Identification Of Metabolites Using UPLC-HESI-MS/MS And QTL Mapping Of Candidate Genes Controlling Key Differential Compounds In Rapeseed Seeds

Rapeseed(Brassica napus L.)is one of the most important oilseed crops in the world,which is considered as an important source of edible vegetable oil and feeding proteins with higher economic value.Rapeseed serves as the main harvesting organ and secondary metabolites are abundant in it,including fiber,lignin and flavonoids.The high levels of these compounds not only influence the color of seedcoat,but also determine the quality of oil and feeding nutritional value.Comparing with black-seeded rapeseed cultivars,the yellow ones possess less fiber,lignin and flavonoids under the same genetic background,providing it with a higher nutritional value.Thus the dissection of yellow-seeded related molecular mechanism is significant to rapeseed breeding.Therefore,breeders hope to further improve the quality by breeding the yellow seed to reduce the husk content and the pigment content.However,pigments,polyphenols and related enzymes are important factors affecting the color of rapeseed.And the flavonoids in Brassica plants have been widely investigated and mainly comprise ep,qn,km,is,and their derivatives.But it is very difficult to carry out qualitative and quantitative analysis on these components,due to the complexity of these components and the influence of genotypes and environmental conditions on flavonoids in different plant seeds.Therefore,in this research,the metabolic components of Brassica napus from different genetic sources at different seed development stages were detected by UPLC-HESI-MS/MS.The metabolic database of Brassica napus seeds was preliminarily formed,and the different components of yellow and black seeds were preliminarily determined.Using the seed of recombinant inbred lines constructed by the self-crossing multi-generation of the hybrid generation,QTL mapping of differential metabolites in yellow-and black-seed rapeseed cultivars.Lastly,according to reference genome information of the?Darmor-bzh?in Brassica napus L.,combined with the results of transcriptome analysis,some candidate genes may be important genes.The main results of our research are as follows:1.The preliminary construction of metabolome library of Brassica napus seeds.This research applied UPLC-HESI-MS/MS to analyze metabolic components of rapeseeds at different stages of development(early,middle,and late,respectively)and analyzed their relative metabolite contents with negative mode.A total of 1610discernible base peak chromatograms were produced.Based on the RTs,accurate mass measurements,MS~2spectral data,authentic standards,previously reported information,together with database,248 features were well characterized,including 31 phenolic acids,54 flavonoids,24 glucosinolates,65 lipid compounds,and 74 other polar compounds.Only 15.40%(248/1610)of metabolites were identified in the present study.2.Preliminary identification of differential metabolites in yellow-and black-seed rapeseed cultivars.To verify the correct identification of these constituents between the yellow-and black-seeded B.napus,we carry out qualitative and quantitative analysis on pigments and polyphenols which causes the difference of yellow and black seeds by using ep,qn,km,and is as standards,including 31 phenolic acids,54 flavonoids.By comparing the metabolic profiles in seeds of various yellow-and black-seeded rapeseed accessions,19compounds with significant differences were selected to be further analyzed.These compounds included is and 5 of its derivatives,ep and 10 of its derivatives and 2phenolic acids.At the same time,these contents in different yellow and black 35DAP seeds were measured,and it was found that they had very significant differences between yellow-and black-rapeseed,indicating that they might be related to the color of seeds.3.QTL mapping of differential metabolites in yellow-and black-seed rapeseed cultivars.In this study,the recombinant inbred lines constructed by the self-crossing multi-generation of the hybrid generation obtained from the yellow seed female parent GH06 and the black seed male parent ZY821.QTL mapping of differential metabolites in yellow-and black-seed rapeseed cultivars was carried out by the composite interval mapping method(CIM).A total of 121 QTLs for differential metabolites of seed were detected in different environments,and distributed on the chromosomes of A01,A02,A03,A04,A05,A06,A07,A08,A09,C01,C02,C04,C05,C06,C07 and C08,respectively.Among them,a single QTL explains 2.52%～53.31%phenotype variability.Although the QTLs detected by different environments,the same linkage marker are repeatedly detected in different environments and different traits,moreover,many QTLs concentrating distribution were detected on chromosomes A09 and C04.And most QTLs on Chr A09 explain more than 10%of phenotypic variation.4.Screening of candidate genes.Based on the results of QTL mapping,firstly,the tightly linked SNP markers were used to determine the physical location of QTLs in the rapeseed?Darmor-bzh?reference genome,and then these candidate QTL intervals were genetically annotated according to the information of the reference genome.As a result,there were 317 annotated genes were detected in different interval of QTLs in 16 chromosomes.At the same time,combined with the results of transcriptome analysis in different developmental seed coat of rapeseed and 19 candidate genes may be important genes involved in pigment metabolism of rapeseed and needed further research and analysis.In this research,we constructed preliminarily metabolome library of Brassica napus seeds and analyze differential metabolites in yellow-and black-seed rapeseed cultivars,to look into the molecular mechanism of yellow seed phenotype.It is beneficial for us to more directly explore the mechanism that affects the seed color of rapeseed,and to develop genetic improvement and new germplasm resources of rapeseed by genetic engineering.