A further analysis of the relationship between yellow ripe-fruit color and the capsanthin-capsorubin synthase gene in pepper (Capsicum sp.) indicated a new mutant variant in C. annuum and a tandem repeat structure in promoter region

Abstract

Mature pepper (Capsicum sp.) fruits come in a variety of colors, including red, orange, yellow, brown, and white. To better understand the genetic and regulatory relationships between the yellow fruit phenotype and the capsanthin-capsorubin synthase gene (Ccs), we examined 156 Capsicum varieties, most of which were collected from Northwest Chinese landraces. A new ccs variant was identified in the yellow fruit cultivar CK7. Cluster analysis revealed that CK7, which belongs to the C. annuum species, has low genetic similarity to other yellow C. annuum varieties. In the coding sequence of this ccs allele, we detected a premature stop codon derived from a C to G change, as well as a downstream frame-shift caused by a 1-bp nucleotide deletion. In addition, the expression of the gene was detected in mature CK7 fruit. Furthermore, the promoter sequences of Ccs from some pepper varieties were examined, and we detected a 176-bp tandem repeat sequence in the promoter region. In all C. annuum varieties examined in this study, the repeat number was three, compared with four in two C. chinense accessions. The sequence similarity ranged from 84.8% to 97.7% among the four types of repeats, and some putative cis-elements were also found in every repeat. This suggests that the transcriptional regulation of Ccs expression is complex. Based on the analysis of the novel C. annuum mutation reported here, along with the studies of three mutation types in yellow C. annuum and C. chinense accessions, we suggest that the mechanism leading to the production of yellow color fruit may be not as complex as that leading to orange fruit production.

Figure 1. Mature red and yellow pepper lines used in this study.

Top two lines (red cultivars): (A) CK6, (B) CK8, (C) R29, (D) R37, (E) R28, (F) R30, (G) R26, (H) R38; bottom two lines (yellow cultivars): (I) P123-1-1, (J) CK4, (K) CK4-1, (L) CK18, (M) CK7, (N) CK26-1, (O) R12, (P) R19.

Figure 2. The clustering pattern obtained for all 156 pepper cultivars examined in this study using SSR analysis.

The novel yellow ripe-color pepper line, CK7, is marked with an asterisk.

Figure 3. PCR amplification of the Ccs gene and its promoters from the genomes of the indicated Capsicum varieties.

The order of the pepper lines is the same as in Figure 1, and the mature fruit colors are indicated with red and yellow highlighting.

Figure 4. Comparisons of the Ccs coding sequences in the indicated Capsicum varieties.

(A) Schematic representation of the mutations in Ccs among different cultivars. The nucleotide sequences were aligned, and the resulting missense mutations in the amino acid sequence are marked with asterisks. The 1-bp deletion in the coding sequence, which leads to early translation-termination, is underlined in the CK7 sequence. ATG and TGA indicate the start and stop codons, respectively. (B) Phylogenetic tree of the Ccs gene generated by multiple alignments of the coding sequence. The sequence of CcsW, which served as the positive control, was obtained from the NCBI (Accession: ×76165), and the sequence of CcsFogo was obtained from Guzman et al. (GenBank GU122933).

Figure 5. Expression pattern of Ccs.

Total RNA obtained from immature and mature pericarps of R28, R30, R26, R38, CK7 and CK18 was used for quantitative RT-PCR analysis. Each result represents the average of three independent biological replicates ± SE (n = 3), with a significance level of P≤0.05.

Figure 6. Sequence comparisons in the Ccs promoter region among the Capsicum varieties examined in this study.

The boxes (red, purple, yellow and blue) indicate the four repeat units found in this region. Major cis–elements as predicted by PLACE and PlantCARE softeare are underlined.