|Schnell Ii, Raymond|
Submitted to: Methods in Enzymology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/3/2004
Publication Date: 1/1/2005
Citation: Kuhn, D.N., Schnell II, R.J. 2005. Use of capillary array electrophoresis single strand conformational polymorphism analysis to estimate genetic diversity of candidate genes in germplasm collections. Methods in Enzymology. 395:238-258. Interpretive Summary: The development of co-dominant molecular markers such as microsatellites is time consuming, expensive, and difficult to justify for minor crop species. An alternative approach is to search for genes that are conserved across species, in order to find and amplify similar genes. These amplification products (alleles) do not differ in size as do microsatellites and other types of markers, but instead differ in sequence. To detect these differences one must use a technique that allows separation based on conformation and this technique is known as Single Strand Conformation Polymorphism Analysis (SSCP). The ability to use DNA sequence information from well studied species and apply it to species with very little DNA sequence information reduces the time and cost of the genetic analysis. For example, resistance gene homologues (RGH) were discovered in cacao based on original sequences from lettuce and Arabidopsis. Using these RGH and SSCP we were able to screen the cacao germplasm collection, estimate genetic diversity, and fingerprint clones. Practical application of the technique is described with information included based on our experiences using SSCP on high throughput DNA fragment analysis machines.
Technical Abstract: Capillary array electrophoresis-single strand conformational polymorphism (CAE-SSCP) analysis provides a reliable high throughput method to genotype plant germplasm collections. Primers designed to highly conserved regions of candidate genes can be used to amplify DNA from plants in the collection. These amplified DNA fragments of identical length are turned into useful markers by assaying sequence differences by CAE-SSCP analysis. Sequence differences affect the electrophoretic mobility of ssDNA under nondenaturing conditions. By collecting the mobility data for both strands assayed at two temperatures, alleles can be defined by mobility alone. For a germplasm collection with an unknown number of alleles at a locus, such mobility data of homozygotes can be used to determine the number of unique alleles without the necessity of cloning and sequencing each allele.