|Fazio, Gennaro - DEPT OF HORT UNIV MADISON|
Submitted to: Cucurbit Genetics Cooperative Report
Publication Type: Research Notes
Publication Acceptance Date: July 15, 2000
Publication Date: N/A
Interpretive Summary: Biotechnology allows for tools that can be used for obtaining better understanding of the genetic nature of plant species One biotechnology, molecular markers (DNA), allows for the characterization of the genetic code (the basic ingredients of biological life) which consists of genes (the basic building block of life). Genes (made up of DNA) are arranged on chromosomes (long lengths of biochemical molecules) which are ingredients in every cell. Genes provide instruction for important biological processes (e.g., maturation, fetal development) in the cell and tissue of an organism. Molecular markers can be synthesized using biotechnology. Molecular markers can be used to interact in biochemical reactions to identify genes and thus give a precise information on the location of genes on chromosomes. Certain types of molecular markers are more valuable than others. Simple sequence repeat markers (SSR) are extremely useful in identifying genes, but very expensive to construct. We communicate a method for the development of SSR markers that is relatively inexpensive and less time consuming that what is now being used in the industry. This method allows the scientist to make more SSR markers at a lower cost so that economically important genes can be identified more quickly, thus increasing the efficiency of genetic analysis of important sections of the chromosome.
Technical Abstract: Genetic markers such as isozymes, RAPI)s and RFLPs have been characterized in cucumber. However, critical documentation of these markers and their usefulness in marker-assisted selection (MAS) for applied breeding programs has been limited. This is at least partially due to the species' low polymorphism level. A higher level of polymorphism has been associated with SSR loci in preliminary studies with C sativus, as well as in other genera (Cucurbita and Citrullus) of the Cucurbitaceae. The development and characterization SSR markers is time consuming and expensive. We report here a method that substantially lowers the cost of SSR development. The method involves library construction, mass excision and plasmid DNA extraction using genomic DNA (cucumber experimental species). This is followed by the capture of plasmids containing microsatellites, and then sequence analysis and primer design. The key element in this procedure is the use of the following: 1) the pZL I plasmid vector (Life Technologies, Gaithersburg, MD); 2) GeneTrapperTM(Life Technologies, Gaithersburg, MD), 3) biotinylated (biotin-14-dCTP) with a terminal transferase enzyme (Life Technologies, Gaithersburg, MD); 4) GeneTool software package (Biotools, Edmonton, Canada), and: 5) primer design software OLIGO 6.0 (Life Science Software, Long Lake, MN).