Abstract

Somaclonal variation arising from tissue culture may provide a valuable resource for the selection of new germplasm, but may not preserve true-to-type characteristics, which is a major concern for germplasm conservation or genome editing. The genomic changes associated with dedifferentiation and somaclonal variation during long-term in vitro culture are largely unknown. Sweet orange was one of the earliest plant species to be cultured in vitro and induced via somatic embryogenesis. We compared four sweet orange callus lines after 30 years of constant tissue culture with newly induced calli by comprehensively determining the single-nucleotide polymorphisms, copy number variations, transposable element insertions, methylomic and transcriptomic changes. We identified a burst of variation during early dedifferentiation, including a retrotransposon outbreak, followed by a variation purge during long-term in vitro culture. Notably, CHH methylation showed a dynamic pattern, initially disappearing during dedifferentiation and then more than recovering after 30 years of in vitro culture. We also analyzed the effects of somaclonal variation on transcriptional reprogramming, and indicated subgenome dominance was evident in the tetraploid callus. We identified a retrotransposon insertion and DNA modification alternations in the potential regeneration-related gene CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 16. This study provides the foundation to harness in vitro variation and offers a deeper understanding of the variation introduced by tissue culture during germplasm conservation, somatic embryogenesis, gene editing, and breeding programs.

中文翻译：

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甜橙体外培养30年后，去分化过程中发生突变，CHH型DNA甲基化增加

 抽象的

组织培养产生的体细胞克隆变异可能为选择新种质提供宝贵的资源，但可能无法保留真实的特征，这是种质保护或基因组编辑的主要问题。长期体外培养期间与去分化和体细胞克隆变异相关的基因组变化在很大程度上是未知的。甜橙是最早进行体外培养并通过体细胞胚胎发生诱导的植物物种之一。我们通过综合测定单核苷酸多态性、拷贝数变异、转座元件插入、甲基组学和转录组学变化，将经过30年持续组织培养的四个甜橙愈伤组织系与新诱导的愈伤组织进行比较。我们在早期去分化过程中发现了一系列变异，包括逆转录转座子爆发，随后是长期体外培养过程中的变异清除。值得注意的是，CHH 甲基化表现出动态模式，最初在去分化过程中消失，然后在体外培养 30 年后恢复超过。我们还分析了体细胞克隆变异对转录重编程的影响，并表明亚基因组优势在四倍体愈伤组织中很明显。我们在潜在的再生相关基因CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 16中发现了逆转录转座子插入和 DNA 修饰改变。这项研究为利用体外变异奠定了基础，并提供了对种质保存、体细胞胚胎发生、基因编辑和育种计划期间组织培养引入的变异的更深入的了解。