MSAP analysis of epigenetic changes reveals the mechanism of bicolor petal formation in Paeonia suffruticosa ‘Shima Nishiki’

Paeonia suffruticosa ‘Shima Nishiki’ is a very valuable bicolor cultivar because of its distinctive and colorful flowers. However, our understanding of the mechanisms underlying bicolor petal formation is limited. In this study, we used the methylation-sensitive amplified polymorphism (MSAP) method to assess the levels and pattern of cytosine methylation in different-colored petals during floral development. Our data showed differences in the methylation levels of red and pink petals. The methylation rate of the red petals was consistently higher than that of the pink petals, with maximum values of 58.45% (red petals) and 44.36% (pink petals) during the S2 developmental stage. However, obvious differences were not observed in the patterns of cytosine methylation in different-colored petals; methylation and demethylation occurred simultaneously and the proportions were similar. In addition, we isolated and sequenced the differentially methylated fragments and found that one fragment was homologous to the bHLH1 gene of P. suffruticosa ‘Luoyang Hong’; its expression pattern suggested that the bHLH1 gene may be involved in the regulation of the formation of bicolor flowers in P. suffruticosa ‘Shima Nishiki’. These results will provide a valuable resource for further investigation of the genetic mechanisms underlying bicolor petal formation in P. suffruticosa ‘Shima Nishiki’.