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Title: COMPARISON OF FOUR FLOW CYTOMETRIC SNP DETECTION ASSAYS AND THEIR USE IN PLANT IMPROVEMENT

Author
item LEE, SUK-HA - SEOUL NATIONAL UNIVERSITY
item WALKER, DAVID - UNIVERSITY OF GEORGIA
item Cregan, Perry
item BOERMA, H - UNIVERSITY OF GEORGIA

Submitted to: Theoretical and Applied Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/27/2004
Publication Date: 9/27/2004
Citation: Lee, S., Walker, D.R., Cregan, P.B., Boerma, H.R. 2004. Comparison of four flow cytometric snp detection assays and their use in plant improvement. Theoretical and Applied Genetics. 110: 167-174.

Interpretive Summary: DNA markers serve as genetic landmarks and are interspersed among the 50,000 or more genes throughout the chromosomes of the soybean. If a marker is located near a gene of interest, the marker can be used to select for the desired form of the gene. For example, the soybean breeder can use a DNA marker to identify plants that carry the form of the gene that gives resistance to a disease rather than the form that leads to susceptibility. It was the objective of this work to report on four different methods to detect a type of DNA marker referred to as a single nucleotide polymorphism or SNP. The reliability, cost and time required to use the four different methods: single base chain extension (SBCE), allele-specific primer extension (ASPE), oligonucleotide ligation (OL) and direct hybridization (DH) were compared. It was concluded that both SBCE and ASPE were very reliable SNP detection methods as they gave results identical to a standard control method. The OL and DH methods were less accurate but less costly and time consuming and with careful optimization could be made to function well. If large number of SNPs were being assayed ASPE was the method of choice. If a few SNPs were being assayed in many soybean lines (such as in a soybean breeding program) DH with careful optimization of the assay would be most appropriate. This research is of use to plant breeders and other plant biologists who are interested in the efficient accumulation of SNP DNA marker data.

Technical Abstract: Single nucleotide polymorphisms (SNPs) are attractive DNA markers due to their abundance and potential for use in automated high-throughput genotyping. Numerous SNP genotyping assays have been developed, but it is unclear which assays are best suited and most efficient for various types of plant improvement research. The objective of this study was to compare the accuracy, efficiency, and cost of four SNP genotyping assays: single base chain extension (SBCE), allele-specific primer extension (ASPE), oligonucleotide ligation (OL), and direct hybridization (DH). All four assay methods used the same Luminex 100 flow cytometer platform. Fifty-eight F2-derived soybean, Glycine max (L.) Merr., lines from a cross between inbred lines G99-G725 and N00-3350 were genotyped at four SNPs. SBCE and ASPE clearly differentiated between the two homozygotes and the heterozygote at each SNP, and the results were in agreement with those identified using the SNaPshotTM minisequencing assay, which was included as a control. In contrast, the OL and DH assays were unable to differentiate between genotypes at some of the SNPs. However, the cost per data point for OL and DH was only about 70% of that for SBCE, and DH requires the least time of the four assays. Based on cost and labor, ASPE is more cost-effective and simpler than SBCE, and would therefore be a good method for genetic mapping and diversity studies which require a large number of markers and a high level of multiplexing. DH would appear the most economical assay for marker-assisted selection (MAS), though optimization for DH would be required for some SNP markers.