Genotyping by Multiplexed Sequencing (GMS) Using SNP Markers.

SNP-based genotyping has become the most effective approach to generate target-specific data for use in genetic studies. In this chapter, we will describe a high-throughput genotyping method that multiplexes hundreds to thousands of SNP markers in a two-step PCR protocol that can be customized to fit the specific needs of a study.

Population structure and genetic diversity of U.S. wheat varieties.

The United States is a major wheat producer with more than a century of wheat (Triticum aestivum L.) research and breeding. Using a panel of 753 historical and modern wheat varieties grown in the United States from the early 1800s to present day, we examined population structure and changes in genetic diversity. We used previously mapped high-quality single-nucleotide polymorphism (SNP) markers from the wheat 90K SNP array for genotyping. The wheat varieties had a slight hierarchical population structure based on growth habit and then by kernel color within spring varieties and by kernel hardness within winter varieties, which corresponds with geographical distribution of the varieties. Classifying varieties by market class, which is a combination of habit, hardness, and color, accounted for the greatest amount of variation (13.3%). We did not find evidence of decreased genetic diversity of either spring or winter varieties after the release of the first semidwarf wheat variety in 1961. On the contrary, northern and Pacific spring varieties, hard red spring (HRS), hard white spring (HWS), and soft white winter (SWW) had increases in both SNP and haplotype genetic diversity after 1961. The soft white spring (SWS) and soft red winter (SRW) market classes already had high genetic diversity in varieties before 1961 and showed some evidence of decreased diversity after 1961. Examination of temporal trends in genetic diversity also did not indicate long-term decline in diversity despite occasional fluctuations.

Genotyping by multiplexed sequencing (GMS): A customizable platform for genomic selection.

As genotyping technologies continue to evolve, so have their throughput and multiplexing capabilities. In this study, we demonstrate a new PCR-based genotyping technology that multiplexes thousands of single nucleotide polymorphism (SNP) markers with high-throughput capabilities in a simple protocol using a two-step PCR approach. The bioinformatic pipeline is user friendly and yields results that are intuitive to interpret. This method was tested on two recombinant inbred line (RIL) populations that had previous genotyping data from the Illumina Infinium assay for Triticum aestivum L. and the two data sets were found to be 100% in agreement. The genotyping by multiplexed sequencing (GMS) protocol multiplexes 1,656 wheat SNP markers, 207 syntenic barley SNP markers, and 49 known informative markers, which generate a possible 2,433 data points (including homoeoalleles and paralogs). This genotyping approach has the flexibility of being sequenced on either the Ion Torrent or Illumina next generation sequencing (NGS) platforms. Products are the result of direct sequencing and are therefore more reliable than scatter plot analysis which is the output of other genotyping methods such as the Illumina Infinium assay, komeptitive allele specific PCR and other like technologies.