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Mutations in the CCD 4 Carotenoid Cleavage Dioxygenase Gene of Yellow-Flesh

The color of fruit flesh is an important factor that affects the preferences of customers. Peaches, one of the major fruits on the world market, show two types of flesh color: white and yellow. In Japan, white-flesh peaches are preferred for eating raw and yellow-flesh peaches are mainly processed as canned fruits. Crossing between peach trees bearing white-flesh fruits sometimes produces trees bearing yellow-flesh fruits, causing a decrease in the breeding efficiency of white-flesh peaches. To date, genetic analysis has revealed that the white color is dominant to the yellow color, and that flesh color is governed by a single locus, named Y . The Y locus was mapped on the middle of the first linkage group. Yellow-flesh peaches contain high amounts of carotenoids, major plant pigments that produce the yellow color. It has been reported that the yellow color of chrysanthemum flowers and potato buds is also due to carotenoids, and that carotenoid accumulation is caused by mutations in CCD4, a carotenoid cleavage dioxygenase gene. In peaches, the expression of CCD4 is repressed in yellow flesh as compared to white flesh. In the present study, we identified mutations in CCD4 in peach cultivars grown in Japan. The sequence data of the peach genome were reported recently. We queried the CCD4 gene against the peach genome using ‘‘Arabidopsis CCD4, AT4G19170’’ as query in the Genome Database for Rosaceae, and found that the peach CCD4 homolog (ppa006109m; 25,639,445 to 25,641,500) is located between two SSR markers, MA036a (20,091,449 to 20,091,716) and pchgms3 (27,691,861 to 27,691,861) on scaffold 1. This position exactly matches the Y locus, suggesting that CCD4 is responsible for the color of peach flesh. To confirm this, we compared the structure of CCD4 between the yellow-flesh peach Yamate-shimizu (genotype y=y) and the white-flesh peach Hakuho by genomic PCR. Hakuho might have a Y=Y genotype, since 26 crossings of Hakuho to various cultivars in breeding by us have never produced yellow-flesh peaches. We designed three sets of primers: 50TTGCTTAGAGAAAGTGAGAGC-30 and 50-GGTAGTGAGGTGTGGGATCA-30 for amplification of the promoter region, 50-GGAAGCGGAAGATATTGCAG30 and 50-AGGCACATCAAACCACCTCA-30 for the N-terminal half of CCD4, and 50-GATGCAAACGGCACAAAGCAG-30 and 50-TGCACTCACCTAGTTTGGGG-30 for the C-terminal half. When we used these primer sets for the promoter region and the C-terminal half, the sizes of the amplified fragments were apparently not different (Fig. 1A). However, when we used the primer set for the N-terminal half, the size of the fragments amplified from the Yamate-shimizu genome was approximately 7 kb longer than that from the Hakuho genome. A comparison of the sequences of the amplified fragments revealed that an extra sequence of 6,253 bp is present in the intron of Yamate-shimizu CCD4 (Fig. 1B). This sequence has tandem repeats of 486 bp at both ends. This indicates that it is a long terminal repeat (LTR)-type retrotransposon, which is widely distributed in the peach genome. LTRs have short inverted repeats (SIR, TGTTATA) at both ends, which are known recognition motifs for a integrase and a TATA box sequence (TATATG) in the middle. These motifs are specific to LTR retrotransposons. The retrotransposon in Yamate-shimizu CCD4 lacks long open reading frames, indicating that it is a nonautonomous element. To determine the distribution of the retrotransposon in the CCD4 gene among peach cultivars, we performed PCR for the genomic DNA of 39 cultivars using three primers: 50-ACCACCTGTTTGACGGAGAC-30 (CCD4-f), 50-TGCTCATGAAGAGCTTGCCA-30 (CCD4-r), and 50-TACCTGAGAGCTTCTCGTGC-30 (RT-f). This PCR can produce 594-bp fragments from the CCD4 and 729-bp fragments from transposon-containing CCD4 at the same time. The result of PCR was that the 594-bp fragment was produced only from the genomes of 10 cultivars, including Hakuho (Fig. 1C), all of which bear whiteflesh fruits. The 729-bp fragment was amplified only