Preliminarily Mapping And Candidate Prediction Of The Variegation Leaf Gene In Brassica Napus L.

Brassica napus is the important oil crop and the one of the large planting area oil crops in China.Because 60% of China's vegetable oil supply relies on import,it is significant that improving the yield per unit area and total yield of Brassica napus for safe supply of crops.The chloroplast development of variegated mutants has defects.It is an ideal material to study the molecular mechanism of chloroplast development.The analysis of the molecular mechanism of chloroplast development will also provide technical support for the theoretical research of high yield of crop.Two variegated lines,ZY-4 and ZY-8,from the same Brassica napus variegated mutant were reported in this study.Genetic,mapping analysis and candidate prediction were conducted with the two lines.The main results were as follows:1.Phenotypic and cytological observation.Under the field planting conditions,the proportion of white parts of ZY-4 leaves was higher than that of ZY-8.In both lines,the cells in the green sector contained normal chloroplasts,while the cells in the white sector contained abnormal plastids.Both chlorophyll content and lutein carotenoid content in ZY-4 were significantly lower than that in ZS11,and that in ZY-8 also decreased compared with ZS11,but was not significantly lower than that in ZS11.As the development of seedlings,the white parts of leaves could change to green.Phenotype observation and chloroplast transmission electron microscopy(TEM)analysis were conducted at five different stages and tissues(cotyledon at 7 days,top shoot at 7 days,first true leaf at 9 days,first true leaf at 11 days,and first true leaf at 13 days)after seeded.It was found that the variegated phenotype gradually appeared in the mutant leaves with the development process.Cytological observation showed that the chloroplast biogenesis process in the variegated parts was blocked.2.Genetic analysis and preliminarily mapping.Two BC2F1 populations from ZS11×ZY-4(designated as BY137,BY142)and one BC2F1 population from ZS11×ZY-8(designated BY56),with an 1:1 segregation at the variegated locus, were successfully constructed.It was found that the variegated phenotypes in the three populations were controlled by a pair of recessive nuclear genes.Three “normal” bulks and three “variegated” bulks were constructed within the three populations,respectively.Combining the bulked segregant analysis(BSA)and Brassica 60 K SNPS chip,it was found that differential SNPs were mainly on C04 chromosome 36Mb-37Mb(BY137),A08 chromosome 10Mb-15Mb(BY142),and A08 chromosome 11Mb-12Mb(BY56),respectively.So it was proposed that there may be two loci in ZY-4(C04,A08)which controlled the variegation,and one locus(A08)was same as the locus controlling the variegation in ZY-8.3.Different expression genes analysis.Transcriptomic sequencing analysis was carried out in the seedling tissues of five stages.Among the three lines,ZY-4,ZY-8 and ZS11,there were a total of 21,379 and 20,322 differentially expressed genes,respectively.In ZY-4,3199 genes were down-regulated and 1349 genes were up-regulated,and in ZY-8,3104 genes were down-regulated and 1420 genes up-regulated compared with ZS11.At each stage,the down-regulated genes were also more than the up-regulated genes.GO and KEGG enrichment analysis of different expressed genes between ZY-4 and ZS11,ZY-8 and ZS11 showed that the significantly changes were happened in photosynthetic and protein translation processes in ZY-4 and ZY-8,and also showed many differences in immune system and stress responses between the two lines.Analysis of the differential gene expression in the relevant differential pathways revealed that the photosynthetic genes were severely inhibited in both lines,and the translational genes required for early chloroplast development were continuously expressed,which was the evidence of inhibition of chloroplast development at transcription level.Among the genes related to the immune system,the expression patterns of oxidized protein proteases and antioxidant proteins were disordered,indicating that the two lines were likely to be in oxidative stress.Chloroplast retrograde signaling genes and downstream genes showed different regulatory patterns in the two lines,which further indicated that the mutants were in oxidative stress due to the destruction of chloroplast structures.ROS histochemical analysis showed that at the five seedling stages,the total ROS content in ZY-4 and ZY-8 leaves were lower than that in ZS11,but the phenotypes of oxidative stress were observed in the two variegated lines,indicating that the oxidative damage of chloroplast was not directly caused by ROS accumulation.4.Candidate genes prediction.The transcriptome analysis of the carotenoid biosynthesis pathway found that the expression patterns of the carotenoid biosynthesis pathway genes were disordered to different degrees in the two lines,and the upstream and downstream genes could not be co-expressed,indicating that ZY-4 and ZY-8 phenotypes were related to the inhibition of carotenoids biosynthesis.Combined with preliminary mapping information and transcriptomic sequencing results,genes expression in the intervals were analyzed and candidate genes were screened.According to the functional annotation of homologous genes in Arabidopsis thaliana,it was found that Bna A08g17160 D and Bna A08g17170 D,homologous genes of phytoene desaturase gene(PDS3),were likely to be mutation loci in ZY-4 and ZY-8.