Fine mapping and candidate gene analysis of the white flower gene Brwf in Chinese cabbage (Brassica rapa L.)

Flower color can be applied to landscaping and identification of the purity of seeds in hybrid production. However, the molecular basis of white flower trait remains largely unknown in Brassica rapa. In this study, an F2 population was constructed from the cross between 15S1040 (white flower) and 92S105 (yellow flower) for fine mapping of white flower genes in B. rapa. Genetic analysis indicated that white flower trait is controlled by two recessive loci, Brwf1 and Brwf2. Using InDel and SNP markers, Brwf1 was mapped to a 49.6-kb region on chromosome A01 containing 9 annotated genes, and among them, Bra013602 encodes a plastid-lipid associated protein (PAP); Brwf2 was located in a 59.3-kb interval on chromosome A09 harboring 12 annotated genes, in which Bra031539 was annotated as a carotenoid isomerase gene (CRTISO). The amino acid sequences of BrPAP and BrCRTISO were compared between two yellow-flowered and three white-flowered lines and critical amino acid mutations of BrPAP and BrCRTISO were identified between yellow-flowered and white-flowered lines. Therefore, Bra013602 and Bra031539 were predicted as potential candidates for white flower trait. Our results provide a foundation for further identification of Brwf and increase understanding of the molecular mechanisms underlying white flower formation in Chinese cabbage.

In nature, flower color was used to attract insect for pollination in plants 1 . There are three chemically distinct pigments, carotenoids, flavonoids, and betalains, responsible for flower color, and among them, carotenoids accumulating in petals can generate yellow, orange, and red flower colors 2,3 . The most common carotenoids in petals are xanthophylls, which show high specificity in composition and quantity among plant species or varieties 4 .
Carotenoid accumulation was modulated by its biosynthesis, degradation, and sequestration [5][6][7][8] . The mutation of key genes involved in the above three processes could result in the conversion of flower and fruit colors. For example, a single-nucleotide mutation in β-carotene hydroxylase 2 (CHYB2) caused orange fruit phenotype in pepper 9 . In Chrysanthemum morifolium, Brassica napus, and B. oleracea, the loss-of-function mutation of carotenoid cleavage dioxygenase 4 (CCD4) led to change in flower color from white to yellow 7,10-14 . The mutation of pale yellow petal (PYP1) that was involved in xanthophyll ester production was responsible for pale yellow petal phenotype in tomato 8 .
In this study, the inheritance pattern of white flower trait was analyzed using an F 2 segregating population developed from the crossing of white flower line 15S1040 and yellow flower line 92S105. Molecular markers designed based on the genome re-sequencing data of 15S1040 and 92S105 were used to map white flower genes, and then the prediction of the candidate genes was performed; the coding sequences of two candidate genes (BrPAP and BrCRTISO) were compared between three white-flowered and two yellow-flowered lines; the expression levels of two candidate genes were tested in different tissues. Our findings provide insights in molecular mechanisms controlling flower color variation in B. rapa.

Results
Genetic analysis of the white flower trait in B. rapa. The flower colors of F 1 plants derived from the cross between white parent 15S1040 and yellow parent 92S105 were all yellow (Fig. 1a-c). Among 1282 F 2 individuals, 718 individuals were yellow flower, 257 individuals were milky yellow flower, 227 individuals were pale yellow flower, and 80 individuals were white flower ( Fig. 1d-g). The F 2 segregation ratio was fitted into an expected ratio of 9:3:3:1 (χ 2 = 1.908, df = 3, P > 0.05) using χ 2 test (Table 1). These results indicated that yellow flower trait was dominant over white flower and the white flower trait was controlled by two recessive genes, Brwf1 and Brwf2, therefore the genotypes of four flower color plants may be yellow flower (BrWF1BrWF1BrWF2BrWF2, BrWF1BrWF1BrWF2Brwf2, BrWF1Brwf1BrWF2BrWF2, or BrWF1Brwf1BrWF2Brwf2), milky yellow flower (Brwf1Brwf1BrWF2BrWF2 or Brwf1Brwf1BrWF2Brwf2), pale yellow flower (BrWF1BrWF1Brwf2Brwf2 or BrWF1Brwf1Brwf2Brwf2) and white flower (Brwf1Brwf1Brwf2Brwf2), respectively.
Carotenoid accumulation and ultrastructural analysis of chromoplasts in yellow and white petals. Carotenoid composition and content in yellow and white petals at the flowering stage were analyzed using high performance liquid chromatography (HPLC). The results showed that the major carotenoids in yellow and white petals were both violaxanthin and lutein, however, the total carotenoid contents of yellow and white petals were 211.69 ± 21.70 μg/g and 10.49 ± 1.21 μg/g (Fig. 2a), respectively, which may result in the difference in color between yellow and white petals.
To study whether there were differences in chromoplast structures between yellow and white petals, the ultrastructural analysis of chromoplasts in the two parents was performed using transmission electron microscopy (TEM). The results indicated that yellow-flowered individuals had normal chromoplasts with numerous fully developed plastoglobules (PGs), however, white-flowered individuals showed abnormal chromoplasts with few PGs (Fig. 2b,c).  Table 1. The segregation of flower colors in the F 1 and F 2 population. a χ 2 > χ 2 (0.05, 3) = 7.815 is considered significant.
trait, 81 insertion/deletion (InDel) markers distributed on 10 chromosomes were developed based on the re-sequencing data of the two parents, and 34 InDel markers (W1-W11, W101-W288) exhibited polymorphism between 15S1040 and 92S105 (Supplementary Table S1). These polymorphic markers were used for bulk segregant analysis (BSA) of flower color trait. As a result, six markers (W101, W105, W107, W112, W114, and W116) on chromosome A01 and three markers (W1, W5, and W11) on chromosome A09 were linked with Brwf genes. Among them, W105 and W112 markers, W5 and W11 markers were randomly chosen to assay 30 milky yellow-flowered and 30 pale yellow-flowered plants from F 2 population. The results showed that W105 and W112 markers were linked with the Brwf1 gene controlling milky yellow flower and W5 and W11 markers were linked with the Brwf2 gene controlling pale yellow flower, which indicated that Brwf1 and Brwf2 were located on chromosomes A01 and A09, respectively.
For preliminary mapping of the Brwf1 and Brwf2 genes, newly designed 36 InDel markers on chromosome A01 and 35 InDel markers on chromosome A09 were screened between the two parental lines, and 14 (W310-W339) and 12 (W23-W60,W67) markers showed polymorphism, respectively (Supplementary Table S1). These polymorphic markers were used for BSA of flower color trait and 14 markers on chromosome A01 and 5 markers on chromosome A09 were linked with the Brwf genes. To preliminarily map the Brwf1 and Brwf2 genes separately, A and B groups that were segregated in BrWF1/Brwf1 and BrWF2/Brwf2 loci, respectively, were selected from F 2 population and A group included 108 yellow-flowered and 36 milky yellow-flowered individuals and B group included 108 yellow-flowered and 36 pale yellow-flowered individuals. Then obtained 19 linkage markers from chromosomes A01 and A09 were used to detect A and B groups, respectively. In A group, the Brwf1 gene, co-segregating with W323 marker, was localized to a region between W322 and W331 markers on chromosome A01, and the genetic and physical distances were 0.74 cM and 186.1 kb, respectively (Fig. 3a). In B group, the Brwf2 gene was mapped to a 0.71 cM interval flanked by W5 and W67 markers with the corresponding physical distance of 216.3 kb on chromosome A09, and one marker W11 co-segregated with Brwf2 (Fig. 3b). fine mapping of the Brwf genes. For fine mapping of the Brwf1 gene, the two markers W322 and W331 were used to detect recombination events in all F 2 plants, and a total of 18 recombinants including 5 recombination events with W322 marker and 13 recombination events with W331 marker were obtained. Using the re-sequencing data of the two parents, 10 new InDel markers were developed from the preliminary mapping region and seven of them (W341-W352) exhibited polymorphism in the two parents (Supplementary Table S1). These polymorphic markers were unceasingly used to screen all the 18 recombinants. The results indicated that the Brwf1 gene was delimited to a shortened interval between W323 and W351 markers with one recombinant and four recombinants, respectively (Fig. 4a-c and Supplementary Fig. S1). To further narrow down the mapping interval, two single-nucleotide polymorphism (SNP) markers (S361 and S371) were developed and used to test recombination events. As a result, one recombination event with S361 marker and two recombination events with S371 marker were found, and then a developed SNP marker S363 on the side of S371 was also used to detect two recombination events (Supplementary Table S1 and Fig. S2). The two SNP markers S361 and S363 further narrowed the Brwf1 gene to an interval of 0.11 cM with the corresponding physical distance of 49.6 kb, Finally, two markers, W348 and W350, co-segregating with Brwf1 were obtained (Fig. 3c).
To fine map the Brwf2 gene, a total of nine recombinants were identified using W5 and W67 markers, which included three recombination events occurring between W5 marker and Brwf2 and six recombination events occurring between W67 marker and Brwf2. Among 15 InDel markers developed from the preliminary mapping interval, five markers (W61, W72-W79) were polymorphic between 15S1040 and 92S105 (Supplementary Table S1). The nine recombinants were screened by five new polymorphic markers. As a result, the Brwf2 gene was restricted to a region between W11 and W78 markers and there were one recombinant with W11 marker and two recombinants with W78 marker (Fig. 4d-f and Supplementary Fig. S1). For further narrow down the mapping region, two more SNP markers (S82 and S83) were developed for detecting recombination events (Supplementary Table S1). www.nature.com/scientificreports www.nature.com/scientificreports/ The result showed that one recombination event was found between each of the two SNP markers and Brwf2 ( Supplementary Fig. S2) respectively, so the Brwf2 gene was delimited to a 0.08 cM region flanked by S82 and S83 markers, and the corresponding physical distance was 59.3 kb. Finally, two markers, W61 and W74, co-segregating with Brwf2 were obtained (Fig. 3d).
Identification and sequence analysis of the candidate genes. According to the B. rapa reference genome in BRAD (Brassica database, http://brassicadb.org/brad), 9 and 12 genes were annotated within the two final mapping intervals of Brwf1 and Brwf2 genes, respectively (Fig. 3e,f). Among 9 annotated genes in the Brwf1 interval on chromosome A01, Bra013602 encodes a plastid-lipid associated protein (PAP) that was previously reported to regulate carotenoid accumulation 39,40 (Table 2). Out of 12 annotated genes in the Brwf2 interval on chromosome A09, Bra031539 was predicted to encode a carotenoid isomerase (CRTISO) that was involved in carotenoid biosynthesis [41][42][43][44] (Table 2). Therefore, Bra013602 and Bra031539 were predicted as the two candidates for Brwf1 and Brwf2 genes, respectively.
The specific primers WY503 was designed for cloning and sequencing of the cDNA sequences of BrPAP (Supplementary Table S1). The gene sequence comparison showed that there were 15 SNPs in the coding region of BrPAP between 92S105 and 15S1040 ( Supplementary Fig. S3a), which resulted in four amino acid residue mutations ( Supplementary Fig. S4a). Based on previous studies 42, 44 , two designed primers, WY571 and WY572, were used to clone the cDNA sequences of BrCRTISO in 92S105 and 15S1040, respectively (Supplementary Table S1). The sequence alignment indicated that there were many SNPs, one small deletion, and one large insertion in the coding region of BrCRTISO in 15S1040. This large insertion had 943 bp that was located at the 3′ end of BrCRTISO ( Supplementary Fig. S3b). After the amino acid sequence alignment, 17 amino acid residue changes and the deletion of two amino acid residues were found in BrCRTISO of 15S1040, however, at the 3′ end, the large insertion resulted in mutations of 15 amino acid residues, one amino acid residue insertion, and three amino acid residue deletions in BrCRTISO of 15S1040 ( Supplementary Fig. S4b).
To identify the key mutations of the two candidate genes between white-flowered and yellow-flowered lines, the genomic sequences of two candidate genes from one yellow-flowered line (09Q5) and two white-flowered lines (15S1001 and 17S690) were cloned using designed specific primers, which included WY503 for BrPAP of yellow-flowered and white-flowered lines, and WY561, WY562, WY563 for the BrCRTISO of yellow-flowered line and WY561, WY562, WY566 for the BrCRTISO of white-flowered lines according to previous studies 42,44 (Supplementary Table S1). The deduced amino acid sequences of BrPAP and BrCRTISO from three yellow-/ white-flowered lines were compared with that from the two parental lines. The results indicated that the deduced amino acid sequence of BrPAP in 09Q5 was same as that in 92S105, while there were seven amino acid residue mutations among 15S1040, 15S1001, and 17S690, but only one mutant amino acid residue (Leu → Pro) was found between two yellow-flowered and three white-flowered lines and it was located in the conserved domain of BrPAP ( Fig. 5a; Supplementary Fig. S4a); the deduced amino acid sequence of BrCRTISO in 09Q5 had 17 amino acid residue mutations and one deletion of two amino acid residues compared with 92S105, while the sequences from 15S1001 and 17S690 were identical to that from 15S1040, however, two amino acid residue mutations (Ile → Val, Leu → Phe) and many amino acid residue changes at the end of sequences were consistent with the flower color and the two amino acid residues were located in the conserved domain of BrCRTISO ( Fig. 5b; Supplementary Fig. S4b). www.nature.com/scientificreports www.nature.com/scientificreports/ Expression analysis of the candidate genes and carotenoid metabolic genes. Expression pattern analysis of BrPAP and BrCRTISO was conducted using Quantitative real-time PCR (qPCR) in different tissues (roots, stems, cauline leaves, and petals) from the two parental lines. BrPAP expressed mainly in petals and could hardly be detected in other tissues with expression level of BrPAP in petals of 92S105 being twofold higher than that in 15S1040 (Fig. 6a); BrCRTISO had relatively higher expression levels in cauline leaves and petals than in roots and stems, however, BrCRTISO did not exhibit significant difference in expression between the petals of the two parental lines (Fig. 6b). Moreover, the expression levels of genes related to carotenoid metabolism in petals were detected. The results indicated that CRTISO and Lycopene ε-cyclase (LCYE) had no significant differences in expression between the petals of the two parental lines, but expression of other seven genes showed down-regulated in petals of 15S1040 compared with 92S105 ( Supplementary Fig. S5).

Discussion
In Brassica species, genetic analysis of flower color traits has been carried out early 16,17,36 . The previous investigations showed that white flower trait was dominant over yellow flower and controlled by a single gene in B. napus 11,45 and B. oleracea 13,14,18,46,47 . However, several studies have reported that white flower trait was a recessive trait controlled by two major genes 20,37,38,48 . In this study, genetic analysis of white flower trait in B. rapa was conducted with F 2 population derived from a cross between white-flowered line 15S1040 and yellow-flowered line 92S105. Our results showed that white flower trait was controlled by two separate loci and the white flower trait is recessive to yellow flower, consistent with previous reports 20, 37,38,48 .
Multiple studies have reported recently on gene mapping of white flower trait in Brassica species. In B. napus, a white flower gene was mapped to a 0.39 cM region on chromosome C03 11 . Ashutosh et al. 46 and Han et al. 12,47 also mapped a white flower gene on chromosome C03 using populations derived from the crosses between broccoli and Chinese kale, cabbage and Chinese kale, respectively. In Chinese kale, a white flower gene was also delimited to chromosome C03 13,14 . The above results indicated that a single gene controlling white flower trait might be the same gene in B. napus and B. oleracea. In B. juncea, two recessive genes that controlled white flower trait were restricted to chromosomes A02 and B04 and the genetic distances were 0.13 cM and 0.25 cM, respectively 37,38 . In this study, we found that white flower trait in B. rapa was also controlled by two genes (Brwf1 and Brwf2), which were mapped to intervals of 0.11 cM and 0.08 cM on chromosomes A01 and A09, respectively. PAP, also called fibrillin, found in the pepper fruit chromoplasts and its homologous protein in chromoplasts of cucumber flower, was named as chromoplast-specific carotenoid-associated protein (CHRC) 49 . In chromoplast, fibrillin and CHRC were positively associated with carotenoid accumulation 39,40 . The suppression of the expression of CHRC gene in tomato flowers resulted in decreased carotenoids 39 , which indicated that CHRC plays a role in mediating carotenoid storage in chromoplasts of flowers. Over-expression of the pepper fibrillin gene in tomato increased the levels of carotenoids in fruit 40 . In this study, the Bra013602 gene encoding PAP was  Table 2. Annotated genes within the mapping intervals of Brwf1 and Brwf2 on chromosomes A01 and A09. a Gene position and annotation based on BRAD B. rapa reference genome data (chromosome v1.5).
located in the final mapping region of Brwf1, which deduced amino acid sequence has four amino acid residue mutations between the two parents and one of the mutations (Leu → Pro) occurred in the conserved domain of BrPAP between yellow-flowered lines (92S105 and 09Q5) and white-flowered lines (15S1040, 15S1001, and 17S690), which might affect the function of BrPAP in white-flowered lines. In addition, fibrillin was involved in plastoglobule formation based on previous investigations 40,50 . Over-expression of the fibrillin gene from pepper in tobacco resulted in the increased number of PGs in plastids of leaves and petals 50 . In this study, ultrastructural analysis of chromoplasts in the two parents revealed that the number of PGs in yellow petal chromoplasts was more than that in white petal chromoplasts. Expression pattern analysis of BrPAP indicated that the expression level of BrPAP in petals was much higher than that in other tissues. These results indicated that BrPAP was the most possible candidate for white flower trait. It was known that the role of CRTISO is the control of the conversion of prolycopene to lycopene. The functional disruption of BrCRTISO gene resulted in the orange head leaf formation in Chinese cabbage [41][42][43][44] . However, Lee et al. 22 reported that 19 amino acid residue changes and deletion of two amino acid residues were found in the amino acid sequence of BrCRTISO from pale-yellow flower cultivar compared with that in yellow  www.nature.com/scientificreports www.nature.com/scientificreports/ flower cultivar. In this study, Bra031539 encoding BrCRTISO was located in the final delimited genomic region of Brwf2. The amino acid sequence analysis of BrCRTISO indicated that two amino acid residue mutations (Ile → Val, Leu → Phe) that were located in the conserved domain of BrCRTISO and many amino acid residue changes at the end of sequences were found between two yellow-flowered lines (92S105 and 09Q5) and three white-flowered lines (15S1040, 15S1001, and 17S690). In addition, although Zhang et al. 42 reported that the amino acid residue mutation (Leu → Phe) of BrCRTISO could not affect the protein function in leaves, this mutation which was found in petals might affect BrCRTISO function in this study. Taken together, two amino acid residue mutations (Ile → Val, Leu → Phe) and many amino acid residue changes in the C-terminal end of BrCRTISO might affect its function, which suggested that BrCRTISO was the most promising candidate for white flower trait.
In B. napus 11 and B. juncea 37 , the major carotenoid in yellow and white petals was violaxanthin, but the total carotenoid contents in yellow petals were forty-twofold and eightfold higher than that in white petals, respectively. In the present study, carotenoid analysis of yellow and white petals showed that violaxanthin and lutein were mainly accumulated in yellow and white petals of Chinese cabbage, however, the total carotenoid content was twenty times higher in yellow petals than in white petals, which were consistent with the previous studies 11,37 . Moreover, because light could partially replace CRTISO activity 22,44 , which combined with the phenotypic observation of F 2 plants and the results of amino acid sequence comparison of BrPAP and BrCRTISO, we hypothesized that the mutations of BrPAP and BrCRTISO and light might jointly affect the prolycopene accumulation and resulted in barely detecting it in 15S1040. In B. napus, a single dominant gene, BnaCCD4, controls the white flower trait and associated with carotenoid degradation 11 . In B. juncea, the white flower trait was jointly controlled by two recessive genes, Bjpc1 and Bjpc2 which encode esterase/lipase/thioesterase family protein and phytyl ester synthase 2, respectively, and were involved in carotenoid esterification 37,38 . In this study, the potential candidate genes for the white flower trait in Chinese cabbage were BrPAP and BrCRTISO that were associated with carotenoid storage and biosynthesis, respectively. The results of TEM analysis and amino acid sequence alignment of BrPAP indicated that the mutation of BrPAP resulted in decrease of carotenoid accumulation by blocking PG formation. The mutant types of BrCRTISO in the present study were incompletely consistent with the previous investigations 42,44 , which indicated that the function of BrCRTISO in 15S1040 might not be complete disruption. In addition, expression analysis of genes associated with carotenoid metabolism showed that the majority of carotenoid biosynthesis pathway genes were down-regulated expression in petals of 15S1040 compared with 92S105. Hence, the mutation of BrCRTISO might decrease the flux of carotenoid biosynthesis pathway. Taken together, we inferred that both mutations of BrPAP and BrCRTISO maybe lead to the white flower formation by decreasing total carotenoid content in 15S1040 (Fig. 7).

Methods
Plant materials. The five Chinese cabbage lines, the white-flowered 15S1040, 15S1001, 17S690, and the yellow-flowered 92S105 and 09Q5, were used in this study. 15S1040 and 92S105 (Fig. 1a,b) were selected as parents for constructing F 2 population. To study the inheritance pattern of white flower trait and fine map the Brwf genes, a cross between the two parental lines, 15S1040 and 92S105, was used to produce the hybrid F 1 , then one F 1 plant was self-pollinated to generate the F 2 population with 1282 individuals. Other white-flowered and yellow-flowered lines were used for the identification of the candidate genes. All materials were bred and provided by the Chinese cabbage research group at the Northwest A&F University, Yangling, China.
All plants used in the present study were grown and naturally vernalized at the experimental field of the Northwest A&F University in 2018. During the flowering stage, the observations of at least ten flowers per plant were performed twice to evaluate the flower color of each individual with an 8-day interval.
Carotenoid extraction and analysis. Carotenoid were extracted from fresh petals at the flowering stage and detected following the methods of Cao et al. 51 . Carotenoid analysis was performed using LC-2010AHT HPLC (Shimadzu, Kyoto, Japan) with C30 column (YMC, Kyoto, Japan). Carotenoids were identified by the typical retention time of the standard compounds, including violaxanthin (Sigma-Aldrich, Saint Louis, America), lutein (Solarbio, Beijing, China), α-carotene and β-carotene (Wako, Osaka, Japan). The identification of prolycopene was performed based on reported the typical retention time and relative order of carotenoid compound peaks 22,43,51 . Carotenoid content was quantified according to Morris' method 52 . The total carotenoid content was the sum of all the detected carotenoid compound contents. Three biological replicates were used for all analyses and the calculation of means and standard deviations were conducted. The significant difference between 92S105 and 15S1040 was analyzed by t-test.
Transmission electron microscopy analysis. Petals from 92S105 and 15S1040 flowers at the flowering stage were cut into 0.3 × 0.6 cm sections, fixed with 2.5% glutaraldehyde. The preparation of observation samples of petals and TEM analysis were performed according to Yi et al. 53

described methods.
DNA and RNA extraction, first-strand cDNA synthesis, and gel electrophoresis. Total genomic DNA was isolated from fresh leaves using the cetyl trimethylammonium bromide (CTAB) method described by Porebski et al. 54  www.nature.com/scientificreports www.nature.com/scientificreports/ Two (yellow and white flowers) and four (yellow, milky yellow, pale yellow, and white flowers) kinds of F 2 individuals were used for BSA and fine mapping, respectively. Two DNA pools, yellow-flowered pool and white-flowered pool, were created by mixing equal amounts of DNA from 8 individuals with yellow flower and 8 individuals with white flower, respectively, which were randomly selected from F 2 population. The PCR reaction and separation of its products were performed as described by Zhang et al. 31 .

Development of InDel and SNP markers.
To develop InDel and SNP markers, the two parental lines, 15S1040 and 92S105, were re-sequenced with HiSeq X Ten (Gene Denovo, Guangzhou, China) at 30-and 91-fold sequencing depths. The re-sequencing data of 15S1040 and 92S105 were mapped to the B. rapa reference genome in BRAD, the genomic variants were found using Genome Analysis Toolkit (GATK), and the annotation of the physical location of each genomic variant was carried out. The insertions/deletions> 3 bp and single-nucleotide polymorphism loci were used to develop InDel and SNP markers, respectively, with the Primer Premier 5.0 (http://www.premierbiosoft.com/primerdesign/) software based on the corresponding flanking sequences in the B. rapa reference genome. The primers used in the present study were synthesized by Sangon Biotech Co., Ltd (Shanghai, China).

Identification of recombination events.
To obtain the DNA fragments that contained SNP loci in the recombinants, the specific primers were designed according to the reference genome of B. rapa. The purification of PCR products and sequencing were conducted using our previous method 55 . The nucleotide sequences were analyzed using the DNASTAR Lasergene 7.1 (http://www.dnastar.com) and Chromas 2.4.1 (http://technelysium. com.au/wp/chromas/) softwares. fine mapping of the Brwf genes and identification of the candidate genes. The polymorphic molecular markers were utilized to assay genotype of plants in the F 2 populations. The linkage analyses were conducted using the genotypic data of the polymorphic markers and phenotypic data of each individual in F 2 segregating population. The linkage map was then constructed using the JoinMap 4.0 (https://www.kyazma.nl/ index.php/JoinMap/) software based on a LOD threshold score of 6.0. The candidate genes in the final delimited region were analyzed based on the annotation data of the B. rapa reference genome in BRAD.
cloning and sequence analysis of the candidate genes. To clone the DNA and cDNA sequences of the putative candidate genes, the primers were designed according to the B. rapa reference genome. The cloning of putative candidate genes and sequencing were performed according to our previous method 55 . The complete coding sequences of two candidate genes from two yellow-flowered and three white-flowered lines were submitted to GenBank, the accession numbers: BrPAP: MN338556 (92S105), MN338557 (09Q5), MN338558