Comparison of four flow cytometric SNP detection assays and their use in plant improvement

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 extension (SBE), 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 F(2)-derived soybean [Glycine max (L.) Merr.] lines from a cross between inbred lines G99-G725 and N00-3350 were genotyped at four SNPs. SBE and ASPE clearly differentiated between the two homozygotes and the heterozygote at each SNP. Results were in agreement with those identified using the SNaPshot minisequencing assay as a control. In contrast, the OL and DH assays were unable to differentiate between genotypes at some of the SNPs. However, when the cost per data point for the four different assays was compared, the cost of OL and DH was only about 70% of that for SBE, with DH requiring the least time of the four assays. On the basis of cost and labor, ASPE is more cost-effective and simpler than SBE, 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 appears to be the most economical assay for marker-assisted selection, though optimization for DH would be required for some SNP markers.

Population structure and genetic differentiation associated with breeding history and selection in tomato (Solanum lycopersicum L.)

“…A total of 300 markers including 149 markers from transcribed (exon) sequences (Yang et al, 2004(Yang et al, , 2005a and 151 markers from intron sequences (Van Deynze et al, 2007) were used to genotype the collection of cultivated tomato varieties. We used multiple genotyping platforms including agarose gel electrophoresis for SNP detection as cleaved amplified polymorphic sequences (CAPS) and the LUMINEX 200 (Luminex, Corp., Austin, TX, USA) for detection of SNPs using allele-specific primer extension (ASPE) (Lee et al, 2004). The LICOR IR2 system (LICOR, Lincoln, NE, USA) was used for fragment analysis of SSR and InDel markers.…”

Fine mapping the soybean aphid resistance gene Rag1 in soybean

“…There are many considerations in selecting a SNP genotyping assay, and the choice usually depends on cost per data point and simplicity of data acquisition (Lee et al 2004). To obtain reliable and reproducible SNP genotyping results, we found that the MCA required relatively high quality and quantiWed DNA extracted using the CTAB extraction method, while the TaqMan assays produced high-quality genotypic data even with quick-extracted DNA (Bell-Johnson et al 1998).…”

“…Single nucleotide polymorphism (SNP) markers are becoming widely used in soybean breeding and research (Hyten et al 2009b) because of their high frequency, widespread distribution throughout the genome (Choi et al 2007), and their suitability for high-throughput automated genotyping (Hyten et al 2008;Lee et al 2004;Schork et al 2000). Zhu et al (2003) found 3.68 SNPs per kilobase pair (kb) in 25 diverse soybean genotypes.…”

BARCSoySNP23: a panel of 23 selected SNPs for soybean cultivar identification

“…The cost of SNP assays varies with SNP assay methods. Lee et al (2004) estimated the cost of reagents per simplex (per data point) reaction among four SNP genotyping assays to be $0.069-0.104. As SNPs continue to gain prominence in plant and animal genetic analysis, the cost effectiveness of SNP detection assays is likely to improve.…”