| Yellow-seeded rapeseed cultivars contain higher oil content and protein content than in black-seeded of Brassica napus with the same genetic background,which has been one of the most important objectives in rapeseed breeding.However,no stable lines of yellow-seeded oilseed germplasm are available due to genetic instability,polygenic inheritance,and complicated inheritance of regulation,which has greatly inhibited the industry breeding application of the yellow-seeded trait in rapeseed.In addition,the yellow-seeded mutant Brassica oleracea was obtained from the ornamental kale,and subsequently bred the yellowseeded B.oleracea varieties through years of systematic selection,containing the major yellow locus in C sub-genome,which provides the foundation for yellow seed rapeseed breeding and is the first yellow-seeded genetic resource of B.oleracea.To date,the molecular mechanism of yellow seed coat color is still not well understood in B.oleracea.Therefore,we preliminarily analyzed the molecular mechanism of yellow seed coat color formation in B.oleracea using the yellow-seeded lines(Y20L903 and Y20L921)and black-seeded lines(B20L926,B20L971).(1)Based on the widely-targeted metabolomics,the differential metabolites were firstly investigated in different development seeds of yellow-and black-seeded B.oleracea by UPLCHESI-MS/MS technology;(2)The integrated Illumina and PacBio sequence data and HiC scaffolding technique were effectively assembled in a high-quality genome of B.oleracea cultivar ’B970’;(3)The differentially expressed genes(DEGs)were confirmed between the yellow-and black-seeded B.oleracea by integrating RNA-Seq and qRT-PCR analysis,and further annotated using GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway,revealing the candidate DEGs function and regulation pathway possibly involved in the seed coat color of B.oleracea;(4)The flavonoid biosynthesis pathway was revealed using the integration of metabolome,transcriptome and genome analysis,providing a foundation for well understanding the molecular mechanism of seed coat color formation in B.oleracea.The main conclusions are summarized in the following.1.Identification analysis of differential metabolites of yellow-and black-seeded B.oleracea seedsUsing UPLC-HESI-MS/MS technology,1162 chromatographic peaks were detected in different development seeds(20,30,40,and 50 DAP)of yellow-and black-seeded B.oleracea(Y20L903,B20L926,and B20L971).Based on the retention time,MS,MS/MS,and published databases,a total of 287 metabolites were identified,including 33 phenolic acids,72 flavonoids,34 glucosinolates,71 lipids,and 77 amino acids and their derivatives.Further,147 of these were then quantitated using the commercial standards(33 phenolic acids,72 flavonoids,34 glucosinolates,and 8 amino acids and their derivatives).Importantly,12 different metabolites showed higher levels in black-seeded than in yellow-seeded and might be involved in the seed coat color,which were classified into the naringenin,pentahydroxyflavan,dihydrokaempferol,eriodictyol,leucocyanidin,epicatechin,catechin,and oligomeric proanthocyanidins and their derivatives.Therefore,we suppose that low levels of epicatechin and proanthocyanidins might be acted as a crucial factor for the formation of the yellow-seeded trait in B.oleracea.2.The high-quality genome of B.oleracea B970B.oleracea cultivar B970 was sequenced and assembled based on the PacBio,Illumina error correction,and HiC sequencing.The high-confidence genomic sequence,524.95 Mb,was produced by HiC correction,including 9 chromosomes with N50 size of 62.44 Mb.Our assembly is more complete compared with Fan’s assembly(89.3% complete BUSCOs).The BUSCO test was used to evaluate the quality of our assembly with 98.2%,indicating that our assembly is more complete.In addition,Annotation revealed 65.14% of the genome was repetitive elements,which were spanning 89.17%(48,291)of the assembly(99.2% complete BUSCOs).Among the predicted genes,85.46% could be annotated in the public database.The assembly of high-confidence B.oleracea genome provides a reference genome for RNA-Seq of yellow-and black-seeded B.oleracea,making RNA-Seq results more accurate and reliable.3.Transcriptome analysis of yellow-and black-seeded B.oleracea seedsTo identify the key genes which are involved in the seed coat color in B.oleracea,we performed transcriptional analysis on different developmental seeds(20,40,and 50 DAP)using the yellow-seeded inbred lines(Y20L903 and Y20L921)and black-seeded inbred lines(B20L926 and B20L971),respectively.The results showed that the DEGs are mainly clustered into the functional categories of flavonoids biosynthesis at 40 and 50 DAP,including the flavonoid and naringenin-chalcone biosynthesis processes.Analysis of DEGs through Kyoto Encyclopedia of Genes and Genomes(KEGG)showed that these DEGs were involved in the isoflavone and flavonoid biosynthesis in the 40 DAP and 50 DAP seeds of Y20L921 and B20L926.Meanwhile,we used the genome sequence of B.oleracea cultivar B970 to perform comprehensive transcript levels of 48 flavonoid biosynthesis genes and found 12 flavonoid biosynthesis pathway genes displayed the different expression patterns in Y20L921 and B20L926.For example,the BolPALb/c,BolC4Hb/c/e,Bol4 CLa,BolTT4b/c,BolTT5 b,and BolTT6 d had relatively high expression levels in black-seeded seeds at 20 DAP;while BolTT3,BolTT18 a,and BolTT10 were expressed at much higher levels in the black-seeded than in yellow-seeded on the whole stages of B.oleracea seeds,indicating that the low levels of structural genes for flavonoid biosynthesis might play an important role in seed coat color of B.oleracea.In addition,we also identified 47 lignin biosynthesis genes,including BolCCR,BolCAD,Bol LAC,and BolPER,which showed relatively low expression levels in both yellowand black-seeded B.oleracea seeds,suggesting that the lignin biosynthesis pathway might have little contribution to the seed coat color of B.oleracea.4.Difference analysis of flavonoid metabolic network between yellow-and black-seeded of B.oleraceaTo further explore the regulatory network of flavonoids in yellow-and black-seeded B.oleracea,we analyzed the identified flavonoids by UPLC-HESI-MS/MS and the expression patterns of differentially expressed genes related to flavonoid biosynthesis by RNA-seq analysis.We found that leucocyanidin,epicatechin and its derivatives,and oligomeric proanthocyanidins were much more abundant in black-seeded(B20L926)than in yellowseeded(Y20L921),and the expression levels of related genes(BolTT3,BolTT18 a,and BolTT10)showed a high positive correlation with the accumulation of these flavonoids,and BolTT3 was almost not expressed in the yellow-seeded Y20L921.However,these differential metabolites also had relatively high accumulation in the yellow-seeded(Y20L903),resulting in the genetic instability of seed coat color in Y20L903.In this study,we supposed that BolTT3 might play an important role in seed coat color,which had significantly higher expression levels in black-seeded than in yellow-seeded B.oleracea.Together,a large difference was found between the yellow-seeded Y20L903 and Y20L921,which may be resulted from that black seeds mixed in Y20L903 due to sampling of mixed materials.In addition,our results showed that the biosynthesis of pigments in yellow-seeded Y20L921 was blocked and located the upstream of leucocyanidin with good blocking effect and less metabolites downstream,resulted in the stable yellow-seeded trait;while the biosynthesis of the pigments in yellowseeded Y20L903 was mostly decreased and located near the oligomeric proanthocyanidins.Y20L903 showed different colors due to the accumulation of oligomeric proanthocyanidins in the mature seed coat.Therefore,the molecular mechanism of seed coat color may be different in B.oleracea. |
