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.

TAMFT-3A and TAMFT-3B2 homeologs are associated with wheat preharvest sprouting.

The phenomenon of preharvest sprouting (PHS), caused by rain after physiological maturity and prior to harvest, negatively affects wheat (Triticum aestivum L.) production and end use. Investigating the genetics that control PHS resistance may result in increased control of seed dormancy. Multiple genes involved in the development of seed dormancy are associated with PHS. In this study, the TaMFT (3A, 3B1, 3B2, 3D), TaMKK3-4A, and TaVP1-3B genes were assessed for association with PHS in a double-haploid line (DHL) hard red winter wheat population derived from a BC1 cross between the cultivars Loma and Warhorse, where Loma was the recurrent and PHS susceptible parent. The 162 BC1 DHL lines were grown over two field seasons and PHS susceptibility was assessed by measuring PHS resistance in physiologically mature heads. The PHS variation was associated with the TaMFT-A and the B2 homeolog with Loma carrying mutant forms of each gene. No sequence variation between Loma and Warhorse was detected in the exons of the TaMFT-B1 and D homeologs. No association between PHS resistance and TaMKK3-4A or TaVp1-3B variation was observed, though Loma and Warhorse vary for TaMKK3-4A and TaVp1-3B mutations reported to be PHS associated. Previous research has shown TaMFT-3A as having a large impact on PHS resistance. In the current study, the TaMFT-3A and TaMFT-3B2 alleles each explained 14% of observed PHS variation. Markers for both TaMFT-3A and TaMFT-3B2 should be used in selecting for increased wheat dormancy and PHS resistance.

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.

Identification and Mapping of Quantitative Trait Loci Associated with Stripe Rust Resistance in Spring Club Wheat Cultivar JD.

Puccinia striiformis Westend. f. sp. tritici, commonly known as stripe rust, is an economically important pathogen of wheat (Triticum aestivum L.). The hexaploid club spring wheat cultivar JD contains both all-stage and adult plant resistance (APR) genes and exhibited consistent high resistance to stripe rust in the field. In this study, we aimed to identify the quantitative trait loci (QTL) for stripe rust resistance using a BC1F7 back-cross inbred-line population derived from the cross of JD and the recurrent parental line 'Avocet'. The population was phenotyped in field plots in Washington State at the Spillman Agronomy Farm in Pullman and Mount Vernon Northwest Washington Research and Extension Center in between 2014 and 2016. A major QTL tentatively designated as QYrJD.wsu-1B, conferring all-stage resistance in JD background, was identified and mapped at the telomere region on the short arm of chromosome 1B using the genotyping-by-sequencing method. This QTL was further characterized with simple sequence repeat (SSR) markers and found to have the greatest logarithm-of-the-odds score and phenotypic effect, using SSR marker wmc798 on chromosome 1BS. Seven additional QTLs associated with APR were identified in the JD background on chromosomes 2D, 3A, 3B, 4A, 6B, and 7A with partial phenotypic effects.

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.