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 年后去分化过程中变异爆发和 CHH 型 DNA 甲基化增加

摘要

组织培养产生的体细胞克隆变异可能为选择新种质提供有价值的资源，但可能无法保留真实类型的特征，这是种质保护或基因组编辑的主要关注点。在长期体外培养过程中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。甜橙是最早体外培养的植物品种之一并通过体细胞胚胎发生诱导。我们通过综合确定单核苷酸多态性、拷贝数变异、转座子插入、甲基组和转录组变化，比较了 30 年恒定组织培养后的 4 个甜橙愈伤组织系与新诱导的愈伤组织。我们在早期去分化过程中发现了一次变异爆发，包括反转录转座子爆发，然后是长期体外培养期间的变异清除。值得注意的是，CHH 甲基化呈动态模式，最初在去分化过程中消失，然后在体外30 年后恢复超过文化。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。我们确定了潜在的再生相关基因CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 16中的反转录转座子插入和 DNA 修饰交替。本研究为利用体外变异奠定了基础，并提供了对组织培养在种质保存、体细胞胚胎发生、基因编辑和育种计划过程中引入的变异的更深入了解。