Determination of flavonoid content
The flavonoid contents of the mature seed coat samples of the two parents were measured. The result showed that the flavonoid content of the yellow seed coat was 3.21 mg·g-1, while that of the brown seed coat was 8.09 mg·g-1 (Fig. 2). The flavonoid content of the brown seed coat was 1.5-fold higher than that of the yellow seed coat.
The flavonoid metabolite profiling analysis of the two parental lines indicated similar results. A total of 19 significantly differentially abundant metabolites, including anthocyanins, flavonols, flavonoids, chalcones, and flavonoid carbonosides, were detected between the IC2508 and IC2518 mature seed coat samples, with most metabolites upregulated (14 upregulated and 5 downregulated) in IC2518. The levels of kaempferol-3-O-sambubioside, apigenin-8-C-glucoside, diosmetin, isovitexin, chrysoeriol-5-O-glucoside, and chrysoeriol-7-O-(6''-acetyl)-glucoside were increased by at least three-fold (Table S3) in IC2518. We mapped the differentially abundant metabolites to KEGG biological pathways to identify the main pathways that the metabolites are involved in. There were three, one, and one significantly differentially abundant metabolites that were assigned to flavone and flavonol biosynthesis (ko00944), anthocyanin biosynthesis (ko00942), and flavonoid biosynthesis (ko00941), respectively, among others (Table S3). The results suggested that the difference in seed coat flavonoid content was responsible for the difference in the seed coat color.
Genetic analysis of melon seed coat color
The seed coat color of all 50 individuals in the F1 (P1 × P2) population was yellow, indicating that the yellow seed coat trait is dominant over the brown seed coat trait. In the F2 population (252 individuals), 186 individuals exhibited a yellow seed coat and 66 individuals produced a brown seed coat. The Chi-square test suggested a 3:1 segregation ratio (Table 1). The analysis of E-value showed that the seed color of F2 population conformed to bimodal distribution (Fig. S1). In the BC1P2 (F1 × P2) population (96 individuals), 58 individuals showed a yellow seed coat and 38 individuals showed a brown seed coat. The Chi-square test suggested that the segregation ratio was 1:1. The seed coat color of all 95 BC1P1 (F1 × P1) individuals was yellow (Table 1). The results confirmed that the yellow seed coat trait was controlled by a single dominant gene, which was named CmBS-1.
Table 1 Genetic analysis of seed coat color trait in six-generation family
Generation
|
Total no. of individuals
|
No. of yellow individuals
|
No. of brown individuals
|
Expected ratio
|
χ2
(α=0.05)
|
P-value
(α=0.05)
|
P1
|
50
|
50
|
0
|
-
|
-
|
-
|
P2
|
50
|
0
|
50
|
-
|
-
|
-
|
F1
|
50
|
50
|
0
|
-
|
-
|
-
|
F2
|
252
|
186
|
66
|
3:1
|
0.1905
|
0.6625
|
BC1P1
|
97
|
97
|
0
|
-
|
-
|
-
|
BC2P2
|
94
|
56
|
38
|
1:1
|
3.4468
|
0.0634
|
Preliminary mapping of CmBS-1 by BSA-Seq
We used BSA-Seq to rapidly identify the genomic region of CmBS-1. A total of 60.06 Gb clean reads were generated by re-sequencing, with an average sequencing depth of 33.37×. The clean reads were aligned to the melon reference genome, and the coverage of the reference genome was 98.02%. A total of 123,456 SNPs and 49,004 small InDels were identified between the two parents, while 30,950 SNPs and 13,894 small InDels were identified between the two DNA bulks. The details of the SNPs and InDels between the two bulks as well as the parents are shown in Tables S4 and S5. We used the ED method (the threshold was 0.29) and ΔSNP index (the confidence value was 99%) to analyze the SNPs and found that CmBS-1 was mapped to a 4.03 Mb region from 11,860,000 to 15,890,000 bp on Chr 6 (Fig. 3). Similarly, we used the ED method (the threshold was 0.30) and ΔInDel index (the confidence value was 95%) to analyze the small InDels, and the result suggested that CmBS-1 was mapped to an interval of 11,770,000–16,270,000 bp (4.50 Mb) on Chr 6 (Fig. 4). The two regions overlapped. Therefore, we confirmed that CmBS-1 was located at the merged region from 11,860,000–15,890,000 bp on Chr 6 at a physical distance of 4.03 Mb.
Fine mapping of CmBS-1 using InDel and dCAPS markers
In the preliminary mapping region, based on the re-sequencing data, we designed 1498 InDel primers and randomly chose 240 InDel primers to screen in the two parents and the two bulks to obtain polymorphic primers. Fifty-two of the 240 primers were polymorphic, and the polymorphism rate was 21.7%. We then chose eight co-dominant polymorphic markers that had clear bands and demonstrated stable amplification to map CmBS-1 in a smaller F2 population (252 individuals) with 12 recombinant individuals (Tables S6 and S7). The result suggested that the CmBS-1 gene was mapped in a 970.78 kb interval between M255 and L274 (11,109,174–12,079,958 bp).
In the interval of M255 and L274, we designed 35 InDel markers and two dCAPS markers based on the re-sequencing data, and eight of them were polymorphic markers. We then used the eight co-dominant polymorphic markers to fine-map the CmBS-1 in a larger F2 population (2270 individuals) with 10 recombinant individuals (Tables S6 and S7). The result indicated that SNP2 and ZH39 were the closest markers to CmBS-1. The physical distance between the two markers was 233.98 kb (11,763,814–11,997,798 bp). We used QTL Icimapping 4.1 software to construct the genetic map with 16 markers (Fig. 5a).
Prediction and sequence analysis of the candidate gene
Based on the melon genome database, 12 genes were identified in the 233.98 kb region. The position and functional annotation of these genes are shown in Table 2. We analyzed the sequences of the 12 genes in the candidate region based on the parental re-sequencing data. The gene sequence alignment of the 12 genes between the yellow seed coat and brown seed coat parental lines reveled that one nonsynonymous SNP and two InDel mutations were present in MELO3C019554. We found that both of the two InDels were in the intron region at 11,965,686 bp (TA→T) and 11,966,210 bp (G→GA) on Chr 6, respectively (Table S8). The one nonsynonymous SNP was in the fifth exon region at 11,967,142 bp (C→T) on Chr 6, resulting in the conversion of an alanine (A) to threonine (T) at residue 534 (Fig. 5 b–d; Table S8). However, we found that there were no mutations in exon of the other 11 genes between the two parental lines. The promoter sequence analysis of the 12 genes between the two parental lines showed that there were no mutations in any of the 12 genes. According to the melon genome database, MELO3C019554 encodes a homeobox protein that is a PHD transcription factor. Therefore, we predicted that MELO3C019554 was the candidate gene for the yellow seed coat and brown seed coat in melon.
Table 2 Annotation of genes in the candidate region
Gene ID
|
Position
|
Functional annotation
|
MELO3C019545
|
11771441..11774714 (- strand)
|
Reticulon-like protein
|
MELO3C019546
|
11775819..11778327 (- strand)
|
PRKR-interacting 1
|
MELO3C019547
|
11783292..11783498 (- strand)
|
MGDG synthase type A family protein
|
MELO3C019548
|
11851782..11853751 (- strand)
|
Alcohol dehydrogenase, putative
|
MELO3C019549
|
11868402..11868584 (- strand)
|
Galactose oxidase/kelch repeat superfamily protein
|
MELO3C019550
|
11892173..11895412 (+ strand)
|
Methylthioribose kinase 1
|
MELO3C019551
|
11897392..11897999 (- strand)
|
FAD-binding Berberine family protein
|
MELO3C019552
|
11900852..11902620 (+ strand)
|
Coffea canephora DH200=94 genomic scaffold, scaffold_1
|
MELO3C019553
|
11924765..11929982 (+ strand)
|
Alpha-L-fucosidase 2
|
MELO3C019554
|
11955708..11969635 (- strand)
|
Homeobox protein
|
MELO3C019555
|
11956237..11956524 (+ strand)
|
Zinc finger protein 1
|
MELO3C019556
|
11972549..11973087 (- strand)
|
DNA polymerase epsilon catalytic subunit A
|
Expression analysis for verifying the candidate gene
To verify the candidate gene, we performed expression analysis of MELO3C019554 using qRT-PCR in the two parents, F1, and 10 melon germplasms with different seed coat colors. As indicated in Fig. 6a, the relative expression level of MELO3C019554 in the seed coat of IC2508 and F1 was significantly lower than that in the seed coat of IC2518 at 5, 10, 15, and 20 d after pollination, while the expression level did not differ significantly between P1(F1) and P2 on the other days. In IC2508, IC2518, and F1, the relative expression level of MELO3C019554 was highest at the color accumulation stage (10 d after pollination). Thus, we analyzed the relative expression of MELO3C019554 using qRT-PCR in five yellow seed coat melon germplasms and five brown seed coat melon germplasms at 10 d after pollination. The result showed that MELO3C019554 exhibited significantly higher relative expression in the brown seed coat melon germplasms than in the yellow seed coat melon germplasms (Fig. 6b). Within the same seed coat color, the differing expression level may be due to the different shades of the coat (Fig. S2). In conclusion, MELO3C019554 exhibited different expression levels in the seed coat, which were associated with the yellow and brown seed coats. The result further revealed that MELO3C019554 was the likely candidate gene associated with seed coat color in melon.
The candidate gene is related to flavonoid metabolites
Combined candidate gene and metabolite analysis was used to better elucidate the relationship between MELO3C019554 and flavonoids. We found that MELO3C019554 was related to 12 metabolites (10 metabolites upregulated and two metabolites downregulated in IC2518; Table S9).
Phylogenetic analysis
We constructed a neighbor-joining tree to evaluate the relationships between the protein (XP 008456177.1) encoded by MELO3C019554 and its homologs. The result showed that the protein encoded by MELO3C019554 was closely phylogenetically related to the Cucurbitaceae family, including Cucurbita moschata, Cucumis sativus, Cucurbita maxima, and Cucurbita pepo (Fig. 7a), which suggested that the protein was highly conserved in the Cucurbitaceae family. The sequence alignment analysis suggested that the region with the amino acid mutation (A→T) at residue 534 was conserved in the homologs (Fig 7b).