Development of Gene-based Markers for capsanthin-capsorubin synthase and zeaxanthin epoxidase Genes to Distinguish Fruit Colors in Paprika (Capsicum annuum L.)

The fruit color of paprika (Capsicum annuum L.) is one of the main traits influencing consumers. Paprika fruit colors include red, yellow, and orange based on the different carotenoid contents. In this study, we aimed to develop gene-based markers to distinguish among three fruit colors, in this case red, yellow, and orange, by identifying genetic variations among the carotenoid biosynthesis genes in paprika. Four candidate genes, phytoene synthase (PSY), β-carotene hydroxylase (BCH), zeaxanthin epoxidase (ZEP), and capsanthin-capsorubin synthase (CCS), were selected, and the gene sequences were obtained from the NCBI database. The genes were sequenced and the genetic variations were uncovered using four red (KNMR, 19BR201, Sirocco, and GINA), three yellow (KNMY, Volante, and Romance Gold), and three orange (KNMOR, DSP7054, and ARO-50R) cultivars. Gene variations depending on the fruit color were detected in CCS and ZEP genes. Red paprika fruits contained the normal CCS gene (Ccs allele), while yellow and orange paprika fruits contained the non-functional deleted CCS gene (ccs allele). One base substitution was found in the ZEP gene. Yellow paprika fruits contained the A base of the SNP (functional Zep allele), while orange paprika fruits contained the G base of the SNP (non-functional zep allele). Red paprika fruits had the A or G base of the SNP. Two codominant high-resolution melting (HRM) markers, CCS-indel-HRM and ZEP1.2-SNP-HRM, were developed using the detected genetic variations of a large indel and a SNP, respectively. The markers were tested in 44 F₁ cultivars, 31 inbred lines, two F₂ populations consisting of 31 and 79 individuals, and 380 germplasms. It was found that red paprika fruits had the Ccs/Ccs or Ccs/ccs genotype regardless of the ZEP genotype, while yellow paprika fruits had the ccs/ccs and Zep/_ genotypes and orange paprika fruits had the ccs/ccs and zep/zep genotypes. The fruit color predictions with the markers were perfectly matched with the fruit color phenotypes, indicating that these markers can be useful for developing new red, yellow, or orange paprika varieties.