Alternative Splicing Variants and a New DNA Marker for an Allelic Selection of Lycopene Beta-cyclase in Watermelon

The carotenoid biosynthetic pathway has been well studied in various crops. One of the pathway genes, lycopene beta-cyclase (LCYB), encodes an enzyme that converts lycopene to beta-carotene. Plants have chromoplast-specific and chloroplast-specific classes of LCYB. At least two copies were reported in tomato, orange, and papaya, but only one copy of LCYB has been identified in watermelon, to date. Our previous study showed that LCYB may be crucial for conditioning flesh color differences between red and canary yellow watermelon varieties. Two different splicing variants were identified during molecular cloning of LCYB. One variant retained an intron in the 5’ untranslated region (UTR) and the other variant did not have an intron. In this study, quantitative real-time PCR (qRT-PCR) was carried out to determine if there was differential gene expression of the coding region. The promoter region was characterized and alternative splicing variants in the 5’UTR were examined using qRT-PCR. A significant difference in mRNA transcripts of the coding region of LCYB was observed between red and canary yellow watermelons. Moreover, it appeared that LCYB transcripts retaining an intron were more abundant than LCYB transcripts without an intron in both flesh colors. Both splicing variants were more abundant in canary yellow than red flesh varieties. Overall, the expression of LCYB in canary yellow was higher than in red regardless of splicing patterns or positions. When promoter regions of the two watermelon varieties were compared, polymorphic sequences were identified and developed into a PCR-based marker for LCYB allelic selection. Genotyping on genetic populations and other genotypes revealed perfect co-segregation of the marker with flesh colors, which indicated that polymorphic sequences in the promoter region may influence transcriptional regulation in red and canary yellow watermelon. Because this newly developed marker is PCR-based, it can be used in watermelon breeding with greater efficiency than our previously reported cleaved amplified polymorphic sequences marker.