Study On The Mechanism Of Seed Coat Color Formation In Brassica Rapa L. By Multi–omics Analysis

Rapeseed is one of the most important oil crops in the world.Compared with common black-seeded rapeseed,yellow-seeded rapeseed has the advantages of higher oil content,higher protein content,lower husk rate and lower secondary metabolite content.It can further improve the quality of rapeseed oil and rapeseed meal,so yellow seed has attracted more attention as an important target of rapeseed quality breeding.However,the wide application of yellow seed trait in rapeseed production was limited due to the complex genetic basis of yellow seed trait,scarcity of yellow seed genes,and unclear regulation mechanism of yellow seed color.Brassica rapa L.is one of the main varieties of Brassica oil crops.It contains a variety of germplasm resources of different seed colors with abundant genetic variations in seed color,such as yellow,brown,seed,mottled and black seed,which is a good material basis for investigating the seed color formation in rapeseed.Therefore,in this study,we performed an integrated metabolome,transcriptome and genome–wide association study(GWAS)on different B.rapa varieties to explore the mechanisms of seed coat color.The results are as follows:1.Transcriptomic analysis was used to analyze the expression profiles of yellow and black seeded of B.rapa with different genetic backgrounds at different developmental stages.A total of 14,868 differentially expressed genes(DEGs)were identified between yellow and black seeds.Based on the clustering analysis results of DEGs,10,717 genes were screened with consistent differentially expressed patterns between yellow and black seeds,among which 2,499 genes were differentially expressed at all developmental stages investigated between yellow and black seeds.GO and KEGG enrichment analyses showed that these common DEGs were significantly enriched in flavonoid metabolic pathway,and their expression were significantly repressed in yellow seeds.2.UPLC-HESI-MS/MS assay was used to detect the metabolic components of seeds between two pairs of materials with extreme seed colors at different development stages.A total of 295 metabolites were identified in yellow and black seeds,including 42 flavonoid metabolites,which could be roughly divided into three categories,including early flavonoid pathway metabolites(mainly flavanones and dihydroflavonols),intermediate metabolites(mainly flavonols and their derivatives)and late metabolites(mainly anthocyanins and proanthocyanidins).Different screening methods were used to identify the differential metabolites between yellow and black seed materials.A total of 116 metabolites were found to be differentially between yellow and black seeds,39 of which were detected by multiple methods simultaneously,and the contents of 11 different flavonoids metabolites in black seeds were significantly higher than those in yellow seeds.Then the metabolic pathway enrichment analysis of 116 different metabolites showed that these differential metabolites were significantly enriched in flavonoid biosynthesis,phenylpropane biosynthesis,flavanone and flavonol biosynthesis pathways.3.Based on the results of transcriptome and metabolome analysis,the weighted gene co-expression network analysis(WGCNA)was performed for different metabolite contents,seed color traits and expression levels of all detected genes,and a total of 56 modules were detected.By analyzing and clustering the correlation between the modules and traits,all metabolites can be classified into four categories,of which 10 flavonoids metabolites(including C122＿Procyanidin B1 、C145＿Procyanidin C1 、 C146＿Procyanidin C1 、 C150＿[DP4] Procyanidin D 、C174＿[DP3]-2 C137＿(-)-Catechin 、 C138＿Epicatechin 、 C140＿3’ 4’ 57-tetrahydroxyflavanone 、 C185＿Taxifolin 、C165＿Isorhamnetin-3-O-sinapoyldiglucoside-7-O-glucoside)with significant difference between yellow and black seeds.They all belong to the fourth category,and were related to 10 modules such as blue,cyan,green,lightcyan,salmon,lightgreen,skyblue,maroon,palevioletred3 and turquoise.The first 30 hub genes in each module were extracted and combined with flavonoid pathway genes to construct the co-expression network analysis,and it was found that blue and green modules had the highest correlation with flavonoid pathway genes.4.Metabolite genome-wide association analysis(m GWAS)of 42 flavonoids in35 days after pollination(DAP)seeds was conducted in a B.rapa natural population that was composed of 159 cultivars.A total of 1,626 quantitative nucleotides loci(QTNs)were detected,which could account for 0.17-61.66% of phenotypic variations.Among these significant loci,306,719 and 699 QTNs were associated with early,middle and late flavonoid biosynthetic pathway,respectively.By calculating the distribution density of QTNs on chromosomes,37 regions on chromosomes where QTNs gathered were selected as important candidate intervals for the regulation of flavonoid metabolism.Combined with m GWAS,transcriptome and genome analysis,241 important candidate genes were obtained in these candidate regions that may be involved in the metabolism of flavonoids.Based on results abovementioned,the possible flavonoid biosynthetic pathway and regulation of related gene expression patterns were systematically depicted in B.rapa.In conclusion,the different flavonoid components,expression regulation genes and metabolic regulation networks that affected the seed color formation were systematically compared by using the method of multi-omics combined analysis.These results will lay a foundation for further elucidate the molecular mechanism of complex regulation of seed color formation in B.rapa.It also provided an important reference for the study of the formation and regulation mechanism of seed color in other plants.