Characterization of RAPD Markers in Vitis

Lodhi, M.A., N.F. Weeden, and B.I. Reisch. 1997. Characterization of RAPD markers in Vitis Vitis 36:133-140

A study was initiated to investigate the possibility of using RAPD markers in related populations. We also sought to design primers that could amplify translation initiation sites (Kozak sequence) as a means to maximize the production of RAPD markers in single copy DNA sequences. RAPD markers were labeled and used as probes on blots with either genomic DNA or RAPD products from 'Aurore', 'Cayuga White', 'Horizon' and Illinois 547-1. Reamplification of excised RAPD products produced either several bands of smaller size, a single band of smaller size or a single band of the same size as the original band. Among 16 probes hybridized to genomic DNA blots, three probes, including one from the Kozak primer amplification, hybridized to 1-2 bands, five of the 16 probes hybridized to 3-8 bands and eight, including two from a Kozak primer reaction, to more than ten bands on the genomic DNA blots. Twelve RAPD bands were also probed on RAPD blots derived from the RAPD reaction that produced each probe. Three of those probes hybridized to 1-2 bands, eight hybridized to 3-8 and one hybridized to more than ten bands indicating the presence of probe sequences in more than one RAPD band as amplified with the same primers. This result and the observations on reamplification of RAPD bands support the hypothesis that some of the longer RAPD fragments harbor internal priming sites that are either not amplified unless the reaction mixture is saturated with longer products or are not present as frequently as the longer sites. RAPD DNA probes from one primer also hybridized to the RAPD products of other primers indicating the amplification from the same sequence but different sized repetitive DNA. RAPD reactions were also run with 16 primers on parental DNA of two crosses used in genetic mapping ('Cayuga White' x 'Aurore' and 'Horizon' x Illinois 547-1). These reactions generated 140 bands; 100 bands were shared by both populations, including 47 polymorphic bands. Ten polymorphic bands in 'Cayuga White' x 'Aurore' and 22 in 'Horizon' x Illinois 547-1 were population specific. The RAPD analysis as well as hybridization of RAPD markers to the genomic blots suggest that linkage analysis could be used in related segregating populations with carefully chosen markers. Tagging single copy regions with Kozak-sequence-derived primers may be possible, but the low number of probes tested and lack of DNA sequence information prevents any definite conclusions.

Key Words. chemiluminescent, hybridization, internal priming sites, Kozak, Southern.