Mapping And Mining Of Major Genes For Seed Vivipary Trait In Chinese Cabbage

Chinese cabbage（Brassica rapa ssp.pekinensis）belongs to the Brassica genus of the cruciferous family.Because of its high yield and rich nutrition,it is popular with the public and is cultivated in most parts of our country.In the process of cultivating Chinese cabbage with disease-resistant,high-quality and high-yielding varieties,it was found that seeds of some materials of Chinese cabbage germinate in advance before maturity,which affects the quality of the seeds and reduces the economic effect.Therefore,it is of great significance to clarify the regulation mechanism of precocious germination in Chinese cabbage seeds.In this study,the vivipary material 8407 and the normal material 72M were selected as parents to construct the F₂ population,and the candidate region was located using the methods of BSA-seq and QTL mapping.At the same time,The genes in the candidate region were mined in combination with transcriptome data in the previous laboratory,the main results were as follows:1.BSA sequencing was performed on the constructed extreme mixing pools and parent pools,A total of 60.23G of raw data was obtained from the parent pools and progeny pools,which 55.96G of valid data was obtained after filtering.After comparing the sequencing data to the reference genome,the comparison ratio of the four sample pools has reached more than88%.CombiningΔSNP-index andΔIn Del-index analysis,the candidate region that a size of2.5Mb was mapped on chromosome 3,containing a total of 436 genes.2.According to the results of BSA-seq,In Del sites and SNP difference sites in the candidate region were screened and used for designing primers.15 In Del primers and 6 d CAPs primers with polymorphisms were used to construct the genetic map in the F2 population with114 individuals and to QTL mapping.The total length of the map was 178.55 c M,and the average genetic distance was 5.25 c M.Combining the vivipary ratio of each plant,the candidate region was narrowed to two pairs of molecular markers In Del-24 and In Del-8,with a genetic distance of 7.73c M and a physical distance of 796kb.3.Based on the analysis of laboratory transcriptome sequencing data before,a total of13,319 differentially expressed genes were found,among which 2,925 genes were up-regulated and 9,823 genes were down-regulated.Through screening and functional annotation analysis of differentially expressed genes,some genes related to seed dormancy and germination were screened,including genes related to seed coat flavonoid synthesis,genes related to embryonic development,genes related to ABA synthesis and regulation,genes of GA metabolism and regulation,and JA synthesis related genes,etc.4.Combining the differentially expressed genes of the transcriptome and the functional annotation of the genes in the candidate region,two candidate genes Bra013201 and Bra012635 with CDS sequences of 615 bp and 576 bp were predicted.They are respectively annotated to two genes involved in ABA regulation in Arabidopsis,IBR5 and PYR1.Two overexpression vectors with a GFP tag were constructed by using the method of homologous recombination,and transient transformation on tobacco found that these two genes were mainly distributed in the cytoplasm and nucleus.