Identification of Genetic Factors Controlling Capsaicinoid Content in Pepper (Capsicum spp.)

Abstract

Peppers (Capsicum spp.) synthesize a pungent metabolite, capsaicinoid. Capsaicinoid biosynthesis pathway has been predicted based on primary and secondary metabolite synthesis pathway of other plants like Arabidopsis. From gene expression studies, multiple genes showed correlation with capsaicinoid content and designated as candidate genes controlling pungency. However, effects of the capsaicinoid biosynthetic genes for capsaicinoid content still unclear. In this study, we conducted two experiments to figure out the quantitative and qualitative genetic factors controlling the level of capsaicinoid in Capsicum. To find the quantitative trait loci (QTL) controlling capsaicinoid content, two recombinant inbred lines (RILs) were used. Capsaicinoid content was evaluated from placental tissue, where capsaicinoid mostly synthesized and accumulated. Furthermore, high-density genetic map using single-nucleotide polymorphism (SNPs) from next-generation sequencing (NGS) based genotyping methods. Based on the C. annuum CM334 reference genome, we found five common QTL located on chromosome 1, 2, 3, 4, and 10 which were detected from both RIL populations. To identify the capsaicinoid biosynthetic genes located on QTL, physical location of QTL were compared with the genome-wide association study (GWAS) conducted from Capsicum core collection. A total of ten regions were commonly detected, and five genes involved in capsaicinoid biosynthesis pathway were proposed to regulate pungency level. Since non-pungent accessions has non-functional capsaicinoid biosynthetic gene, they were widely used to identify the genes. RIL and F2 population derived from a cross between non-pungent accession C. annuum YCM334 and pungent accession C. annuum Tean were used to find a novel locus controlling presence of capsaicinoid. High-density genetic map was constructed using genotyping-by-sequencing (GBS) method, and the novel locus designated as Pun3 was genetically mapped on chromosome 7. To find the candidate genes on the Pun3 locus, High-resolution melting (HRM) markers were designed and the locus was mapped between 192.2-193.1 Mbp and 199.3-200.1 Mbp on Zunla-1 and PI159236 reference genome, respectively. Additionally RNA-Seq of parental lines was performed in three developmental stages, and expressions of the genes in fatty-acid biosynthetic pathway were extremely reduced in non-pungent parent YCM334. Three genes including Capana07g001603, Capana07g001604, and Capana07g001614 showed significant difference in expression level. They were only expressed in pungent parent Tean, and all three genes were predicted to encode MYB transcription factors. One of the genes, Capana07g001604, was reported to be related with capsaicinoid level in previous research. Therefore, we proposed that one of the predicted MYB transcription factors will be the candidate gene for Pun3. Taken together, we identified capsaicinoid biosynthetic genes controlling capsaicinoid content and a transcription factor regulates presence of pungency. Although the exact function of genes should be validated in multiple accessions, this result could be helpful to understand the capsaicinoid biosynthetic pathway as well as understanding the genetic control and breeding of pungent pepper.