Molecular Mapping And Candidate Genes For Oil And Protein Content In Brassica Napus L.

Brassica napus is one of the most important oil crops in the world possessing abundant oil and protein that they are the important traits.It is also an important source of edible oil and protein fodder in our country.In this study,we analyzed the relationship among quality traits of 520 accessions were cultivated in three environments.To identify the basic gene-group of oil and protein contents,we used QTL mapping with the high density SNP genetic map which were constructed with 60 k Brassica Illumina SNP of RIL population.Then,combining the result of genome-wide association with 31,832 SNP markers of rapeseed genome sequence and the BLUP values of 520 accessions were cultivated in three environments.To identify the significantly associated signals of oil and protein contents responsible for environment,We calculated the D-value of two traits between Yunnan and Chongqing in 2014(denoted as DV)by the formula: DV=2×(YN-CQ)/(CQ+YN).DV was used for genome-wide association to identify the Potential gene-group.Finally,we analyzed the expression of candidate genes.The main conclusions are as follows:1.Phenotypic analysis of quality traits in RIL population and natural populationDescriptive statistics ofthese quality traits showed that the largest CV(coefficient of variation)was found in different environments,indicating extensive variation in the panel of RILs population and 520 accessions.And these quality traits had a nomal distribution in RILs and 520 accsessions of natural population,as expected for the quantitative traits.The results showed that these traits could be used for further study.2.Correlation and path analysis of the quality traits of natural population cultivated at three environmentsThe correlation analysis were performed and showed that the significant or extremely significant level were found among these traits in different environments.But some of the correlation is different in three locations.The result of path analysis of three locations indicated: there is co-controlled between oil and protein contents.The direct path coefficients of yellow degree on oil content and protein content were all positive.Adversely,glucosinolate and palmitic acid were negative onoil and protein contents.At Chongqing in 2013 and 2014,the direct path coefficients of erucic acid on oil content and protein content were all negative,but they were positive at Yunnan in 2014.Oleic acid has great difference of the direct path coefficients in three locations on oil content.Which indicated contribution of oleic acid on oil contentis influenced by the environment,Therefore,further research is needed,but oleic acid has considerable negative effects on protein content.3.QTL mapping for oil and protein contents in RIL population.Using the composite interval mapping(CIM),a total of 41 QTLs for oil content were detected in fourteen environments,accounting for 3.28%~17.19% of the phenotypic variation,of which 9 QTLs were simultaneously detected and distributed on chromosome A07,A09,C01,C03 and C06,respectively.16 QTLs for protein content were detected in seven environments,explaining 4.30%~19.55% of the phenotypic variation.But only 1 QTLs(qPCA09-1)were continuously detected in different environments.In addition,2 QTLs for oil and protein contents were detected and mapped in the chromosome C04 and C06.4.Genome-wide association study for oil and protein contentsUsing the Brassica 60 K SNP array,we performed a genome-wide association study of oil and protein contents in a population of 520 accessions.Using the model of GLM,MLM and A-D test(P<1/31832),we identified 360 SNP loci were significantly associated with of the oil and protein contents of 520 accessions,which were cultivated at Chongqing in 2013,Chongqing in 2014 and Yunnan in 2014,respectively.3 peaked SNP loci for oil content were congruously detected using different models,explaining 4.93%~5.08% of the phenotypic variation;14 peaked SNP loci for protein content were detected using different models,accounting for 4.43%~7.26% of the phenotypic variation.Using the model of GLM and MLM,3 peaked SNP loci for oil+protein content were detected,explaining 3.94%~5.00% of the phenotypic variation.At the same time,oil+protein content detected 2 peaked SNP loci,which were detected repeatedly for oil content or protein content.Additionally,67 and 1 peaked SNP loci for D-value of oil and protein contents were simultaneously detected by different model,explaining 6.79%~12.75% and 9.74% of the phenotypic variation.5.The peaked SNP loci for the identification of candidate genesBased on the results of QTL mapping and genome-wide association analysis,5 peaked SNP for oil content and 20 peaked SNP for protein content were detected in the QTL overlapping intervals.Using different models,1 SNP for oil content,20 SNP for protein content and 5 SNP for oil+protein content were simultaneously detected in the QTL overlapping intervals.These peaked SNP markers would be used as the candidate genes of basic gene-group for identifying the oil and protein contents.Additionally,67 and 1 SNP for the D-value of oil and protein contents were simultaneously detected in different models,indicating that they would be an important potential gene-group for identifying the candidate genes associated with oil content and protein content.6.The prediction of candidate genesBased on the reference genome of B.napus(http://www.genoscope.cns.fr/ brassica napus/),we annotated 9 homologous genes(AT5G58330,CER3,SVL5,AT5G56350,DS2,AT2G31690,AHB2,SDP6 and IDH-VI)of Arabidopsis thaliana with in the confidence intervals of LD,which are mainly involved in the tricarboxylic acid cycle,glycolytic pathway,the synthesis of fatty acid and the development of sperm cells to regulate oil content of B.napus.And we then found 13 annotated homologous genes(eg.BS14 A,CUL1)that associate with protein content of B.napus,which are mainly involved in protein ubiquitination,protein modification and translation;5 homologous genes(AHB2,SDP6,IDH-VI,AT2G23070 and AT5G05580)were detected in the basic gene-group,contributing to the accumulation of oil+protein content.Moreover,three candidate genes(AHB2,SDP6 and IDH-VI)had been confirmed in Arabidopsis thaliana that are associated with oil content.These findings showed that above-mentioned homologous genes are involved in the basic metabolism of oil and protein content,which could be the target genes for improving the high oil content or protein content in B.napus.In the potential gene-group,we detected 10 homologous genes(PAHX,AT2G03980,KASI,PLC4,ALPHA-DOX2,PFK7,MES18,AT5G58660,GAPC1,LAG1 and PMDH1)of Arabidopsis thaliana in the LD confidence intervals,which are associated with oil content in B.napus.And 4 homologous genes(CYCT1,AT3G56370,AT4G19580 and AT4G19570)of Arabidopsis thaliana.which are associated with protein content in B.napus.These genes are contributed to the high oil and protein contents in the specific environment,affected by environments.7.The detection of the candidate gene expressionBased on the transcriptom datasets of different tissues and organsin ZS11(data not published),the transcription information of 40 candidate genes were extracted analyzed in this study.The results showed that 10 candidate genes have the highest expression levels in all various tissues and organs;which are AT5G58330,AT5G5635,SDP6?AT3G16560,SERK1,RUB1,HDA6,AT3G17090,AT2G23070 and CYC1.6 candidate genes(CER3,DS2,SVL5,AT5G62560,scpl33 and AT5G05580)showed a higher expression levels in the part of tissues and organs;others had the lower or harder expression in all tissues and organs.Therefore,the further study on these highest expression genes,including gene cloning and gene function analysis,will lay a foundation for elucidating the molecular mechanism of oil and protein content in B.napus.