Construction Of Genetic Map Of Chinese Cabbage And Genetic Location Of Flower Color Trait Gene

Chinese cabbage（Brassica rapa L.ssp.pekinensis）,originated in China,is one of the most widely distributed vegetable crops in China.The cabbage breeding involves more agronomic traits,such as head type,cohesion type,plant height,yield,etc.Although the conventional breeding techniques of Chinese cabbage are more mature,the application of molecular assisted selection breeding（MAS）in breeding is fewer.The preparation of genetic linkage groups and the construction of high density genetic maps are important foundations for QTL mapping and gene map cloning of agronomic traits,and are of great significance for improving the application of MAS technology in Chinese cabbage breeding.In this study,F₂ segregation populations were prepared from C hinese cabbage pure breeds ZHB and G291 as parents,and EST-SSR molecular markers were used to construct the genetic linkage map of C hinese cabbage.Combined with field phenotypic data,plant height,plant breadth,total weight,head weight,head height,height of compressed stem and other related traits were analyzed and QTL mapping,and the genes controlling the flower color traits of Chinese cabbage were further finely mapped.Candidate genes in the interval were cloned and analyzed.The main results are as follows:1.Preparation and phenotype identification of ZHB×G291 F₂ populationUsing ZHB and G291 as parents,a F₂ segregation population consisting of 240 individuals was formulated.ZHB is a yellow flower that is not resistant to twitching Cabbage variety,and G291 is a white flower that is more resistant to twitching Cabbage variety.There are significant differences in plant height,plant breadth,total weight,head weight,head height,head width,height of compressed stem and width of compressed stem,maximum leaf area,total number of leaves,maximum leaf rib area and flower color in two parents.We used 240 F₂ individuals as materials for planting in the normal sowing season of C hinese cabbage.We systematically investigated the plant height,plant breadth,total weight,head weight,head height,head width,height of compressed stem,width of compressed stem,maximum leaf area,total number of leaves,maximum leaf rib area and flower color at mature stage.The results showed that the plant height,plant breadth,total weight,head weight,head height,and head width,etc.all met the normal distribution and indicated quantitative traits.At the same time,there was a significant correlation between head weight and plant height,plant breadth,total weight,head height,head width,height of compressed stem,width of compressed stem,maximum leaf area,total number of leaves,maximum leaf rib area.There was a significant correlation between height of compressed stem and plant height,plant breadth,total weight,head weight,head height,head width,width of compressed stem,maximum leaf area,total number of leaves,maximum leaf rib area.Through the investigation and statistical analysis of the flower color traits of C hinese cabbage,it was found that the ratio of the number of yellow flower and white flower plants was in line with the separation ratio of 3:1,indicating the quality traits.2.Construction of genetic mapWe selected 500 pairs of EST-SSRs that were detected in a number of studies and had a high number of repetitions in the EST-SSR developed by the cabbage SSR database and the Vegetable and Flower Research Institute of Shandong Academy of Agricultural Science.The first step was that we conducted polymorphism verification in the parents.A total of 107 pairs of EST-SSR primers with polymorphism between the two parents were screened out.A F ₂population was used to construct a C hinese cabbage genetic linkage map containing 107EST-SSR molecular markers and 10 linkage groups（corresponding to 10 chromosomes of Chinese cabbage respectively）.The map covers a genome length of 2277.9 cM,and the average genetic distance is 23.63 c M,of which the SSR markers are the most on chromosome 6,a total of 16.3.The QTL mapping of important traits of Chinese cabbageUsing the above genetic map combined with field phenotypic data,the plant height,plant breadth,total weight,head weight,head height,head width,height of compressed stem and width of compressed stem,maximum leaf area,total number of leaves,maximum leaf rib area and flower color was QTL mapping,and 19 Q TL loci were detected on 10 chromosomes.One of the QTLs associated with plant height was located on chromosome 2;four Q TLs related to plant breadth were located on chromosomes 1,3,9 and 10,respectively;One of the QTLs associated with total weight was located on chromosome 3;two QTLs related to the height of compressed stem were located on chromosomes 3 and 9,respectively;five QTLs related to the maximum leaf area were located at on chromosomes 2,3,6,9 and 10,respectively;two QTLs related to the total number of leaves,located on chromosomes 2 and 5,respectively;and three QTLs associated with maximum leaf rib area were located on chromosomes 2,3,and 10,respectively.One QTL site associated with flower color of Chinese cabbage was located on chromosome 2.4.Fine mapping of flower color gene,candidate gene cloning and sequence and expression analysisUsing the above genetic linkage maps,we have initially mapped the genes controlling the flower color traits of Chinese cabbage on the A02 chromosome,located between marker A2S58and A2S38.We have finely mapped this trait by encrypting molecular markers and expanding the F₂ population.The genes that regulate flower color traits are located between A2S106-A2S89and their physical positions are between 20998808bp and 22979049bp.After comparing with the database of C hinese cabbage,four genes related to the carotenoid synthesis were found in the region:Bra020718（LYCE）,Bra008358（NCED9）,Bra023603（PSY）and Bra026656（LYCE）.We cloned and analyzed the ZHB、G291 genomic sequence and promoter sequence of these four genes and found that there was no difference in the genomic sequence and promoter sequence of Bra020718 and Bra023603 of the white flower cultivar G291 compared to the yellow flower cultivar ZHB.There is 9 base insertion on the promoter of Bra008358 of ZHB and 17 base insertion on the promoter of Bra026656 of G291.At the same time,we used RT-qPCR technology to analyze the temporal and spatial expression patterns of these four candidate genes in different tissues（buds,petals）of C hinese cabbage.The results showed that three genes,Bra020718,Bra023603 and Bra026656,were no difference in the expression levels of the petals and buds of the parental ZHB and G291,but there was a significant difference in the expression of the gene Bra008358 in the petals and buds of the parental ZHB and G291,and,its expression level in petals and buds of yellow flower variety ZHB was higher than that in petals and buds of white flower variety G291.The result indicated that Bra008358 may be related to the flower traits of Chinese cabbage,which needs to be further studied.