Identification and assessment of alleles in the promoter of the Cyc-B gene that modulate levels of β-carotene in ripe tomato fruit.

Novel diversity may be mined from databases and de novo sequencing, but functional characterization remains a limiting step to identifying new alleles. Classical breeding approaches augmented by marker-assisted selection offer a means to rapidly assess the function of new variation in coding or regulatory regions to modulate traits. We used the Cyc-B gene (B) of tomato (Solanum lycopersicum L.) for a proof of concept because of its role in the production of β-carotene, a provitamin A carotenoid with importance to human nutrition. We measured carotenoid content in vintage and contemporary varieties and the profiles had a range of β-carotene from 0.2 to 4.06 mg 100 g fresh weight. We characterized variation in B from 84 sequences recovered from public databases and from an additional 29 high β-carotene tomato, S. galapagense S. C. Darwin & Peralta, and S. cheesmaniae (L. Riley) Fosberg accessions. Thirteen unique haplotypes across 1600 bp of sequence 5' to the first ATG were identified with 11 occurring in high β-carotene accessions we sequenced, and additional haplotypes were identified in public data. Phylogenetic analysis suggested that the alleles in high β-carotene varieties were derived from wild species. Association analysis suggested two single nucleotide polymorphisms (SNPs) as the most likely causes of high β-carotene, presumably through their influence on transcription of B that is elevated in ripening fruit. A marker-assisted backcross breeding scheme leveraging SNPs for background genome selection was used to rapidly develop germplasm resources containing different alleles of B in a uniform genetic background. Evaluation demonstrated that distinct promoter haplotypes function as different alleles that can be used to modulate the levels of β-carotene in tomato.