Genome analysis of tissue culture-derived variations in regenerated Brassica rapa ssp. pekinensis plants using next-generation sequencing

Genetic variations have often been detected in plants regenerated from tissue culture, which is an essential step of molecular breeding and tissue culture techniques. The incidence of unintended genetic variations should be kept low to ensure the stability of biotechnological crops. In this study, regenerated Chinese cabbage plants (Brassica rapa ssp. pekinensis) were produced and sequenced to identify the genetic variation generated during tissue culture. The sequencing data of each regenerated plants were compared with reference genome sequences. A total of 6644 genetic variations, including single-nucleotide polymorphisms as well as insertions or deletions in the regenerated plants, were detected using bioinformatic analysis, and 109 mutation candidates that are expected to consistently occur in the regenerated R0 group were identified. To investigate the inheritance of the variations, the R1 and R2 lines of the two regenerated plants were also sequenced and analyzed. The results indicated that 39 of the regenerant-specific mutation candidates were expected to be maintained despite the progress of the generation. Among those, five of the SNP candidates occurred in exons, and sequence analysis was performed on these mutations. For detailed analysis, we validated the nucleotide sequences and deduced the amino acid sequences of the genes with five base substitution candidates in the exonic regions. Among the five SNP candidates, three were found to be synonymous mutations that resulted in no changes in the amino acid; however, two mutations were nonsynonymous mutations that altered the amino acid compositions. Our findings suggested a genetic diversity of the regenerated plants and revealed the mutation candidates that consistently occurred in the regenerated plants and were maintained in the progeny lines.