Project Number: 1910-21220-005-02
Project Type: Reimbursable

Start Date: Sep 01, 2008
End Date: Aug 31, 2010

Objective:
This is a large project with many collaborators. The overall focus of the proposal is in the area of applied functional and structural genomics in both grape and yeast. The objectives of the proposal conducted at GGRU will be 1) SNP discovery in 35 candidate genes related to volatile aroma synthesis in grape, and 2) SNP genotyping of these 35 candidate genes in 376 accessions from the grape germplasm collection.

Approach:
SNP discovery ¿ candidate genes: SNP genotyping of the candidate genes will occur in two phases. Based on our previous work we expect that 24 or fewer individuals will be sufficient to capture the majority of common nucleotide variation in grape. But because the frequency spectrum of variation in grape can vary from locus to locus, this must be tested empirically. Therefore, we will sequence approximately 2 reads, one each on the 5¿ and 3¿ ends in each of 35 candidate loci from 24 individuals. Subsets of the data from fewer individuals will be analyzed to see how much of the total variation they capture. Our final sample size will be one that captures at least 90 percent of the non-singleton variation; we anticipate that this number will be less than 24. Our calculations of the amount of sequencing required to discover a given number of SNPs are based on our previous work in which we have sequenced a small number of loci in 100 individuals and a larger number of loci in diverse samples of 16-24 individuals. Sequence diversity in grape is relatively high: a SNP on average every 60 bp. We are anticipating that, on average, 5 SNPs will need genotyped per gene to accurately define the majority of haplotypes in the sample. To identify alleles associated with variation in volatile aroma production and other genes of physiological interest, we will survey genes in the following trait-related pathways: monoterpene and sesquiterpene synthesis, upstream isoprenoid and carotenoid pathway genes, LOXs, hydroperoxide lyase, ABA singaling, and vacuolar transport. The specific candidate genes have been previously identified by our collaborators at U. of British Columbia. SNP genotyping in 376 grape accessions: For candidate genes, tag-SNPs will be chosen to capture all non-singleton variation observed in the SNP discovery sample using the software package Haploview. Sequence flanking the chosen SNPs will be provided to Illumina for development of a Golden-Gate based SNP genotyping assay. Genotyping assays will be done by the staff of the BioResource Center at Cornell on a pay-for-service basis.