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

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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 F2-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.

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Acknowledgements

This research was supported by grants from the United Soybean Board (St. Louis, Mo.), from the Crop Functional Genomic Center (code no. CG3131) of the Twenty-first Century Frontier Research Program funded by the Ministry of Science and Technology (MOST) and Rural Development Administration (RDA) of the Republic of Korea, and support provided by the Georgia Agricultural Experiment Stations.

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Authors and Affiliations

  1. Department of Crop and Soil Sciences, Center for Applied Genetic Technologies, University of Georgia, 111 Riverbend Road, Athens, GA, 30602, USA

    S.-H. Lee, D. R. Walker & H. R. Boerma

  2. School of Plant Science, Seoul National University, San 56-1, Shillim-dong, Kwanak-gu, Seoul, 151-742, Republic of Korea

    S.-H. Lee

  3. Soybean Genomics and Improvement Laboratory, USDA-ARS, Beltsville, MD, 20705, USA

    P. B. Cregan

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  1. S.-H. Lee
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  2. D. R. Walker
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  3. P. B. Cregan
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  4. H. R. Boerma
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Correspondence to D. R. Walker.

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Communicated by F.J. Muehlbauer

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Lee, SH., Walker, D.R., Cregan, P.B. et al. Comparison of four flow cytometric SNP detection assays and their use in plant improvement. Theor Appl Genet 110, 167–174 (2004). https://doi.org/10.1007/s00122-004-1827-1

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  • DOI: https://doi.org/10.1007/s00122-004-1827-1

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