GWAS for the visual evaluation of root color and evaluations of color components and carotene contents in roots of F2 populations A and B
F2 populations A and B both showed a normal distribution in all root color evaluations (Suppl. Fig. S1), suggesting the involvement of multiple associations in carrot root color. The ddRAD-seq analysis detected 3,106 and 1,901 high-confidence SNPs in F2 populations A and B, respectively. The GWASs were performed using these genotypic data and values from the visual evaluation and evaluations of the color components and carotene contents in the carrot roots. In F2 population A, significant associations were detected for the visual evaluation of root color (Fig. 3a); color components a* (Fig. 3c) and b* (Fig. 3d); α-carotene (Fig. 3e), β-carotene (Fig. 3f), and lutein contents (Fig. 3g); and the β/α-carotene ratio (Fig. 3h) in root (Table 1). No significant associations were detected for color component L* (Fig. 3b).
The associations for visual evaluation, color components a* and b*, and α- and β-carotene contents on chromosome 1 were detected at close physical positions, and the highest associations were detected at a physical position around 31 Mb (Fig. 3, Table 1), suggesting that these associations are caused by an identical locus. The physical positions of the associations for the α- and β-carotene contents on chromosome 3 were close, and the highest associations were detected at a physical position around 6 Mb (Fig. 3, Table 1). An association detected in population B for visual color evaluation on chromosome 3 showed the highest association at physical position 5.4 Mb, and this physical position was similar to those of the associations detected in population A for α- and β-carotene contents (Figs. 3, 4, Table 1). These results suggest that the associations are caused by an identical locus. Interestingly, the association detected on chromosome 5 (showing the highest association for visual evaluation in F2 population A) was not detected in any other evaluations (Fig. 3, Table 1).
Table 1
Significant associations for carrot root color identified by GWAS
|
Trait
|
F2_population
|
physical positiona
|
-log10P
|
Chr
|
bp
|
Visual evaluation
|
A
|
5
|
39,247,011
|
5.80
|
A
|
1
|
30,782,032
|
5.31
|
A
|
4
|
20,589,731
|
5.01
|
A
|
6
|
6,440,506
|
4.72
|
B
|
3
|
5,419,538
|
9.69
|
a*
|
A
|
1
|
32,693,618
|
8.42
|
A
|
3
|
1,689,065
|
6.37
|
b*
|
A
|
1
|
31,112,534
|
12.49
|
α-carotene
|
A
|
1
|
30,704,558
|
11.08
|
A
|
1
|
15,832,013
|
8.22
|
A
|
3
|
5,849,853
|
7.77
|
β-carotene
|
A
|
1
|
30,704,558
|
13.72
|
A
|
1
|
15,832,013
|
11.82
|
A
|
3
|
6,455,250
|
5.79
|
Lutein
|
A
|
5
|
11,540,998
|
8.38
|
A
|
2
|
10,281,204
|
5.09
|
A
|
6
|
32,679,396
|
4.75
|
β/α-carotene ratio
|
A
|
6
|
4,640,387
|
11.02
|
A
|
1
|
12,574,447
|
8.78
|
A
|
5
|
29,337,124
|
6.63
|
A
|
2
|
34,903,955
|
5.04
|
aphysical position of the SNP showing the most significant association in the GWAS based on carrot genome Daucus carota v2.0 (10).
|
Correlations among visual evaluation, color components, and carotene contents in root of F2 population A
The Pearson correlation between each phenotype showed that three color components, i.e., L*, a* and b*, the α-carotene content, and the β-carotene content were highly correlated (Table 2). The lutein content was slightly correlated with L*, a* and b* and highly correlated with the α-carotene content. As lutein is biosynthesized downstream of the α-carotene (Fig. 1), this high correlation of lutein and α-carotene is consistent with the biosynthesis pathway. The visual evaluation was not highly correlated with any other phenotypes.
Allelic effects of associations detected by GWAS on chromosomes 1 and 3 for the α-carotene and β-carotene contents in F2 population A
We examined the allelic effects of the associations detected by the GWAS for the α- and β-carotene contents. At the median, the carrots with AA allele on the SNP showing the highest association for α-carotene (DCARV2_CHR1_30704558) had approx. 1.5-fold higher contents of α- and β-carotene than those with GG allele (Fig. 5a, b). Similarly, at the median, the carrots with GG allele on the SNP showing the highest association for α-carotene (DCARV2_CHR3_5849853) had approx. 1.3-fold higher contents of α-carotene and approx. 1.2-fold higher contents of β-carotene compared to those with AA allele (Fig. 5c, d). A clear genetic interaction such as epistasis was not observed between the associations detected on chromosomes 1 and 3 (Suppl. Fig. S2). Together with both associations detected on chromosomes 1 and 3, at the median, the carrots that had alleles showing higher carotenoid content in both associations also had approx. 2.6-fold higher α-carotene and approx. 1.8-fold higher β-carotene contents in carrot surface compared to those with alleles showing lower carotenoid contents in both associations (Suppl. Fig. S2).
Possible candidate gene for the association detected on chromosome 3 by the GWAS and sequence comparison between parents in F2 populations A and B
Significant associations were detected around the physical position at 5–6 Mb on chromosome 3 for α-carotene and β-carotene contents in F2 population A and for visual evaluation in F2 population B (Figs. 3, 4, Table 1). Within this region, the reported Or gene (DCAR_009172), which affects carotenoid contents in carrot [16], is located at 5.2 Mb on chromosome 3. To examine the involvement of Or, we performed Sanger sequencing of Or in the parents of populations A and B. The Sanger sequencing detected only one non-synonymous amino acid substitution at the fourth amino acid from the end, which was caused by an SNP between both parents of F2 populations A and B (Fig. 6a). A thymine which was identical to that in the carrot reference genome [10] in Fs001 and Fs003 was changed to guanine in Fs002, which resulted in a change from Tyr309 in the Fs001 and Fs003 to aspartic acid in the Fs002.
To examine the effect of the SNP on Or causing the non-synonymous amino acid substitution, we developed a KASP marker which could genotype the SNP. We applied the developed KASP marker to breeding line C whose root color was segregated and that is the progeny of Fs002 (Fig. 2). The root color of breeding line C was visually evaluated into three grades (Fig. 6c). The genotype of KASP marker on Or was clearly correlated with the visual evaluation (Fig. 6b). All of the carrots whose root color was bright and middle orange had a heterozygote for the SNP on Or, and all of the carrots whose root color was slightly light orange had a TT homozygote for the SNP. We thus speculate that the associations detected in F2 population A for α-carotene and β-carotene contents on chromosome 3 and the association detected in F2 population B for visual evaluation were responsible for the SNP causing the non-synonymous amino acid substitution on Or.
Possible candidate gene for the β/α-carotene ratio in population A, and the amino acid comparison between parents of population A
In the GWAS of F2 population A, the association for the β/α-carotene ratio was detected on chromosome 6 and showed the highest association on the physical position at around 4.6 Mb. Iorizzo et al. [10] summarized the carrot orthologous and homologous candidate genes involved in the plastid 2-C-methyl-D-erythritol 4-phosphate (MEP) and carotenoid pathways in a table. According to the table, DCAR_022896 (which has a lycopene cyclase domain) is located on a physical position at 4.1 Mb on chromosome 6, which is between the SNP showing the highest association for the β/α-carotene ratio and the next SNP (Suppl. Table S2). Carotenoid biosynthesis bifurcates after lycopene to produce e- and β-carotenoids by enzymatic activity of the two lycopene cyclases, lycopene e-cyclase (LCYE) and lycopene β-cyclase (LCYB) [28] (Fig. 1). In addition, it is known that the proportions of β-carotene and α-carotene are determined mostly by the comparative amounts and/or activities of the LCYB and LCYE enzymes [20, 29–32].
However, the genes annotated as carrot LCYB and LCYE are not DCAR_022896 [10, 24]. Our BLAST search (NCBI; http://www.ncbi.nlm.nih.gov) of amino acids of DCAR_022896 showed sequence homology with neoxanthin synthase (NYS) and capsanthin-capsorubin synthase (CCS). The Phytozome database annotated DCAR_022896 as NYS, CCS, and lycopene cyclase (https://phytozome.jgi.doe.gov/pz/portal.html#). It is known that LCYB, NSY (which catalyzes violaxanthin into neoxanthin), CCS (which catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively) (Fig. 1), and chromoplast-specific lycopene β-cyclase (CYC-B) have high sequence homology and similar putative catalytic mechanisms [20, 21, 33, 34].
Our phylogenic analysis of DCAR_022896, LCYE, LCYB, NSY, CCS, and CYC-B in carrot and Arabidopsis as well as Solanum lycopersicum, Carica papaya, Citrus sinensis, Capsicum annuum, and Lillium lancifolium showed that DCAR_022896 belonged to the same clade as CYC-B in C. sinensis and C. papaya (Fig. 7a). At the amino acid level, DCAR_022896 had 76.9% identity to CYC-B in C. sinensis and 62.1% to CYC-B in C. papaya. CYC-B is a LCYB, and it converts lycopene to β-carotene in chromoplasts, where carotenoids are accumulated [35, 36], in a specific manner [20] (Fig. 1). Moreover, our BLAST search of primers for the reported LCYB2 in carrot showed that CYC-B (DCAR_022896) in the present study is identical to LCYB2 [5, 6, 37, 38]. We thus presume that DCAR_022896 is a possible candidate gene for the β/α-carotene ratio, and we compared the amino acid sequences between the parents of F2 population A by Sanger sequencing. The amino acid comparison revealed five amino acid substitutions between the parents of F2 population A (Fig. 7b). These results suggested the possibility of the involvement of CYC-B in the β/α-carotene ratio in carrot root.