The Reference Genome And Full-Length Transcriptome Of Pakchoi Reveal Mechanisms Of Cuticle Formation And Heat Adaption

Brassica include a variety of vegetables with high economic value.Among them,B.rapa ssp.chinensis,also known as bok choy or pakchoi,is a cruciferous plant originating from East Asia.As a widely planted and consumed vegetable,pakchoi exhibits excellent growth in tropical and subtropical regions.Natural selection and artificial breeding have made it more adaptable to these regions.Compared to closely related Brassica vegetables,pakchoi has higher heat tolerance and adaptability,partly due to its rich wax layer on the surface.However,research on the formation mechanism and adaptive evolution of the wax layer in pakchoi is still relatively limited.Therefore,this study used multiple omics approaches to investigate the formation of the wax layer in pakchoi and its dynamic changes in transcriptional and post-transcriptional modifications during heat adaptation.The aim is to provide scientific basis for breeding pakchoi varieties with stronger stress resistance.The main results are as follows,This study collected a type of pakchoi that is highly heat-tolerant and rich in waxes,and is widely used as a parental material in breeding.Through third-generation sequencing technology combined with Illumina sequencing and Hi-C technology,a high-quality pakchoi genome was obtained.The size of the genome is 411Mb,with contig and scaffold N50s of 4.7Mb and 39.6Mb,respectively.63.3%of the genome consists of repetitive sequences,and it contains 52,511 protein-coding genes.Through comparative genomic and transposon analysis,a common phenomenon was discovered that,compared to other B.rapa vegetable genomes,Chinese cabbage has longer terminal repeat（LTR）retrotransposon insertions in collinear regions of the genome.Further investigation into the differences in transposon insertion between Chinese cabbage and pakchoi revealed a common occurrence of LTR insertions in the collinear regions of the Chinese cabbage genome compared to other B.rapa vegetables.Multiple wax-related genes in pakchoi were found to lack transposon insertions,whereas homologous genes in Chinese cabbage exhibited widespread LTR insertions,which correlated with gene expression.Through subgenome analysis,it was found that after whole-genome triplication（WGT）,over-retained pakchoi genes escape stringent selection pressure,and among them a set of cuticle-related genes are retained.To elucidate the genetic features underlying the deposition of wax layer in pakchoi,two cultivars that exhibit opposite wax phenotypes and varying heat tolerance were collected,and an F₂ population was constructed.Using the bulked segregant analysis（BSA）to identify candidate genes related to wax traits,it was found that the third exon and adjacent intron of the wax biosynthesis gene Brc CER1 were affected by a frameshift deletion in the glossy cultivar.Finally,functional validation of the gene was performed through transgenic methods,indicating that the gene regulates waxy deposition.This study conducted full-length transcriptome analysis of two varieties with opposite high-temperature sensitivity based on nanopore long-read sequencing.We find that the heat-resistant pakchoi cultivar can mitigate heat-caused leaf damage by activating an unfolded protein response,as well as by inhibiting chloroplast development and energy metabolism,which are presumably mediated by both transcriptional regulation and splicing factors.