Analysis of flesh color-related carotenoids and development of a CRTISO gene-based DNA marker for prolycopene accumulation in watermelon

Abstract

Fourteen watermelon cultivars with different fruit flesh colors (red, salmon yellow, orange, and canary yellow) were analyzed for carotenoid contents (prolycopene, lycopene, β-carotene, ζ-carotene, and neoxanthin). Genes encoding the carotenoid biosynthesis enzymes carotenoid isomerase (encoded by CRTISO), which catalyzes the isomerization of prolycopene to lycopene, and β-carotene hydroxylase (CHYB), which catalyzes the conversion of β-carotene to xanthophyll, were also analyzed. High-performance liquid chromatography showed that the salmon yellow and orange flesh accumulated either prolycopene (orange-P flesh) or β-carotene (orange-β flesh), whereas lycopene and neoxanthin were the main carotenoids in the red and canary yellow flesh, respectively. Quantitative real-time polymerase chain reaction indicated that CRTISO and CHYB were mainly expressed during fruit maturation, regardless of the flesh color, and there was no significant association between differential gene expression and flesh color. Importantly, transcript sequencing revealed a non-synonymous single-nucleotide mismatch (T > C1976) in exon 13 of CRTISO between orange-P-fleshed and other cultivars, suggesting CRTISO as a candidate gene for high prolycopene accumulation. However, in β-carotene-accumulating cultivars, there were no mutations in CHYB transcripts. A cleaved amplified polymorphic sequence marker was developed for T > C1976, and its applicability for marker-assisted selection of orange-P flesh was validated in 105 watermelon accessions.