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.

中文翻译：

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组织培养再生的大白菜 (Brassica rapa ssp. pekinensis) 植物 DNA 甲基化模式的改变

植物组织培养是生物技术中必不可少的工具。然而，组织培养的植物通常表现出起源于遗传或表观遗传的变异，称为体细胞克隆变异。在这些变异中，DNA 甲基化是一种重要的可遗传表观遗传修饰，在包括基因表达在内的多种生物过程中发挥作用。在本研究中，我们对再生的大白菜 (Brassica rapa ssp. pekinensis) 品系进行了亚硫酸氢盐测序，以确定组织培养诱导的 DNA 改变。将每个再生品系的测序数据与参考基因组序列进行比较，并在再生体中检测到常见的差异甲基化区域 (DMR)。为了确定含有 DMR 的基因表达水平的变化，我们对目标基因进行了定量逆转录聚合酶链反应 (qRT-PCR)，并使用亚硫酸氢盐转化的 DNA 进行 PCR 扩增。我们确定了非再生系和再生系之间的 DMR，并选择了 10 个在拟南芥或芸苔中提供注释信息的 DMR 相关基因。通过 qRT-PCR 验证它们的表达水平以确定甲基化状态与基因表达之间的关系。我们观察到位于 DMR 中的基因与差异基因表达显着相关。我们还通过基于 PCR 的甲基化分析在选定的 DMR 中观察到类似的甲基化模式。本研究的结果是再生品系表观遗传分析的宝贵资源，尤其是大白菜。