|Fazio, Gennaro - UNIVERSITY OF WISCONSIN|
|Katzir, Nurit - NEWE YA'AR RESEARCH CENTE|
Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 15, 2002
Publication Date: December 10, 2002
Citation: Fazio, G., Staub, J.E., Katzir, N. 2002. Development and characterization of pcr markers in cucumber (cucumis sativus l.). Journal of the American Society for Horticultural Science. Interpretive Summary: Molecular biology (the study of the biology of DNA) has provided opportunities for unlocking secrets of life. Science has benefited from a development of new biotechnologies (biological methods for studying DNA) that have broadened our understanding of how genes (groups of DNA molecules on chromosomes) interact to produce productions that are important to the consuming public (i.e., cancer treatments). Cucumber is a vegetable that is grown widely in the U.S. and throughout the world. Although the basic biology of this crop species (what we can see visually such as fruit yield and quality) has been studied in depth, scientific advances at the molecular DNA level have not been as great. What is known is that cucumber has relatively little amounts of DNA compared to other crop species that makes DNA analysis of cucumber an attractive scientific endeavor. However, it is clear from recent attempts to study the DNA of cucumber, that the DNA of this species is not easy to examine. Thus, a study was designed to create new biotechnologies for cucumber that would allow for easier examination of cucumber DNA in order to allow plant geneticists and breeders to be more efficient in developing improved varieties. The experiments allowed for the creation of a DNA examination technology that greatly increases the ability for scientists to understand the genetics of this economically important crop species. The development of these new biotechnologies will benefit the consuming public directly by allowing scientists to develop better varieties more rapidly.
Technical Abstract: Highly polymorphic microsatellites or simple sequence repeats (SSR), along with sequence characterized amplified regions (SCAR) and single nucleotide polymorphisms (SNP) markers are reliable, cost-effective, and amenable for large scale analyses. 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 di-nucleotide repeats (CT, CA, TA), 21 (19%) possessed tri-nucleotide repeats (CTT, ATT, ACC, GCA), 3 (2.7%) contained tetra-nucleotide repeats (TGCG, TTAA, TAAA), 4 (3.6%) enclosed penta-nucleotide repeat (ATTTT, GTTTT, GGGTC, AGCCC), 3 (2.7%) contained hexa-nucleotide 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) were also developed. The SNP markers were developed from otherwise monomorphic SCAR markers. 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 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.