Evaluation Method
The purpose of this project is to 1) document Vitis genetic diversity present in the Geneva repository 2) ascertain the clonal identity of replicate vines that represent each accession. This project runs in parallel with the fingerprinting effort of the Vitis germplasm repository in Davis, California. Markers, standards and DNA extraction were coordinated between the sites, while electrophoresis and analysis was done on different genetic analyzers and slightly different programs. The genetic data collected in Geneva during 2007/2008 timeframe is the result of the first phase and includes genetic diversity data for eight SSR markers for the second vine of each accession (if present). In 2008/2009 the process is repeated for the first vine of each accession. Gaps in the current data due to lack of replicate vines will then be filled in and any discrepancies between readings of the two clonally propagated vines per accession will be dealt with. A potential future third phase may add additional diversity data for a subset of accessions.
Assay Method
DNA Extraction: Genomic DNA was extracted from very young leaves using the kit DNeasy96 Plant (Qiagen, Valencia, CA). PCR: Forward PCR primers were fluorescently labeled with PET, VIC, NED, 6_FAM by ABI (http://www.appliedbiosystems.com/). Reverse PCR primers were synthesized by Integrated DNA Technologies (www.idtdna.com) with a 5? `GTTTCTT? tail. Fragments were PCR amplified on two BioRad iCyclers or an MJ Tetrad in two four-plexed primer sets: 1) VrZag79, VVMD34, VVMD27, VVS2 and 2) VVMD31, VrZAG62, VVMD7, VVMD5. The 15ul reactions contained 15ng genomic DNA, 2.5mM MgCl2, 200uM ea dNTP, 0.6U FlexiTaq (Promega), 1x FlexiTaq reaction buffer (0 MgCl2) and the following primer amounts: VrZAG79 (0.9pmol ea), VVMD34 (1pmol ea), VVMD27 (1.1pmol ea), VVS2 (0.7pmol ea or 0.9pmol ea) and VVMD31 (1.8pmol ea), VrZAG62 (0.4pmol ea), VVMD7 (0.8pmol ea), VVMD5 (1.8pmol ea). The cycling program for all amplifications: 5min initial denat at 94C, followed by 30 cycles of 30s denat at 94C, 1min annealing at 54C and 1min extension at 72C, followed by a final 7min extension (72C). Each plate had one negative control (water instead of DNA) and three positive controls: V. labrusca `Lady? (DVIT 0087), V. vinifera `Muscat of Alexandria? (DVIT 0466) and V. vinifera `Salt Creek? (Foundation Plant Services, UC Davis, ID# 40122). 2ul PCR products were added to 9.6ul Hi-Di Formamide (ABI) and 0.4ul Genescan 600LIZ size standard (ABI) and electrophoresed at the Cornell University Core Laboratory on an ABI 3730xl Genetic Analyzer. For maximum consistency all plates from a multiplex were run consecutively on the same instrument over the same capillaries. Data analysis: fragment sizes were analyzed in GeneMapper4.0 (Applied Biosystems, Inc.). Bin sizes (range of sizes that were called the same allele) and individual allele calls were determined automatically and adjusted manually by individually inspecting each electropherogram and allele call.
Scoring Method
Automated sizing of fragments was determined using ABI GeneMapper V4.0 software and calibrated by the high-density internal lane standard GeneScan 600LIZ.
Control Value
V. labrusca 'Lady' (DVIT 0087):264, 270; V. vinifera 'Muscat of Alexandria' (DVIT 0466): 253, 260; V. vinifera 'Salt Creek' (FPS ID# 40122): 243, 260
Number of Observed Alleles
21