amplification. The methylation patterns were estimated by classifying the markers based on the number of MethylationSusceptible Loci (MSL) and Non-Methylated Loci (NML). The dissimilarity relations and variability distribution within and between the accessions was evaluated. A total of 1341 loci were obtained and the diversity was significantly higher in MSL, resulting in a higher variability in the epigenome. Most of the polymorphisms observed in the group of accessions was due to external cytosine methylation.The frequency of methylated loci significantly varied among the groups according to the selective primer combination. The accessions were divided in two subpopulations, with a higher epigenetic variability within the investigated groups. Moderate epigenetic difference among the species was observed. Our results signals differences in methylation patterns among the species that constitute the sugarcane basic germplasm and commercial cultivars, being that this epigenetic modification can be exploited by the sugarcane breeding programs.
The search for renewable energy sources has promoted the interest in one of the main crops explored in Brazil, the sugarcane. Modern sugarcane cultivars derived from interspecific crosses, mainly between Saccharum officinarum and S. spontaneum in which few assessions of these species were involved leading to a narrow genetic base. However, there is a huge genetic and epigenetic variability among the accessions of the “Saccharum complex” that has not yet been explored in the breeding programs. The DNA methylation, one of the main epigenetic modifications that occur in plants, can be quantified by using the MSAP (Methylation sensitive amplification polymorphism) technique. In this context, this study aimed to estimate the epigenetic diversity of S. officinarum, S. spontaneum, S. robustum, S. barberi, Erianthus sp. species and commercial cultivars by using MSAP derived markers. Two DNA samples of each accession were separetly digested with the restriction enzymes EcoRI-MspI and EcoRI-HpaII, ligated to adapters followed by pre-selective and selective amplification. The methylation patterns were estimated by classifying the markers based on the number of MethylationSusceptible Loci (MSL) and Non-Methylated Loci (NML). The dissimilarity relations and variability distribution within and between the accessions was evaluated. A total of 1341 loci were obtained and the diversity was significantly higher in MSL, resulting in a higher variability in the epigenome. Most of the polymorphisms observed in the group of accessions was due to external cytosine methylation.The frequency of methylated loci significantly varied among the groups according to the selective primer combination. The accessions were divided in two subpopulations, with a higher epigenetic variability within the investigated groups. Moderate epigenetic difference among the species was observed. Our results signals differences in methylation patterns among the species that constitute the sugarcane basic germplasm and commercial cultivars, being that this epigenetic modification can be exploited by the sugarcane breeding programs.
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Palavras-chave
Cana-de-açúcar, Metilação de DNA, MSAP, Epigenética