Valencene Deficiency in Mandarin Hybrids Is Associated with a Deletion in the Promoter Region of the Valencene Synthase Gene

We have discovered that in Murcott mandarin (Citrus reticulata hybrid) expression of the gene encoding valencene synthase (CsTps1; EC:4.2.3.22 4.2.3.75) is severely reduced. Valencene synthase (CsTps1) catalyzes the biosynthesis of linear and cyclic compounds from the 15-carbon substrate farnesyl diphosphate. The product of CsTps1, valencene, is a major sesquiterpene in citrus oils, and the second most abundant volatile compound typically found in mandarin juice. We have investigated a segregating population between Fortune and Murcott mandarins. Fortune produces abundant valencene, whereas valencene is undetectable in Murcott. Quantitative PCR analysis showed that the expression level of CsTps1 gene in Fortune was over 3000-fold higher than in Murcott. Phenotypic segregation was determined from 80 individuals and was 34:46 (non-detected: valencene), not significantly different from a 1:1 ratio (χ2=0.134). There was a clear correlation between level of CsTps1 transcript, measured by QRT-PCR, and the level of valencene in fruit from parents and their progeny. The pattern of gene expression of CsTps1 and valencene quantification is inherited as a simple Mendelian locus. The results indicated that valencene synthase is a single gene which controls the production of valencene. Cloning and sequencing the CsTps1 gene in Fortune and Murcott revealed that they are both composed of seven exons and six introns in identical length, and encodes a 548-amino acid protein with 97% similarity. The upstream regulatory regions of CsTps1 were isolated by genome walking in Fortune and Murcott. Sequence alignment showed a deletion of 12 nucleotides in the Murcott CsTps1 promoter compared to Fortune. Transient expression analysis of the Fortune CsTps1 promoter in sweet orange showed significant GUS expression, whereas the Murcott CsTps1 promoter did not show any. In addition, functional activity of the CsTps1 promoter in Murcott is recovered by re-inserting the deletion of 12 nucleotides. By genotyping the 12 nucleotide deletion allele among 115 individuals, the ratio between deletion and non-deletion in the promoter region is 57:58. The genotype matches the production of valencene in these individuals. This result suggests the deletion of 12 nucleotides in the upstream regulatory promoter regions of the CsTps1 gene is associated with the production of valencene deficiency in hybrid mandarins.