title: Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat.
abstract: With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat (Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat.
authors.affiliation: Department of Plant Pathology, Kansas State University, Manhattan, Kansas, United States of America.
authors.forename: Amy
authors.initials: A
authors.lastname: Bernardo
authors.affiliation: Department of Agronomy, Kansas State University, Manhattan, Kansas, United States of America.
authors.forename: Shan
authors.initials: S
authors.lastname: Wang
authors.affiliation: United States Department of Agriculture, Agricultural Research Service, Hard Winter Wheat Genetics Research Unit, Manhattan, Kansas, United States of America.
authors.forename: Paul
authors.initials: P
authors.lastname: St Amand
authors.affiliation: Department of Agronomy, Kansas State University, Manhattan, Kansas, United States of America.|United States Department of Agriculture, Agricultural Research Service, Hard Winter Wheat Genetics Research Unit, Manhattan, Kansas, United States of America.
authors.forename: Guihua
authors.initials: G
authors.lastname: Bai
mesh_terms: D000483:Alleles; D002874:Chromosome Mapping / Q000379:methods; D018556:Crops, Agricultural / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics*; D008040:Genetic Linkage / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D040641:Quantitative Trait Loci / Q000235:genetics*; D016324:Sequence Tagged Sites; D014908:Triticum / Q000235:genetics*
chemical_list: D005819:Genetic Markers