Genotyping by Multiplexed Sequencing (GMS) protocol in Barley

Abstract

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.

BarPlex v1.0: a multiplex PCR-based enrichment of genome-wide short segments that enable genetic studies in barley

“…That might be explained by ascertainment bias, similar to an earlier study that genotyped barley landraces by SNP array (Moragues et al 2010). By applying the polymorphic sites revealed by BarPlex v1.0, we were able to detect minor alleles as identi ed by GBS (Darrier et al 2019), thus overcoming the bias of genotyping assays (i.e., SNP array) that only rely on pre-identi ed SNPs (Eagle et al 2021;Moragues et al 2010;Rasheed et al 2017). We tested BarPlex v1.0 to identify the genetic factor controlling the adherence of caryopsis (hulled vs. naked) by both linkage mapping and GWAS.…”

High resolution mapping of a novel non-transgressive hybrid susceptibility locus in barley exploited by P. teres f. maculata

“…A total of 83 CI5791 × Golden Promise F 2 individuals were screened for disease response as described in biological materials and phenotyping. After DNA extraction, accessions were genotyped using a GMS marker panel [ 60 ] using Illumina sequencing. SNP calling preceded as described in exome derived high-resolution mapping of CI5791 × Tifang except the reads were trimmed using fastp [ 61 ].…”

High resolution mapping of novel non-transgressive hybrid susceptibility in barley exploited byP. teresf.maculatamaps to a single pentatricopeptide repeat-containing protein

“…CI5791 × Golden Promise Genotype-by-Multiplex Sequencing Mapping A total of 83 CI5791 × Golden Promise F 2 individuals were screened for disease response as described in biological materials and phenotyping. After DNA extraction, accessions were genotyped using a genotype-by-multiplex sequencing marker panel [60] using Illumina sequencing. SNP calling preceded as described in exome derived high-resolution mapping of CI5791 × Tifang except the reads were trimmed using fastp [61].…”