|Novy, Richard - Rich|
Submitted to: American Journal of Potato Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/11/2000
Publication Date: 8/1/2000
Citation: Rojas-Cifuentes, G.A., Kianian, P.M., Novy, R.G. 2000. Potato rapd heteroduplexes: their impact on genetic distance estimates andtheir use in developing codominant sequence tagged site (sts) markers. American Journal of Potato Research.77:416-417.
Technical Abstract: Codominant RAPDs result from the amplification of "allelic" DNA segments that differ by the insertion, deletion, or substitution of base pairs between primer annealing sites. During PCR amplification, the DNA strands of codominant RAPDs may reanneal to form two homoduplexes and two heteroduplexes. The migration of hybrid heteroduplex molecules during non-denaturing electrophoresis differs from the homoduplex bands from which they originate, leading to their misinterpretation as unique RAPDs. The DNA of 38 haploid clones were amplified using 48 RAPD primers. The DNA of haploids lacking a given RAPD marker were intermixed in equal parts, and re-amplified. Synthesis of a RAPD, previously absent in the haploids, was evidence that it was a probable heteroduplex artifact. Based on this assay, 39 of 263 (15%) RAPDs were identified as heteroduplexes. Genetic distance matrices were calculated for the haploid and a tetraploid population using (1) all RAPDs, and (2) only non-heteroduplex RAPDs; the matrices were then compared to assess the impact heteroduplexes have on genetic distance estimates. Two sets of codominant RAPDs were identified, cloned, and sequenced after their formation of heteroduplex bands following haploid DNA intermixing. Each pair of RAPDs was found to have high sequence homology (>80%) to each other. Primary sequence differences observed among the codominant RAPDs were nucleotide substitutions and deletions. Twenty-mer primers have been developed for the conversion of the codominant RAPDs to sequence tagged site (STS) markers. STS markers developed through this approach are similar to simple sequence repeats