Multi-omics analysis reveals the mechanism of seed coat color formation in Brassica rapa L.

KEYMESSAGE: Multi-omics analysis of the transcriptome, metabolome and genome identified major and minor loci and candidate genes for seed coat color and explored the mechanism of flavonoid metabolites biosynthesis in Brassica rapa. Yellow seed trait is considered an agronomically desirable trait with great potential for improving seed quality of Brassica crops. Mechanisms of the yellow seed trait are complex and not well understood. In this study, we performed an integrated metabolome, transcriptome and genome-wide association study (GWAS) on different B. rapa varieties to explore the mechanisms underlying the seed coat color formation. A total of 2,499 differentially expressed genes and 116 differential metabolites between yellow and black seeds with strong association with the flavonoid biosynthesis pathway was identified. In addition, 330 hub genes involved in the seed coat color formation, and the most significantly differential flavonoids biosynthesis were detected based on weighted gene co-expression network analysis. Metabolite GWAS analysis using the contents of 42 flavonoids in developing seeds of 159 B. rapa lines resulted in the identification of 1,626 quantitative trait nucleotides (QTNs) and 37 chromosomal intervals, including one major locus on chromosome A09. A combination of QTNs detection, transcriptome and functional analyses led to the identification of 241 candidate genes that were associated with different flavonoid metabolites. The flavonoid biosynthesis pathway in B. rapa was assembled based on the identified flavonoid metabolites and candidate genes. Furthermore, BrMYB111 members (BraA09g004490.3C and BraA06g034790.3C) involved in the biosynthesis of taxifolin were functionally analyzed in vitro. Our findings lay a foundation and provide a reference for systematically investigating the mechanism of seed coat color in B. rapa and in the other plants.

Metabolite Profiling and Transcriptome Analysis Provide Insight into Seed Coat Color in Brassica juncea.

The allotetraploid species Brassica juncea (mustard) is grown worldwide as oilseed and vegetable crops; the yellow seed-color trait is particularly important for oilseed crops. Here, to examine the factors affecting seed coat color, we performed a metabolic and transcriptomic analysis of yellow- and dark-seeded B. juncea seeds. In this study, we identified 236 compounds, including 31 phenolic acids, 47 flavonoids, 17 glucosinolates, 38 lipids, 69 other hydroxycinnamic acid compounds, and 34 novel unknown compounds. Of these, 36 compounds (especially epicatechin and its derivatives) accumulated significantly different levels during the development of yellow- and dark-seeded B. juncea. In addition, the transcript levels of BjuDFR, BjuANS,BjuBAN, BjuTT8, and BjuTT19 were closely associated with changes to epicatechin and its derivatives during seed development, implicating this pathway in the seed coat color determinant in B. juncea. Furthermore, we found numerous variations of sequences in the TT8A genes that may be associated with the stability of seed coat color in B. rapa, B. napus, and B. juncea, which might have undergone functional differentiation during polyploidization in the Brassica species. The results provide valuable information for understanding the accumulation of metabolites in the seed coat color of B. juncea and lay a foundation for exploring the underlying mechanism.

Comparative genomic analyses reveal the genetic basis of the yellow-seed trait in Brassica napus.

Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47aZY821, but not the yellow-seed allele BnA09MYB47aGH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops.

Genome-Wide Association Study of Phenylalanine Derived Glucosinolates in Brassica rapa.

Glucosinolates (GSLs) are sulfur-containing bioactive compounds usually present in Brassicaceae plants and are usually responsible for a pungent flavor and reduction of the nutritional values of seeds. Therefore, breeding rapeseed varieties with low GSL levels is an important breeding objective. Most GSLs in Brassica rapa are derived from methionine or tryptophan, but two are derived from phenylalanine, one directly (benzylGSL) and one after a round of chain elongation (phenethylGSL). In the present study, two phenylalanine (Phe)-derived GSLs (benzylGSL and phenethylGSL) were identified and quantified in seeds by liquid chromatography and mass spectrometry (LC-MS) analysis. Levels of benzylGSL were low but differed among investigated low and high GSL genotypes. Levels of phenethylGSL (also known as 2-phenylethylGSL) were high but did not differ among GSL genotypes. Subsequently, a genome-wide association study (GWAS) was conducted using 159 B. rapa accessions to demarcate candidate regions underlying 43 and 59 QTNs associated with benzylGSL and phenethylGSL that were distributed on 10 chromosomes and 9 scaffolds, explaining 0.56% to 70.86% of phenotypic variations, respectively. Furthermore, we find that 15 and 18 known or novel candidate genes were identified for the biosynthesis of benzylGSL and phenethylGSL, including known regulators of GSL biosynthesis, such as BrMYB34, BrMYB51, BrMYB28, BrMYB29 and BrMYB122, and novel regulators or structural genes, such as BrMYB44/BrMYB77 and BrMYB60 for benzylGSL and BrCYP79B2 for phenethylGSL. Finally, we investigate the expression profiles of the biosynthetic genes for two Phe-derived GSLs by transcriptomic analysis. Our findings provide new insight into the complex machinery of Phe-derived GSLs in seeds of B. rapa and help to improve the quality of Brassicaceae plant breeding.

Mutations in the CDS and promoter of BjuA07.CLV1 cause a multilocular trait in Brassica juncea.

Multilocular trait has recently attracted considerable attention for its potential to increase yield. Our previous studies indicated that two genes (Bjln1 and Bjln2) are responsible for multilocular siliques in Brassica juncea and the Bjln1 gene has been delimited to a 208-kb region. In present study, the Bjln1 gene was successfully isolated using the map-based cloning method. Complementation test indicated that the BjuA07.CLV1 (equivalent to BjLn1) could rescue the multilocular phenotype and generate bilocular siliques. Two amino acids changes at positions 28 and 63 in BjuA07.clv1 as well as a 702-bp deletion in its promoter have been proved to affect the carpel numbers. Microscopic analyses suggested that BjuA07.CLV1 is involved in the maintenance of shoot and floral meristem size. The expression level of BjuA07.clv1 was significantly reduced in the SAM. Furthermore, WUS, CLV2, CLV3, RPK2 and POL, key genes in the CLV/WUS signal pathway, showed lower expression level in the multilocular plants. These data suggest that the mutations in the CDS and promoter of BjuA07.clv1 reduced its function and expression level, which disturbed CLV/WUS signal pathway, thereby leading to the enlargement of the shoot and floral meristem and resulting in the multilocular siliques.

Mapping and Identifying a Candidate Gene (Bnmfs) for Female-Male Sterility through Whole-Genome Resequencing and RNA-Seq in Rapeseed (Brassica napus L.).

In oilseed crops, carpel and stamen development play vital roles in pollination and rapeseed yield, but the genetic mechanisms underlying carpel and stamen development remain unclear. Herein, a male- and female-sterile mutant was obtained in offspring of a (Brassica napus cv. Qingyou 14) × (Qingyou 14 × B. rapa landrace Dahuang) cross. Subsequently, F2-F9 populations were generated through selfing of the heterozygote plants among the progeny of each generation. The male- and female-sterility exhibited stable inheritance in successive generations and was controlled by a recessive gene. The mutant kept the same chromosome number (2n = 38) as B. napus parent but showed abnormal meiosis for male and female. One candidate gene for the sterility was identified by simple sequence repeat (SSR) and insertion deletion length polymorphism (InDel) markers in F7-F9 plants, and whole-genome resequencing with F8 pools and RNA sequencing with F9 pools. Whole-genome resequencing found three candidate intervals (35.40-35.68, 35.74-35.75, and 45.34-46.45 Mb) on chromosome C3 in B. napus and candidate region for Bnmfs was narrowed to approximately 1.11-Mb (45.34-46.45 M) by combining SSR and InDel marker analyses with whole-genome resequencing. From transcriptome profiling in 0-2 mm buds, all of the genes in the candidate interval were detected, and only two genes with significant differences (BnaC03g56670D and BnaC03g56870D) were revealed. BnaC03g56870D was a candidate gene that shared homology with the CYP86C4 gene of Arabidopsis thaliana. Quantitative reverse transcription (qRT)-PCR analysis showed that Bnmfs primarily functioned in flower buds. Thus, sequencing and expression analyses provided evidence that BnaC03g56870D was the candidate gene for male and female sterility in the B. napus mutant.