Development and characterization of PCR markers in cucumber

Highly polymorphic microsatellites; or simple sequence repeat (SSR), along with sequence characterized amplified region (SCAR) and single nucleotide polymorphisms (SNP), markers are reliable, cost-effective, and amenable for large scale analyses. Molecular polymorhisms are relatively rare in cucumber (Cucumis sativus L.) (3% to 8%). Therefore, experiments were designed to develop SSR, SCAR and SNP markers, and optimize reaction conditions for PCR. A set of 110 SSR markers was constructed using a unique, strategically applied methodology that included the GeneTrapper (Life Technologies, Gaithersburg, Md.) kit to select plasmids harboring microsatellites. Of these markers, 58 (52%) contained dinucleotide repeats (CT, CA, TA), 21 (19%) possessed trinucleotide repeats (CTT, ATT, ACC, GCA), 3 (2.7%) contained tetranucleotide repeats (TGCG, TTAA, TAAA), 4 (3.6%) enclosed pentanucleotide repeat (ATTTT, GTTTT, GGGTC, AGCCC), 3 (2.7%) contained hexanucleotide repeats (CCCAAA, TAAAAA, GCTGGC) and 21 possessed composite repeats. Four SCARS (L18-3 SCAR, AT1-2 SCAR, N6-A SCAR, and N6-B SCAR) and two PCR markers based on SNPs (L18-2H19 A and B) that are tightly linked to multiple lateral branching (i.e., a yield component) were also developed. The SNP markers were developed from otherwise monomorphic SCAR markers, producing genetically variable amplicons. The markers L18-3 SCAR and AT1-2 SCAR were codominant. A three-primer strategy was devised to develop a codominant SCAR from a sequence containing a transposable element, and a new codominant SCAR product was detected by annealing temperature gradient (ATG) PCR. The use of a marker among laboratories can be enhanced by methodological optimization of the PCR. The utility of the primers developed was optimized by ATG-PCR to increase reliability and facilitate technology transfer. This array of markers substantially increases the pool of genetic markers available for genetic investigation in Cucumis.