Genetic Analysis of Genomic and Methylomic Variation and Construction of Multi-Trait Mutant Library in Rice Carried on Chang'e-5

Stable yield and improved quality in rice are crucial for ensuring food security. Traditional breeding techniques are unable to meet the urgent demand for breakthrough germplasm resources, while space mutagenesis breeding provides a new approach for crop genetic improvement. Existing research lacks sufficient understanding of the genetic patterns of variation and has not fully explored the genetic diversity within mutant populations. In this study, rice seeds were carried by the Chang'e-5 lunar probe, upon their return to the ground, were propagated to establish lineages for genetic analysis of space-induced mutagenesis effects and for the construction of a multi-trait mutant library. The whole-genome sequencing results showed that the number of mutations was higher in the space mutation second generation than in the first generation. The SNPs, homozygous, and coding sequence mutations were more likely to be inherited compared to other types and positions of mutations. The whole-genome bisulfite sequencing results showed that the methylation level in the space mutation second generation was also higher than that in the first generation, with CG type showing the highest genetic stability. Differentially methylated cytosines in genebody were more heritable than those in gene upstream and downstream. Conversely, differentially methylated regions in gene upstream and downstream exhibited higher heritability compared to those in genebody. A total of 277 mutants covering multiple traits were screened. The mutant library constructed from these mutants showed a high gene coverage rate of 96.69% and a high functional mutation ratio of 59.39%. Based on RNA sequencing and the mutant library from representative mutants, 9 candidate genes related to nitrogen utilization, drought tolerance, cold tolerance, and grain type were identified. This study systematically reveals the genetic patterns of genomic and methylomic variation in rice induced by the space environment. The high-quality mutant library constructed here provides direct support for rice functional gene cloning and the development of new breeding materials.