Variation burst during dedifferentiation and increased CHH-type DNA methylation after 30 years of in vitro culture of sweet orange

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.

Comparative Restriction Enzyme Analysis of Methylation (CREAM) Reveals Methylome Variability Within a ClonalIn VitroCannabis Population

“…There have also been cases where methylation levels initially decrease in early generations and then recover over longer periods of time, as shown in sweet orange after 30 years (X. Wang et al, 2022). Considering that our study is the first investigation of the cannabis methylome, it is plausible that methylation patterns stabilized in our clonal population over several subcultures.…”

Comparative restriction enzyme analysis of methylation (CREAM) reveals methylome variability within a clonal in vitro cannabis population

“…Some studies have reported increases in methylation levels during subculturing ( Rival et al., 2013 ; Fraga et al., 2016 ), while others have observed decreases ( Xu et al., 2004 ; Huang et al., 2012 ; Machczyńska et al., 2014 ) over different subculturing durations. There have also been cases where methylation levels initially decrease in early generations and then recover over longer periods of time, as shown in sweet orange after 30 years ( Wang et al., 2022 ). Considering that our study is the first investigation of the cannabis methylome, it is plausible that methylation patterns stabilized in our clonal population over several subcultures.…”

Establishment of an efficient and wide-spectrum droplet-vitrification cryopreservation for raspberry (Rubus idaeus L.) germplasm and assessments of genetic integrity and vegetative growth in the regenerants

“…However, it is important to note that these techniques only study a small portion of the genome, which may not fully re ect the true extent of genetic variation. Wang et al (2022) recently reported the use of next-generation sequencing (NGS) to compare four sweet orange callus samples after 30 years of constant tissue culture with newly induced callus, providing new insights into assessing genetic variation. In our present study, we performed SNPs and InDels analysis using NGS, by sequencing the entire Raspberry 'Stiora' genome.…”