Alterations in DNA methylation patterns in regenerated Chinese cabbage (Brassica rapa ssp. pekinensis) plants derived from tissue culture

Abstract

Plant tissue culture is an essential tool in biotechnology. However, tissue-cultured plants often exhibit variations that are either genetic or epigenetic in origin, termed somaclonal variations. Among these variations, DNA methylation is an important heritable epigenetic modification that plays a role in a wide variety of biological processes, including gene expression. In this study, we performed bisulfite sequencing of regenerated Chinese cabbage (Brassica rapa ssp. pekinensis) lines to identify DNA alterations induced by tissue culture. Sequencing data from each regenerated line were compared with reference genome sequences, and common differentially methylated regions (DMRs) were detected in the regenerants. To determine changes in expression levels of DMR-containing genes, we performed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of the target genes and PCR amplification with bisulfite-converted DNA. We identified DMRs between a non-regenerant line and regenerant lines and selected 10 DMR-associated genes that presented annotation information in Arabidopsis or Brassica rapa. Their expression levels were verified by qRT-PCR to determine the relation between methylation state and gene expression. We observed that genes positioned in DMRs significantly correlated with differential gene expression. We also observed similar methylation patterns in the selected DMRs by PCR-based methylation analysis. The results of this study are a valuable resource for the epigenetic analysis of regenerated lines, especially for Chinese cabbage.