CHARACTERIZATION OF RAPD MARKERS IN VITIS

A study was initiated to investigate the possibility of using RAPD mar kers in related populations of Vitis. We also sought to design primers that could amplify translation initiation sites (Kozak sequence) as a mean to maximize the production of RAPD markers from single copy DNA sequences in the genome. RAPD bands were labeled and used as probes on blots with either genomic DNA or RAPD products from cvs Aurore, Cayug a White, Horizon and Illinois 547-1. Reamplification of excised RAPD p roducts produced either several bands of smaller size, a single band o f smaller size or a single band of the same size as the original band. Among 16 probes hybridized to genomic DNA blots, three probes, includ ing one from the Kozak primer amplification, hybridized to 1-2 bands, 5 probes hybridized to 3-8 bands and 8, including two from a Kozak pri mer reaction, to more than 10 bands on the genomic DNA blots. Twelve R APD bands were also probed on RAPD blots derived from the RAPD reactio n that produced each probe. Three of those probes hybridized to 1-2 ba nds, 8 hybridized to 3-8 and one hybridized to more than 10 bands indi cating the presence of probe sequences in more than one RAPD band as a mplified with the same primers. This result and the observations on re amplification of RAPD bands support the hypothesis that some of the lo nger RAPD fragments harbor internal priming sites that are either not amplified unless the reaction mixture is saturated with longer other p rimers indicating amplification from the same sequence but different s ized repetitive DNA. RAPD reactions were also run with 16 primers on p arental DNA of 2 crosses used in genetic mapping (Cayuga White x Auror e and Horizon x Illinois 547-1). These reactions rated 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 Il linois 547-1 were population specific. The RAPD analysis as well as hy bridization of RAPD markers to the genomic blots suggest that linkage analysis could be used in related segregating populations with careful ly chosen markers. Tagging single copy regions with Kozak-sequence-der ived primers may be possible, but the low number of probes tested and lack of DNA sequence information prevents any definite conclusions.