Chunji Zho ,Luqmn Bin Sfr ,Meili Xie ,Meijun Shi ,Zhixue Dong ,Li Yng,c ,Xiohui Cheng,Yueying Liu,Zeto Bi,Yng Xing,Chobo Tong,Junyn Hung,*,Lijing Liu,*,Shengyi Liu

a Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences/The Key Laboratory of Biology and Genetic Improvement of Oil Crops,The Ministry of Agriculture and Rural Affairs,Wuhan 430062,Hubei,China

b National Key Laboratory of Crop Genetic Improvement,National Center of Rapeseed Improvement,Huazhong Agricultural University,Wuhan 430070,Hubei,China

c Biosystematics Group,Experimental Plant Sciences,Wageningen University and Research,Wageningen,Netherlands

d Guizhou Rapeseed Institute,Guizhou Academy of Agricultural Sciences,Guiyang 550008,Guizhou,China

Keywords:Yellowish-white flower BSA-seq RNA-seq BnaA08.PDS3 Carotenoid biosynthesis

ABSTRACT Oilseed rape(Brassica napus)with yellow flowers is an attractive ornamental landscape plant during the flowering period,and the development of different petal colors has become a breeding objective.Although yellowish flower color is a common variant observed in field-grown oilseed rape,the genetics behind this variation remains unclear.We obtained a yellowish-white flower (ywf) mutant from Zhongshuang 9 (ZS9) by ethyl methanesulfonate mutagenesis (EMS) treatment.Compared with ZS9,ywf exhibited a lower carotenoid content with a reduced and defective chromoplast ultrastructure in the petals.Genetic analysis revealed that the yellowish-white trait was controlled by a single recessive gene.Using bulked-segregant analysis sequencing (BSA-seq) and kompetitive allele-specific PCR(KASP),we performed map-based cloning of the ywf locus on chromosome A08 and found that ywf harbored a C-to-T substitution in the coding region,resulting in a premature translation termination. YWF,encoding phytoene desaturase 3(PDS3),was highly expressed in oilseed rape petals and involved in carotenoid biosynthesis.Pathway enrichment analysis of the transcriptome profiles from ZS9 and ywf indicated the carotenoid biosynthesis pathway to be highly enriched.Further analyses of differentially expressed genes and carotenoid components revealed that the truncated BnaA08.PDS3 resulted in decreased carotenoid biosynthesis in the mutant.These results contribute to an understanding of the carotenoid biosynthesis pathway and manipulation of flower-color variation in B.napus.

1.Introduction

The flower is one of the most adaptive tissues in plant evolution and flower color is the most typical phenotype.Because flowers have had a strong influence on human civilization,owing to their ornamental values and societal impact,the trade volume of flowers increases annually.From the mid-20th to early 21st centuries,the global flower trade has increased one hundredfold in volume [1].For plants,flower color is a visual signal to attract insects,and different insects prefer different flower colors [2,3].Flower color thus affects plant reproduction and evolution.

Carotenoids are C40secondary metabolites involved in many plant physiological processes in plants,including coloring of flowers and fruits,growth and development,and response to environmental stimuli [4].In higher plants,the carotenoid biosynthesis pathway is well established [5].Briefly,the pathway begins when the upstream methylerythritol (MEP) pathway precursor geranylgeranylpyrophosphate (GGPP) is converted by phytoene synthase(PSY) to a C4015-cis-phytoene.The phytoene is desaturated and isomerized to produce lycopene by the activity of phytoene dehydrogenase(PDS),carotene dehydrogenases(ZSD),and carotene isomerases (CRTISO).Lycopene is cyclized to produce carotenes,which are hydroxylated and cyclized to xanthophylls.PSYs and PDSs are considered the rate-limiting enzymes in this pathway.Understanding of their roles has benefited from quantitative trait loci mapping,diverse germplasm resources,and mutant phenotypes in maize (Zea maysL.).For instance,PSY1andPSY3are reportedly responsible for carotenoid accumulation in maize seeds and roots,respectively [6,7].Similarly,functional inactivation of thePDSgene leads to an albino phenotype due to reduced phytoene content in the leaves or seeds of the maize mutantsvp2,vp5,andw3[8–10].The degradation of carotenoids in plants is catalyzed by special enzymes called carotenoid cleavage dioxygenases(CCDs) [11].Mutation ofCCDs can lead to a change in carotenoid content,as has been reported [12] in seeds of theArabidopsis ccd1mutant.The metabolic regulation of carotenoids includes regulation not only of transcription level(synthesis and degradation),but of post-transcriptional storage[13].Because the chromoplast is the main organelle for the synthesis and storage of carotenoids in petals,the accumulation of carotenoids is also affected by chromoplast development.

Brassica napusL.(2n=38,genome formula AACC) is a major world oil crop.In China,from southwest to northwest,the flowering period of oilseed rape lasts from January to August,attracting much tourism for its ornamental potential.Because petal color in oilseed rape is usually stable and little influenced by the external environment [14],it serves as a potential morphological marker for eliminating hybrids,selecting parents,improving seed purity,and identifying outcrossing rate in cross breeding [15].

Genetic analyses[16–18]of petal color inB.napusindicate that petal color is little affected by environmental factors and shows dominant or incompletely dominant inheritance.However,few genes influencing petal color have been studied inB.napus.To our knowledge,only one gene,BnaC3.CCD4,which encodes a carotenoid cleavage dioxygenase and is involved in carotenoid degradation,has been cloned inB.napus[19].In this study,we characterized an oilseed rape mutant namedyellowish-white flower(ywf),and isolatedBnaA08.YWFlocus by map-based cloning.Inywfmutant,a C-to-T substitution occurs in the coding region ofBnaA08.PDS3,generating a premature stop codon.Based on RNAseq and carotenoid component analysis,the truncatedBnaA08.PDS3disrupts the carotenoid biosynthesis inywfpetals.The results will promote the understanding of the carotenoid biosynthesis pathway inB.napus.

2.Materials and methods

2.1.Plant materials and growth conditions

Aywfmutant was obtained fromB.napuscv.Zhongshuang 9(ZS9) by ethyl methanesulfonate mutagenesis (EMS) treatment.Two F2populations of 544 and 278 individuals were developed from a reciprocal cross between ZS9 andywffor genetic analysis.A large F2population consisting of 3360 individuals derived from ZS9 ×ywfwas used for fine mapping.Several tissues of Zhongshuang 11 (ZS11) were used to characterize the expression patterns of target genes.A set of 629 oilseed rape accessions collected worldwide was used to identify the allelic SNP mutation site inBnaA08.YWF.All materials and populations were grown in the field at Wuhan,Hubei province,China.

2.2.Chromoplast ultrastructure and carotenoid profile analysis in petals

Blooming petals of ZS9 andywfwere sampled in the field and fixed in 2.5%(w/v)glutaraldehyde with 0.1 mol L-1phosphate buffer.Sample preparation and scanning of transmission electron microscopy were as described previously [20],and employed a Tecnai G220 TWIN transmission electron microscope (FEI,Hillsboro,OR,USA).

Similarly,blooming petals of ZS9 andywfwere sampled and immediately frozen in liquid nitrogen to measure the content of total carotenoids and compositions.Total carotenoid content was determined by spectrophotometry following Wrolstad [21].Highperformance liquid chromatography was performed [22] to identify the qualitative and quantitative characteristics of carotenoid components.

2.3.Bulked-segregant analysis sequencing and positional cloning

Bulked-segregant analysis sequencing (BSA-seq) based on whole-genome resequencing (WGS) was performed to rapidly define the primary candidate region linked to theywflocus.Briefly,20 yellow-petal and 20 yellowish-white-petal individuals were collected from the F2population of ZS9 ×ywf.Equal amounts of high-quality genomic DNA from these plants were combined into two pools:Y and W pools,representing yellow-petal plants and yellowish-white-petal plants,respectively.Four DNA libraries of the two pools and two parental lines were sequenced on to the Illumina HiSeq 2500 platform (Beijing,China).Subsequent steps,including reads alignment,filtering,and BSA-seq analysis were performed as previously described [23].

For fine mapping,835 yellowish-white-petal individuals in the F2population of 3360 plants were screened for recombinants.A high-throughput genotyping technique,kompetitive allelespecific PCR (KASP) [24],was employed to genotype the mapping population.Homozygous and polymorphic variation points(single nucleotide polymorphism,SNPs) were verified by sequencing PCR products using the Sanger method and converted into KSAP markers(Fig.S1) [23].Statistical analysis and classification of polymorphic SNPs in the candidate region were performed with the OmicShare tools (https://www.omicshare.com/tools/).All primers used in fine mapping are listed in Tables S1 and S2.

The full-length genomic DNA including the promoter region of the candidate gene was amplified from ZS9 and theywfmutant using gene-specific primers (Table S3),and cloned into the pBI121 vector using ClonExpress II One Step Cloning Kit C112-01(Vazyme,Nanjing,China).Recombinant plasmids were transformed into DH5α competent cells for sequencing.Multiple sequence alignment of the candidate gene was performed with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and Color Align Conservation (http://www.bioinformatics.org/sms2/color_align_cons.html).

2.4.Phylogenetic and synteny analysis of PDS3 genes in B.napus,B.rapa,and B.oleracea

The amino acid sequence of PDS3 inBrassicawas retrieved from theBrassicaDatabase(http://brassicadb.org/brad/index.php).ClustalW [25]was used to align amino acid sequences.A phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap trials and 111 random seeds with MEGA 5 [26].

The orthologous regions covering thePDS3genes (extending 20 kb upstream and downstream)were extracted from the genome region inB.napus,B.rapa,andB.oleracea.Diagrams of alignments were constructed with custom Perl scripts.Alignment images were exported from the Artemis Comparison Tool (ACT) 11.0.0 (https://www.sanger.ac.uk/tool/artemis-comparison-tool-act/).

2.5.Plasmid construction and subcellular localization

To identify the subcellular localization of BnaA08.PDS3,we obtained 1629-and 162-bp coding sequences lacking the stop codons ofBnaA08.YWFandBnaA08.ywfby PCR using the primers listed in Table S3.The two fragments were separately fused with green fluorescent protein (GFP) in the vector pBWA(V)HSGlosgfp,resulting in the two plasmids35S::YWF-GFPand35S::ywf-GFP.For the fusion of all plasmids and fragments,we used ClonExpressMultiS One Step Cloning Kit C113-01 (Vazyme).These fusion proteins were expressed inArabidopsisprotoplast cells by transient transformation,and the subcellular localization of the target protein was determined by observing the position of the GFP displayed in the cell under a Nikon C2-ER confocal microscopy(Nikon,Japan).Both 4′,6-diamidino-2-phenylindole(DAPI)and farred fluorescence protein reporter(mKate)fused with nuclear localization signal were used as the control for nuclear localization.

2.6.RNA-seq and data analysis

Three biological replicate samples from ZS9 andywfblooming petals were collected at the full blooming stage.Total RNA was isolated using RNA prep pure Plant Kit (Tiangen,Beijing,China).An RNA-seq library was constructed with 10 μg purified total RNA from each sample and sequenced on the Illumina HiSeq platform.To ensure the quality of information analysis,raw reads were filtered to obtain clean reads,and subsequent analysis was based on clean reads.TopHat2 was used to map clean reads to theB.napusgenome (http://www.genoscope.cns.fr/brassicanapus/data/)[27–29].Then the mapped reads were assembled and quantified using StringTie [30].As an indicator,fragments per kilobase of transcript per million fragments mapped(FPKM)values were used to measure transcript or gene expression levels [31].Principal component analysis (PCA) and Spearman correlation coefficient was used as the index of correlation between biological replicates[32].Fold change was used to represent the ratio of expression levels between the two groups.The false discovery rate (FDR)was obtained by correcting for theP-value of significant difference[33].|Fold change| ≥2 and correctedP-value <0.01 were used as the screening criteria for detection of differentially expressed genes (DEGs).The Kyoto Encyclopedia of Genes and Genomes(KEGG)of DEGs and heat map were performed with the OmicShare tools (https://www.omicshare.com/tools/).

2.7.Synthesis of cDNA and quantitative real-time PCR analysis

Total RNA was extracted from roots,stems,leaves,buds,siliques,sepals,stamens,pistils,fresh petals,blooming petals,and withered petals of ZS11.First-strand cDNA was generated using the PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio,Beijing,China).The relative expression level of genes was assessed by quantitative real-time PCR (RT-qPCR) with theB.napus actingene as an internal control.All RT-qPCR reactions were performed with a CFX Connect Real-time PCR system (Bio-Rad,Hercules,CA,USA)and each reaction mixture (20 μL) consisted of 1 μL reversetranscribed product and 10 μL 2×SYBR Green Real-time PCR Master Mix (Bio-Rad).The relative quantification of gene expression was calculated using the 2-ΔΔCTmethod[34].The RT-qPCR results were expressed as the mean ± standard deviation (SD) by three biological replicates (each with three technical repeats).The primer sequences are listed in Table S4.

2.8.Metabolome analysis

Blooming petals of ZS9 andywfwith three biological replicates were collected and frozen in liquid nitrogen.Metabolome analysis was performed following the method described previously [35].Fold change ≥2 or fold change ≤0.5 while variable importance in project (VIP) ≥1 were taken as standards to assign differential metabolites abundance in ZS9 andywf.

3.Results

3.1.Phenotypic characteristics of the ywf mutant

The EMS mutantywfderived from ZS9 showed a visually distinct phenotype with yellowish-white petals (Fig.1A–C).The distinctive color of petals was observed throughout flower development including fresh,blooming,and withering flowers(Fig.1C).Plant architectural traits were most affected in theywfmutant,in which the primary branch number was increased while plant height and initial branch height were reduced (Fig.1D;Table S5).However,there were no significant differences in main inflorescence length,silique number of the main inflorescence,and 1000-seed weight (Fig.1D;Table S5).

Total carotenoid content inywfpetals was only one-third that in ZS9 petals(Fig.1E).Of six carotenoid components,all were uniformly reduced inywf,and all were reduced at least twofold but zeaxanthin (Table 1).These results suggested that the carotenoid biosynthesis pathway was blocked in theywfmutant.Chromoplast structures in petals of ZS9 andywfshowed marked differences(Fig.1F–I).In ZS9 petals,the whole chromoplast was well developed,deeply stained,and plastoglobule-filled (Fig.1F and G),whereas the development of chromoplast in theywfpetals was defective (Fig.1H and I).Plastoglobules were much plumper in ZS9 than inywfpetals (Fig.1F–I).These observations suggested that theywfmutant was defective in chromoplast development and carotenoid contents.

3.2.Map-based cloning of YWF

Map-based cloning was used to identifyYWFand investigate the genetic basis of theywfphenotype.We constructed a reciprocal cross between ZS9 andywf.Petal color in the reciprocal cross F1generation was the same as in ZS9(Fig.1A and B).Both F2populations developed from the reciprocal cross F1lines segregated for yellow and yellowish-white petals,and the ratio fitted a segregation of 3:1 (yellow,yellowish-white,χ2=0.12 and 0.31,1=3.84) (Table 2).Thus,the petal color of theywfmutant was caused by a single recessive nuclear gene.

Based on the above genetic analysis,BSA-seq was performed for primary mapping.The statistical analysis of WGS reads (described in Table S6)suggested that the WGS data were reliable and suitable for mutation detection.The WGS reads harbored homozygous polymorphic sites (73150 SNPs and 19030 InDels) (Fig.2A).Based on the ΔSNP index[36],a single candidate region on chromosome A08 was identified (Fig.2B).This region,covering 11.2–16.8 Mb,was assigned as the initial candidate region of theYWFlocus(Fig.2C).

Table 1 The contents of carotenoid compositions (μg g-1 fresh weight) determined by HPLC.

Table 2 Segregation of the two F2 populations.

TheYWFlocus was fine-mapped to an 857-kb region between markers ywf-14 and ywf-16,based on 152 recombinants(Fig.3A).Among the 4743 SNPs in the 857-kb region,there was only one homozygous site (ywf-15) polymorphic between the two parents (Fig.S2).Thus,the region was not narrowed further,even though numerous recombinants were linked to the 857-kb region.The SNP ywf-15 contained a substitution of C by T inBnaA08g17170Dgene,generating a premature stop codon (Fig.3B and C).The allelic SNP distinguishedywffrom the other 628 oilseed accessions (Fig.3D).According to the functional annotation of theB.napusgenome database[27],BnaA08g17170Dencodes phytoene desaturase (PDS3),which is involved in carotenoid biosynthesis.BecauseBnaA08.YWFwas truncated inywf,the same RT-qPCR primer was designed forBnaA08.YWFandBnaA08.ywf,uniquely matching the first exon ofBnaA08g17170D.The relative expression level ofBanA08.ywfwas down-regulated significantly inywfpetals(Fig.3E).These results suggested that the truncatedBnaA08g17170Dwas responsible for theywfmutant phenotype.

Fig.1.Phenotypic characterization of the ywf mutant.(A-B)Phenotypes of blooming petals in ZS9,ywf,F1 lines of ZS9×ywf,and ywf×ZS9.Scale bar,1 cm(A)and 5 cm(B).(C) Whole plants of ZS9 (right) and ywf (left),Scale bar,30 cm.(D) Statistical analysis of agronomic traits in ZS9 and ywf plants.Thirty plants were measured.Error bars indicate SD.Significance level of the Student’s t-test,**, P <0.001.(E) Total carotenoid content in fresh blooming petals of ZS9 and ywf,in mg g-1 fresh weight.Error bars indicate SD from three biological replicates,significance level of Student’s t-test,**, P<0.001.(F–I)Ultra-structure of plastids in blooming petals of ZS9(F,G)andywf(H,I).PG,plastoglobule.Scale bar,2 μm (F,H) and 0.5 μm (G,I).

3.3.Comparative genomics of PDS3 genes in B.rapa,B.oleracea,and B.napus

Using theBrassicadatabase,twoPDS3genes were identified in each ofB.rapa(Bra032770,Bra010751) andB.oleracea(Bol016089,Bol009962).Overall,there is high synteny between theB.napussub-genomes(A and C)and the genomes of their progenitorsB.rapaandB.oleracea[27,37].However,according to the reference genome of Darmor-bzh,protein and cDNA sequence analysis indicated that there were four otherPDS3genes(BnaA04g06150D,BnaA08g17160D,BnaC03g76050D,andBnaC04g28970D) inB.napus(Fig.S3A).Evolutionary relationships indicated thatBnaA08g17160D,BnaA08g17170D,andBnaC03g76050Dwere orthologous toBol016089inB.oleraceaandBra010751inB.rapa(Fig.S3A).The syntenic regions harboringPDS3genes on chromosomes A04 and C04 ofB.napuswere delineated,and the syntenic relationship was in accord with the evolutionary relationship(Fig.S3B).Curiously,the two contiguousPDS3genes(BnaA08g17160DandBnaA08g17170D)were located on chromosome A08 inB.napus(Fig.S3C),and the two contiguousPDS3genes showed higher synteny withBol016089andBra010751(Fig.S3C).However,there was only onePDS3gene(chrA08g002187) on chromosome A08 in the Ningyou7 (NY7) genome,which was assembled by super-scaffolding with highthroughput chromosome conformation capture (Hi-C) data [38].Using the NY7 reference sequence,the full-length coding sequence ofBnaA08.PDS3andBnaA08.pds3was isolated from the petals of ZS9 andywfusing gene-specific primers.After sequencing validation,the full-lengthBnaA08.PDS3consisted of twelve exons(1632 bp) in ZS9 and premature termination ofBnaA08.PDS3was established inywf(Fig.4A;Fig.S4A).

Fig.2.BSA-seq approach applied for mapping a genomic region.(A)The innermost two circles display the genome-wide densities of respectively SNPs and InDels between ZS9 and ywf based on WGS data.The outermost circles show the physical sizes of 19 chromosomes.The yellow and green circles show the SNP index of ywf-type petal color pool and ZS9-type petal color pool,respectively.The red circle shows the ΔSNP index.(B)SNP index plot of ywf-type petal color pool(top),ZS9-type petal color pool(middle)and ΔSNP-index plot (bottom) of chromosome A08.(C) The candidate region is in chromosome A08 at 11.2–16.8 Mb.

3.4.BnaA08.PDS3 encodes a nucleus-localized protein and is highly expressed in oilseed rape petals

As described above,four corresponding orthologous genes were divided into two syntenic blocks includingBnaA04g06150D,BnaC04g28970D,BnaC03g76050D,andBnaA08.PDS3.Although the fourPDS3genes were expressed in all tissues ofB.napus,subfunctionalization was still present,based on the RT-qPCR analysis(Fig.4B).The transcript levels ofBnaA04g06150DandBnaC04g28970Dfrom the same syntenic block were markedly lower than those of the other twoPDS3genes in the other syntenic block (Fig.4B).Still,BnaA08.PDS3showed higher expression in petals thanBnaC03g76050D(Fig.4B).This result suggested thatBnaA08.PDS3might play a more pivotal role in than other orthologous genes.

Subcellular localization using confocal microscopy showed that both YWF-GFP and ΔYWF-GFP were localized in the nucleus,as verified by DAPI and mKate(Fig.4C).Thus,BnaA08.PDS3 was predicted to be a nucleus-localized protein.

3.5.Global transcriptome analysis of ZS9 and ywf

To characterize the gene expression of theBnaA08.PDS3-associated pathway,transcriptome profiles of fresh blooming petals from ZS9 andywfwere generated with three quality biological replicates based on PCA and Spearman correlation(Fig.5A and B).Global transcriptome analysis generated a total of 82.22 Gb clean data and the Q30 base percentage of each sample was at least 94.68% (Table S7).The reads map rate to the reference genome of each sample was between 90.45% and 91.23% (Table S7).

A total of 2789 DEGs were identified,including 1565 (56.11%)up-regulated and 1224(43.89%)down-regulated in theywfmutant(Fig.5C).Of these,20 DEGs (10 up-regulated and 10 downregulated) were selected to verify the expression levels by RTqPCR,the results of which were highly consistent with the FPKM value based on RNA-seq (Fig.5D).To discern the functional distribution of DEGs,the KEGG enrichment was conducted.The DEGs were enriched in carotenoid biosynthesis,nitrogen metabolism,circadian rhythm,plant hormone signal transduction,protein processing in the endoplasmic reticulum,and flavonoid biosynthesis pathways (Fig.5E).Carotenoid biosynthesis was the most significantly enriched pathway and plant hormone signal transduction pathway contained the most DEGs (Fig.5E).

3.6.BnaA08.PDS3 required for carotenoid biosynthesis

Fig.3.Map-based cloning of the ywf gene.(A)The ywf locus was initially mapped to 11.2–16.8 Mb of chromosome A08 based on BSA-seq.Finally,the ywf locus was limited to a 857-kb region linked with ywf-14 and ywf-16 using 855 F2 mutant-phenotype individuals.(B)Gene structure of BnaA08g17170D.ATG and TGA are start and stop codons.Black boxes and gray box represent exons and 5′ UTR region,respectively.(C) Confirmation of the mutation by Sanger sequencing.Yellow shading indicates the C-to-T substitution and codon change.(D) Mutation site variation in 629 accessions.Red and blue dots represent two types of SNP variation (T:T and C:C).Black dots show the negative control.(E)The mRNA levels of BnaA08g17170D in the blooming petals of ZS9 and ywf.Error bars indicate SD of three biological replicates.***,significant at P<0.001 by Student’s t-test,

Because carotenoid biosynthesis was the most significantly enriched pathway and the contents of total carotenoids and carotenoid components were significantly lower in the petals of theywfmutant,we investigated the DEGs involved in carotenoid biosynthesis.In higher plants,carotenoid biosynthesis is divided into four steps:the MEP pathway and the formation of lycopene,carotenes,and xanthophylls (Fig.6) [5].A total of 100 genes involved in carotenoid biosynthesis inB.napuswere identified (Fig.6;Table S8).According to the transcript level of RNA-seq,no DEGs were identified in the MEP pathway or carotene formation (Fig.6;Table S8).Six genes,two up-regulated and four down-regulated,were found in the process of lycopene formation,and three up-regulated genes were identified in the formation of xanthophylls(Fig.6;Table S8).Having already verified that there was only onePDS3gene (BnaA08.PDS3,rather thanBnaA08g17160DandBnaA08g17170D) on chromosome A08 (Fig.4A),we recalculated the FPKM value ofBnaA08.PDS3in ZS9 andywfbased on the number of reads and gene length,finding it consistent with the result of RT-qPCR (Fig.3E;Table S8).Thus,the DEGs of this pathway suggested that abnormalities may occur during the formation of lycopene and xanthophylls.

The level of upstream product of β-carotene in the six components in theywfmutant petals was significantly lower than that in ZS9,suggesting that carotenoid biosynthesis was interrupted upstream of β-carotene (Fig.6;Table 1).Only twoPDS3genes involved in the formation of β-carotene were down-regulated significantly (Fig.6).The down-regulatedPDS3genes were from the same syntenic block of the fourPDS3orthologous genes(Fig.S3C).The expression pattern of the twoPDSgenes indicated thatBnaA08.PDS3played a more active role thanBnaC03g76050Din blooming petals (Fig.4B).The results of positional cloning showed that the candidate geneBnaA08.PDS3was prematurely terminated inywf(Fig.4A).Taken together,these findings suggested that the truncatedBnaA08.PDS3interrupted carotenoid biosynthesis inywf.

4.Discussion

4.1.Identification and evolution of BnaA08.PDS3 in B.napus

BSA-seq combined with fine mapping has been used for rapid delimitation of candidate regions and gene cloning in several crops including polyploid rapeseed.Cloned genes include the boron uptake efficiency regulating geneBnaA3.NIP5;1[39],the leaf color controlling geneBnaA03.CHLH[23],and the petal color controlling geneBjpc2[15].In the present study,after an initial candidate region was identified by BSA-seq,it was further narrowed to 857 kb on chromosome A08 using polymorphic markers.However,because in this region only one homozygous mutation site was found based on SNP mutation site analysis,no polymorphic marker could be effectively developed in this region.This lone mutation site led to the premature termination ofBnaA08g17170D,suggesting thatBnaA08g17170Dwas the candidate gene forywf.

The reference genome of Darmor-bzhis the first one assembled by next-generation sequencing inB.napus[27].According to this reference genome,there were twoBnaA08.PDS3(BnaA08g17170DandBnaA08g17160D) genes on chromosome A08 (Fig.S3C);however,the reference genomes of NY7 and other rapeseed cultivars including ZS11,Gangan,No2127,Shengli,Westar,and Zheyou,assembled by Pac-Bio method,showed only onePDS3gene on chromosome A08 (http://cbi.hzau.edu.cn/bnapus/index.php)[38,40].We also used a specific primer to amplify and sequence the target gene to examine theBnaA08.PDS3gene structure in Darmor-bzh(Fig.4A,S4).We observed that the Darmor-bzhgenome had assembly errors forPDS3on chromosome A08,and accordingly we assignedBnaA08.PDS3(notBnaA08g17170D) as the candidate gene forywf.

4.2.PDS3 acts as a multifunctional gene

BnaA08.PDS3was highly expressed in floral tissues,especially in blooming petals (Fig.4B).WhenBnaA08.PDS3was mutated,the petal color changed but not the leaf color.Apart from the different petal colors,ywfmutant also displayed a dwarf,multi-branched phenotype in contrast to ZS9 (Fig.1D;Table S5).Previous studies have also suggesed multifunctional roles of thePDS3gene.For example,in theArabidopsisT-DNA insertion mutantpds3,a mutation inPDS3disrupted the biosynthesis of chlorophyll,carotenoids,and gibberellins,resulting in albinism and dwarfing phenotypes[41].Similarly,in transgenic tobacco,RNAi-mediated suppression of thePDSgene led to varying degrees of albinism [42].Because this phenotype is easily observed with the naked eye,thePDSgene can be used as a reporter gene in a virus-induced gene silencing system [43].In a previous study [44],specific site mutations in thePDSgene improved resistance to herbicides.These reports and the present findings reflect the potentially wide-ranging application of thePDSgene in genetic engineering.

4.3.The possible mechanism of petal color variation in oilseed rape

The color of flower petals is determined mainly by the composition of three major pigments including anthocyanin,betalain,and carotenoids [45].In petal coloring,anthocyanin controls the orange,red,purple,and blue colors,and betalain controls the deep red and yellow colors [46].The carotenoids most abundant in petals are violaxanthin and β-carotene,which control the yellow,orange,red,and purple colors.InBrassicaceae,the carotenoid content is always higher in yellow than in white petals [15,19].In the present study,the contents of total carotenoids and compositions were significantly reduced in the petals of theywfmutant(Fig.1E;Table 1).

The carotenoid biosynthesis pathway is well established and the responsible genes have been identified in higher plants [5].The composition and content of carotenoids are correlated with the expression level of carotenoid biosynthesis genes.In white petals ofB.rapa,with the decrease of carotenoid content,the expression levels of the genes (PSY,PDS,ZDS,ZEP,andLCYB)involved in carotenoid biosynthesis were down-regulated [47].KEGG analysis showed that the carotenoid biosynthesis pathway was most significantly enriched (Fig.5E).Inywfpetals,upstream of β-carotene,BnaA08.PDS3was the most down-regulated gene(Fig.6).Combined with the results of map-based cloning,BnaA08.PDS3was confirmed as the candidate gene.Thus,the truncatedBnaA08.PDS3interrupted the carotenoid biosynthesis pathway inywf,resulting in the decrease of carotenoid content and a yellowish-white petal phenotype.All these results indicate that the change of flower color inBrassicaceaefrom yellow to white is associated mainly with the composition and content of carotenoids.

Fig.6.Simplified carotenoid biosynthetic pathway.Heat map displays the expression levels of genes involved in carotenoid biosynthesis pathway.log2[fold change-FPKM(ywf/ZS9)] was calculated by FPKM value.The FPKM value of BnaA08.PDS3 was recalculated based on read abundances of BnaA08g17160D and BnaA08g17170D.Dots and circles indicate respectively up-regulated and down-regulated DEGs.Red star indicates the most highly differentially expressed gene in the pathway.

4.4.The ywf mutant may offer resistance to Sclerotinia sclerotiorum

Sclerotiniastem rot is a highly infectious and devastating disease inB.napus.Petals can provide nutrients for ascospores ofS.sclerotiorum,forming a primary infection.When infected petals fall on the leaves or stem,they can infect these tissues and produce large disease spots [48].Oilseed rape blooming time is the most important period forSclerotiniaspores to infect petals [49].The ascospores ofS.sclerotiorumcan germinate only on petals to form hyphae,whereas spores falling directly on oilseed rape leaves of cannot survive for a long time [50].Flavonols are ubiquitous in plants and play an important role in plant defense against pathogen infection [51–53].InArabidopsis,S.sclerotiorumstrains with lower efficiency to degrade flavonols were less virulent,suggested that flavonols increased resistance againstS.sclerotiorum[54].In our study,of 59 significantly differentially expressed metabolites,hesperetin,kaempferol,quercetin,and flavonol were increased in the petals ofywf(Table S9).The sclerotinia disease index ofywfwas significantly lower than that of ZS9 (Table S10),suggesting the potential of theywfmutant for disease resistance and plant defense.

CRediT authorship contribution statement

Shengyi Liu,Chuanji Zhao,Lijiang Liu,and Junyan Huang:designed the research.Chuanji Zhao:conducted the experiments,performed the data analysis,and wrote the manuscript.MeijuanShi,Zhixue Dong,Xiaohui Cheng,Yueying Liu,and Yang Xiang:managed and provided materials.Li Yang,Zetao Bai,Meili Xie,and Chaobo Tong:assisted in data analysis.Luqman Bin Safdar,Lijiang Liu,Junyan Huang,and Shengyi Liu:revised the manuscript.All authors reviewed and approved this manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was supported by the National Key Research and Development Program Of China (2016YFD0101007 and 2018YFE0108000),National Natural Science Foundation of China(31770250),the Natural Science Foundation of Hubei Province(2019CFB628),China Agriculture Research System(CARS-12),Agricultural Science and Technology Innovation Program (ASTIP) of Chinese Academy of Agricultural Sciences,and The Agricultural Scientific and Technological Research Projects of Guizhou Province(No.Qiankehezhicheng [2019] 2397).

Data availability

Raw RNA-seq reads generated in this study are available from the Sequence Read Archive(SRA)at National Center for Biotechnology Information(NCBI)(https://www.ncbi.nlm.nih.gov/sra/)under accession number PRJNA601012.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.10.012.