| Mature pepper (Capsicum spp.) fruits come in a variety of colors, including red, orange,yellow, brown, and white. The color found in ripe pepper fruit (Capsicum spp.) mainlydepends on the variable accumulation of capsanthin and capsorubin. The yellow pepper fruitcolor phenotype, which is recessive to red, is controlled by the y locus. Linkage analysisindicated that the candidate gene of the y locus is also the Ccs gene. To better understand thegenetic and regulatory relationships between the yellow fruit phenotype and thecapsanthin-capsorubin synthase gene (Ccs), we examined156Capsicum varieties, most ofwhich were collected from Northwest Chinese landraces. The main results of the study are asfollows:1. Most of the cultivars showed a diverse degree of red coloring in fruit, which includeddark red, red and light red. In addition, some lines producing orange or brown ripe fruit, andeight cultivars producing yellow ripe fruit, were also observed.2. PCR was performed to analyze the genomic coding sequences of Ccs in the156pepper varieties. All non-yellow lines produced positive PCR results after electrophoresis.Only one yellow line, CK7, produced the same banding pattern as that of the red cultivars.Sequence analysis revealed that CK7contains a new variant of Ccs, with two types of earlytranslation termination of the Ccs gene. One mutation is a frame shift derived from a1-bpdeletion (1,265bp downstream of the start codon). The second mutation, a novel prematurestop codon produced by a point mutation (1,095bp downstream of the start codon, cytosine toguanine) was found upstream of the deletion described above.3. We examined the promoter regions of this gene in all of the varieties using PCR. Allred mature C. annuum lines produced a similar920-bp band. Two larger bands were detectedin two C. chinense species (R28and R30), and no PCR product was detected in two C.baccatum accessions (R26and R38). The yellow mutant cultivar evaluated in this study, CK7,produced the similar PCR band as in red-fruited C. annuum lines, while none of the otheryellow peppers produced a positive PCR result. We used semi-quantitative RT-PCR to test theexpression of Ccs in the lines that produced positive PCR results for the Ccs CDS. Transcriptsfor Ccs were not detectable in the immature fruits of any of the pepper lines examined. Wedetected some accumulation of Ccs transcript in CK7, which has a mutant Ccs gene and yellow fruit, although the expression level was lower than that detected in the red fruit lines.4. Cluster analysis revealed one main cluster (C. annuum) and four genetically distinctspecies. The majority of the cultivars evaluated fell into the main cluster, which could besubdivided into two large (I, II) and one small group. All of the unknown local yellow pepperlines were grouped in the main cluster, revealing that these lines are all C. annuum cultivars.Among the eight yellow cultivars, seven are in group II, which mainly includes sweet or bellpepper. One exception is the yellow fruit line, CK7, which is found in group I. Most chilipeppers in this study are found in this group, and CK7is also a hot pepper.5. The promoter sequences of Ccs from some pepper varieties were examined, nosequence differences in the two C. annuum lines (CK8and CK7) were likely to be associatedwith the differences in Ccs gene transcription. And we detected a176-bp tandem repeatsequence in the promoter region. In all C. annuum varieties examined in this study, the repeatnumber was three, compared with four in two C. chinense accessions. The sequence similarityranged from84.8%to97.7%among the four types of repeats, and some putative cis-elementswere also found in every repeat. This suggests that the transcriptional regulation of Ccsexpression is complex. |
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