QTL Location And Candidate Gene Analysis For Seed Germination Traits In Brassica Napu

Rapeseed is one of the four major oil crops in the world and an important source of edible vegetable oil and high-quality feed protein in China.At present,China’s rapeseed production is still dominated by artificial planting,facing problems such as low germination percentage,large seed consumption,and lack of seedlings in the later stage,which hinders the development of rapeseed industry.Selecting seeds with high germination quality is an effective way to reduce the cost of seed use and improve the production efficiency of rapeseed.At present,there are few studies on the genetic basis and molecular mechanism of seed germination traits in Brassica napus,which brings obstacles to the breeding improvement of germination traits.In this study,a recombinant inbred line(AH-RILs)population of Brassica napus composed of 189 lines with low germination rate line APL01 and high germination rate line Holly as parents was used as material.The seed germination energy(GE)and germination percentage(GP)were measured by seed germination test.The genetic model of GE and GP was analyzed by the mixed model of major gene + polygene.The molecular basis of regulating seed germination of Brassica napus was revealed by means of transcriptomics research.The results of QTL mapping,DNA sequence variation analysis and transcriptomics analysis were integrated to explore the genetic loci and candidate genes regulating seed germination.Candidate genes were cloned,gene sequence variation analysis and expression analysis were carried out.The function of candidate genes was preliminarily identified,which could provide new gene resources and research basis for the cultivation of new rape varieties with high germination quality.The main results are as follows:1.Through phenotypic variation analysis,it was found that the GE and GP of Brassica napus seeds were quantitative traits controlled by multiple genes,and had obvious main gene effects.Correlation analysis showed that GE was significantly positively correlated with GP,and GE largely determined the level of GP.2.It was found that the GE and GP of AH-RILs population had the same genetic law and were controlled by 4 pairs of additive-epistatic major gene(4MG-AI)models.The main gene heritability of GE and GP in the three environments was greater than 90%,and the environmental variance was less than 0.5%,that is,the GE and GP were regulated by the main gene,which was little affected by environmental factors and could be stably inherited in the offspring.3.Additive QTL mapping analysis showed that 7 QTLs related to GE were detected in three environments,and the phenotypic contribution rate was 2.04%～13.61%.The main QTL qGE.C3 explained 8.28%～13.61% of the phenotypic variation.A total of 8 QTLs related to GP were detected in three environments,and the phenotypic contribution rate was 2.31%～16.37%.The main QTL qGP.C3 explained 8.28%～13.61% of the phenotypic variation.Epistatic QTL mapping analysis showed that six epistatic effects affecting GE were detected in three environments,explaining 3.39%～13.01% of phenotypic variation.A total of 9 epistatic effects affecting GP were detected in three environments,explaining 2.41%～10.1% of phenotypic variation.QTL additive effect and epistatic effect jointly regulate seed germination of Brassica napus.4.Compared with APL01 seeds before germination,the number of up-regulated genes in Holly seeds before germination was 4685.GO enrichment and KEGG enrichment analysis showed that these differentially expressed genes were mainly related to sugar,protein,lipid,cell components,amino acid metabolism,tricarboxylic acid cycle,electron transport and signal transduction,indicating that the difference between APL01 and Holly in these anabolic processes may be the molecular basis for Holly seed germination quality higher than APL01.5.According to the results of QTL physical mapping,109 candidate genes that may be related to germination traits were identified in the qGE.C3(qGP.C3)mapping interval.Combined with the results of transcriptome analysis,5 differentially expressed genes before and after seed germination were screened from 109 genes.Combined with the results of DNA sequence variation analysis,29 genes with sequence variation between APL01 and Holly were screened from 109 genes.Finally,13 most likely candidate genes were screened by gene function annotation.Among them,the BnaC03g10380 D gene encodes a putative small heat shock protein(s Hsps)in Brassica napus,which plays a positive regulatory role in seed germination.6.The DNA and CDS sequences of BnaC03g10380 D were cloned from APL01 and Holly.After comparative analysis,it was found that there were 6 single base mutations in the gene between the two strains,of which 5 were located in the CDS region and 1 was located in the3’UTR region,causing 4 amino acid mutations.According to the prediction of protein structure and function,the small heat shock protein is a transmembrane secreted protein and plays its role through the ribosome-endoplasmic reticulum-Golgi apparatus-extracellular secretory pathway.The 4 amino acids mutated between APL01 and Holly were located outside the functional domain of the protein,indicating that the protein could function normally in both parents.Phylogenetic analysis showed that the sequence of BnaC03g10380 D gene was highly conserved in Brassica relatives.It had the highest homology with the Bo3g015470 gene of cabbage(97.4%)and the AT5G20970 gene of Arabidopsis thaliana(88.5%).Further analysis of the correlation between 6SNPs and germination traits in BnaC03g10380 D showed that these SNPs had no significant correlation with germination traits,indicating that these SNPs did not lead to changes in gene function.Analysis of the expression level of BnaC03g10380 D showed that the expression level of the gene in the seeds of the high germination strain before germination was higher than that of the low germination strain,indicating that the gene regulated the seed GP by changing the expression level in the AH population.In summary,this study determined the genetic model of seed germination traits(germination energy and germination percentage)of Brassica napus,excavated the main genetic loci regulating seed germination,and screened a potential candidate gene,which is conducive to the application of high germination traits through transgenic technology to the cultivation of new varieties of Brassica napus with high germination quality,which is of great significance to the development of rapeseed industry in China.