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.

Loss of crop genetic diversity due to modern plant breeding is an enduring global concern. The U.S. Pacific Northwest (PNW) is an important wheat (Triticum aestivum L.)‐producing region. We examined population structure and changes in genomic‐level and agroecosystem‐level genetic diversity of PNW wheat over the past 120 yr. Wheat varieties were slightly structured by spring and winter growth habit, but not by geographic origin or period of variety release. At the genomic level, we did not find long‐term shifts in gene diversity in either spring or winter wheat, but fluctuations were significant within market classes and within the most widely grown spring and winter varieties. Diversity in hard red spring (HRS) wheat peaked in 1970–1999 and since dropped below the level of varieties before 1930. Hard red winter (HRW) in 2000–2019 is more diverse than before 1930, whereas soft white spring (SWS) and soft white winter (SWW) are at the same level of diversity in 2000–2019 as they were before 1930. The most widely grown spring varieties had the lowest diversity in 1930–1969, whereas the most widely grown winter varieties had the lowest diversity in 2000–2019. At the agroecosystem level, Simpson's index of diversity (1 − D) has been on a rising trend since the 1990s as the dominance of acreages by a few varieties has become less common. Cultivation of multiple market classes and periodic incorporation of new germplasm by breeding programs have been able to maintain the quantitative level of genetic diversity in PNW wheat over time.

Wheat near-isogenic line AvSYr17NIL carrying Yr17 originally from Aegilops ventricosa for all-stage resistance to Puccinia striiformis f. sp. tritici (Pst) also shows non-race specific, high-temperature adult-plant (HTAP) resistance to the stripe rust pathogen. To separate and identify the HTAP resistance gene, seeds of AvSYr17NIL were treated with ethyl methanesulfonate (EMS). Mutant lines with only HTAP resistance were obtained, and one of the lines, M1225, was crossed with the susceptible recurrent parent Avocet S (AvS). Field responses of the F2 plants and F3 lines, together with the parents, were recorded at the adult-plant stage in Pullman and Mount Vernon, WA under natural Pst infection. The parents and the F4 population were phenotyped with an Yr17-virulent Pst race in the adult-plant stage under the high temperature profile in the greenhouse. The phenotypic results were confirmed by testing the F5 population in the field under natural Pst infection. The F2 data indicated a single recessive gene, temporarily named YrM1225, for HTAP resistance. The F4 lines were genotyped with Kompetitive allele specific PCR (KASP) markers converted from single nucleotide polymorphism (SNP) markers polymorphic between M1225 and AvS. The HTAP resistance gene was mapped on the short arm of chromosomal 2A in an interval of 7.5 cM using both linkage and quantitative trait locus mapping approaches. The separation of the HTAP resistance gene from Yr17 should improve the understanding and utilization of the different types of resistance.

Genotyping by sequencing (GBS) and single nucleotide polymorphism (SNP) chip technologies are the primary SNP genotyping technologies used today. However, these genotyping technologies have some drawbacks that limit their usefulness in analysis. We have developed a robust protocol called genotyping by multiplexed sequencing (GMS) using SNP markers, providing informative genotypic data with greater flexibility. The genotypes derived from direct sequence reads reduce ambiguity in genetic analysis. The advantages of this protocol include: (1) This PCR-based direct sequencing protocol generates information from markers of interest and provides a more streamlined and accurate analysis process, by multiplexing hundreds of informative markers into a single sequencing run. (2) The marker sets are easily customized to the species of interest and can readily be changed. In this study we have taken the GMS protocol developed in wheat and adapted it to barley. We have identified 577 SNP markers that work well using this protocol providing adequate genome coverage for genomic selection and tag 267 QTL’s for genes of interest. Good markers have an adequate read depth of at least 5 amplicons and are reliably present across the population.