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  • Author or Editor: Diane J. Wilson x
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A total of eight random amplified polymorphic DNA (RAPD) markers were generated in a screen of 77 primers of 10-base length and were detected reproducibly among nine different grape (Vitis) rootstocks. Occasional failed amplifications could not be explained rationally nor easily corrected by systematic replacement of individual reaction components. In an effort to improve their reliability, the RAPD markers were cloned, their termini sequenced, and new sequence-specific primer pairs were synthesized based on addition of 10 to 14 bases to the 3' termini of the original 10-mers. Six pairs of the new primers were evaluated at their optimal and higher-than optimal annealing temperatures. One primer pair amplified a product the same size as the original RAPD marker in all rootstocks, resulting in loss of polymorphism. Post-amplification digestion with 7 different restriction endonucleases failed to reveal restriction site differences. Three primer pairs amplified an unexpected length variant in some accessions. Two other pairs of primers amplified a number of unexpected bands. Better approaches for exploiting the sequence differences that account for the RAPD phenomenon will be discussed.

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Randomly amplified polymorphic DNA (RAPD) markers were generated for identifying grape (Vitis) rootstocks. Seventy-seven primers (10 bases long) were screened using CsCl-purified leaf DNA derived from several field samples of nine rootstocks sampled in successive years. Nine RAPD markers were detected from six primers and, in combination, distinguished all nine rootstocks tested. Because inconsistencies were encountered in performing the RAPD assay, sequence-specific primers were derived from cloned RAPD bands for use under more stringent amplification conditions. 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 some cases, bands of the same size generated by the same primer in different rootstocks-normally scored as the same marker-failed to cross-hybridize, implying lack of homology between the bands. More commonly, bands scored as absent based on ethidium bromide staining were detected by hybridization. Six of the nine cloned RAPD bands were partially sequenced, and sequence-specific primer pairs were synthesized. Two primer pairs amplified a product the same size as the original RAPD band in all rootstocks, resulting in loss of polymorphism. Two other pairs of sequence-specific primers derived from the same marker failed to amplify the expected band consistently. Three of the most useful primer pairs amplified apparent length variants in some accessions and will have value as polymerase chain-reaction markers for fingerprinting.

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