Carotenoid Presence Is Associated with the Or Gene in Domesticated Carrot

Abstract

Carrots are among the richest sources of provitamin A carotenes in the human diet, but genetic variation in the carotenoid pathway does not fully explain the high levels of carotenoids in carrot roots. Using a diverse collection of modern and historic domesticated varieties, and wild carrot accessions, an association analysis for orange pigmentation revealed a significant genomic region that contains the Or gene, advancing it as a candidate for carotenoid presence in carrot. Analysis of sequence variation at the Or locus revealed a nonsynonymous mutation cosegregating with carotenoid content. This mutation was absent in all wild carrot samples and nearly fixed in all orange domesticated samples. Or has been found to control carotenoid presence in other crops but has not previously been described in carrot. Our analysis also allowed us to more completely characterize the genetic structure of carrot, showing that the Western domesticated carrot largely forms one genetic group, despite dramatic phenotypic differences among market classes. Eastern domesticated and wild accessions form a second group, which reflects the recent cultivation history of carrots in Central Asia. Other wild accessions form distinct geographic groups, particularly on the Iberian peninsula and in Northern Africa. Using genome-wide F_st , nucleotide diversity, and the cross-population composite likelihood ratio, we analyzed the genome for regions putatively under selection during domestication and identified 12 regions that were significant for all three methods of detection, one of which includes the Or gene. The Or domestication allele appears to have been selected after the initial domestication of yellow carrots in the East, near the proposed center of domestication in Central Asia. The rapid fixation of the Or domestication allele in almost all orange and nonorange carrots in the West may explain why it has not been found with less genetically diverse mapping populations.

Keywords: Daucus carota; GWAS; carotenoids; domestication; population structure; selective sweep.

Figure 1

Carrot accessions illustrating the stages of carrot domestication and improvement. Carrots shown are fully mature (100 days). (A) Wild, (B) Eastern landrace, (C) Western historic open pollinated, and (D) modern hybrids (left: processing type and right: imperator type). Photo courtesy of Matthew Mirkes.

Figure 2

Population structure of 463 carrot accessions with $<30\text{\%}$ admixture (D2-LowAd). (A) STRUCTURE groups. Percentage of membership (q) for each group identified at K$=$6. (B) Geographic distribution of accessions, each represented by a point on the map colored according to STRUCTURE group. Current commercial varieties not shown. (C) Principal component (PC) analysis plot of the first two principal components. PC1 and PC2 account for 12.4 and 4.6% of the total variation, respectively. (D) Maximum-likelihood tree of carrot accessions. Numbers on the branches indicate bootstrap support. Black branch represents outgroup D. syrticus. (E) Color key. Total number of accessions in each STRUCTURE Group. LowAd, low admixture.

Figure 3

Genome-wide association analysis of orange pigmentation and identification of the candidate gene Or on chromosome 3. (A) Manhattan plot for orange carrot root color. SNPs with empirically-adjusted P-values < 0.05, were defined as significant and are colored orange. (B) Allele frequency of SNP S3_5228434 and pigmentation classification superimposed on the principal component analysis from Figure 2C. o, orange; p, purple; r, red; w, white; y, yellow. (C) SNP S3_5228434 genotype frequency separated by STRUCTURE classification. Or and NOr indicates orange or nonorange root pigmentation. W, D, or HI is wild, domesticated, or Hybrid Imperator, respectively. (D) SNP S3_5228434 genotype frequency separated by root pigmentation and domestication status. Quantities indicate the number of samples in each group. (E) Open reading frame of Or and the nonsynonymous mutation in exon 5 at position 3350 (T3350C). (F) Box plots for total carotenoids for the three Or genotypes (C/C, T/C, and TT) at position 3350. Center line $=$ median, box limits $=$ upper and lower quartiles, whiskers = 1.5× the interquartile range, dots $=$ outliers. Different letters indicate significant differences between genotypes (P < 0.05, Tukey’s honest significant difference). Reported values are in $\mu g/g$ dry weight.

Figure 4

Regions of the carrot genome that likely underwent a selective sweep during domestication. (A) Venn diagram represents the overlapping of 500-kb regions tested for selection signatures: top 5% of F_st and nucleotide diversity difference between wild and domesticated carrot accessions, and top 1% of cross-population composite likelihood ratio (XP-CLR) values. (B) Genomic location of potential selective sweeps identified by F_st, nucleotide diversity, and XP-CLR. The asterisk signifies the genome region carrying the candidate orange pigmentation gene Or. (C) Genome-wide linkage disequilibrium averaged across sliding windows of 100 SNPs in domesticated carrots. Regions identified as significant in (A) and (B) are highlighted in orange. The region containing the Or candidate gene for orange pigmentation in carrot is marked “Or.”