Quantitative Trait Loci Identification Uncovers Zinc Finger Protein CONSTANS-LIKE 4 as the Key Candidate Gene of Stigma Color in Watermelon (Citrullus lanatus)1

Stigma color is a critical agronomic trait in watermelon and plays an important role in pollination. However, there are few reports the regulation of this process to date. In this study, a genetic analysis of the F2 population derived from ZXG1553 (P1, with orange stigma) and W1-17 (P2, with yellow stigma) indicated that stigma color is a quantitative trait and orange stigma is recessive compared with yellow stigma. BSA-seq (Bulk segregant analysis sequencing) determined a chromosome segment related to stigma color in a 3.75 Mb on chromosome 6, and a major stable effective quantitative trait locus (QTL) Clqsc6.1 (QTL stigma color) was detected in two years between CAPS markers Chr06_8338913 and Chr06_9344593 spanning ~1.01 Mb interval harboring 51 annotated genes. Cla97C06G117020 (annotated as Zinc finger protein CONSTANS-LIKE 4) was identified as the best candidate gene for stigma color trait through RNA-seq, quantitative real-time (qRT)–polymerase chain reaction (PCR), and gene structure alignment analysis among the nature watermelon panel. The expression level of Cla97C06G117020 in orange stigma accession was lower than yellow stigma accessions with a significant difference. A nonsynonymous SNP site of Cla97C06G117020 coding region causing amino acid variation was related to stigma color variation among nine watermelon accessions according to their re-sequencing data. Stigma color formation is often related to carotenoid, and we also found that the expression trend of ClCHYB (annotated as Beta-carotene hydroxylase) in the carotenoid metabolic pathway was consistent with Cla97C06G117020, which was expressed in low amounts in the orange stigma accession. These data indicated that Cla97C06G117020 and ClCHYB may have interacted to form stigma color. This study provided a theoretical basis for gene fine mapping and mechanisms for the regulation of stigma color in watermelon.