Development of PCR-based markers for identifying grape rootstocks Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/dn39x505b

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  • Sequence-specific PCR markers were derived from one new and eight previously identified random amplified polymorphic DNA (RAPD) markers for the purpose of identifying grape (Vitis) rootstocks. The markers were developed because the RAPD assay was found to be inconsistent and the original RAPD markers unreliable. Southern hybridization analysis of the RAPD gels with cloned RAPD bands as probes revealed deficiencies of scoring RAPD bands based solely on ethidium bromide staining. In one case, bands of the same size generated by the same primer in different rootstocks -- normally scored as the same marker -- failed to cross-hybridize, implying a lack of homology between the bands. In another case, a band scored as absent based on ethidium bromide staining was detected by hybridization. One of nine RAPD bands was cloned in the present study. All nine were partially sequenced and sequence-specific pairs of primers were synthesized for each for use under stringent PCR conditions. Three of the primer pairs generated products only from the rootstocks from which the original RAPD bands had been cloned; three others produced products from additional rootstocks while still generating polymorphisms; two others generated apparent length variants from some accessions; and one primer pair resulted in a loss of polymorphism. Based on the eight polymorphic markers, five of nine rootstocks could be unambiguously distinguished.
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