2012 Annual Report
1a.Objectives (from AD-416):
Adapt a genotyping strategy to obtain 50,000 markers per seedling and apply this strategy for discovering markers associated with powdery mildew resistance and other important traits. Develop a pipeline for applying molecular markers in public grape breeding programs at no cost to the breeder.
1b.Approach (from AD-416):
DNA will be provided by USDA-ARS from 400 individuals of the Vitis aestivalis mapping population. Whole genome amplification (WGA) will be completed. To select a restriction enzyme for preferential sequencing of gene-rich regions, an in silico digestion of the V. vinifera ‘Pinot noir’ genome sequence will be completed. Then the chosen restriction enzyme will be used to apply established restriction sequencing protocols for pooled, barcoded samples. A computational pipeline modified from the one used in the USDA-ARS maize hapmap project will be used to assign SNPs to individual seedlings and filter out low quality SNPs.
For the new SNP markers developed during this project, a redundant, locus-based assay will be developed and applied using SNPlex or a similar 24-48 multiplex technology. Additional publicly available markers can be immediately applied for marker assisted breeding. DNA will be provided by USDA-ARS for up to 1000 progeny and by Cornell for up to 1000 progeny. After WGA, the following existing markers will be screened for segregation and then across the entire population when appropriate: Run1, Rpv1, V. cinerea, V. romanetii, Ren1, and Regent.
Molecular DNA markers can be used to tag traits in the genome, providing optimal selection of parents and early selection of elite progeny with multiple desirable traits and/or multiple resistance genes for improved durability. The purpose of this project was to develop a pipeline for applying molecular markers in public grape breeding programs. Grape breeders provided populations focused on powdery mildew resistance and seedlessness for validation of known marker-trait associations.
During this project, we guided marker technology advances from.
1)labor-intensive, targeted SSRs to.
2)high-cost, genome-wide SNP microarrays to.
3)low-cost, targeted SNP assays to.
4)low-cost, genome-wide genotyping-by-sequencing (GBS). Our current GBS pipeline provides genome-wide information at similar cost per plant to using SSRs to track 2-3 genes. This GBS advance equates to an approximately 1000-fold decrease in cost per marker relative to SSRs.
Specifically, we tested three markers linked with seed development inhibitor (SdI) and found that combining the marker data resulted in enhanced selection of seedlings with little or no seed trace. Using stringent selection in three populations, 100% of progeny with SdI markers were seedless. These markers would enable breeders, depending on their preferred stringency, to discard up to three-quarters of young seedlings solely based on predictions of seedlessness. This would enable the focused evaluation of more crosses and more elite progeny.
For powdery mildew resistance, we focused on resistance genes from V. rotundifolia (Run1), V. cinerea (Ren2), and V. romanetii (Ren4). Of those progeny with Run1 markers, 99.8% were resistant, and more than 98% of progeny with Ren4 markers were resistant. We had technical issues with markers for the quantitative resistance gene Ren2 in most populations, but for two populations in which the markers were easy to score, 76% and 97% of resistant individuals had the Ren2 marker. This underscores the value in development of improved markers for this resistance source.
Based on our results, breeders could currently combine Run1, Ren4, and SdI using these marker sets and could eliminate nearly 90% of young seedlings. Among those progeny that are kept, out data suggest that an impressive 98% would have all three genes.