Location: Hard Winter Wheat Genetics ResearchTitle: Using next generation sequencing for multiplexed trait-linked markers in wheat
|BERNARDO, AMY - Kansas State University|
|WANG, SHAN - Kansas State University|
|St Amand, Paul|
Submitted to: PLoS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/10/2015
Publication Date: 12/1/2015
Publication URL: https://handle.nal.usda.gov/10113/5454535
Citation: Bernardo, A., Wang, S., St Amand, P.C., Bai, G. 2015. Using Next Generation Sequencing for Multiplexed Trait-linked Markers in Wheat. PLoS One. 10(12):e0143890. doi:10.1371/journal.pone.0143890.
Interpretive Summary: Single nucleotide polymorphisms (SNPs), are differences between individuals at a single position in their DNA sequence. SNPs are abundant in wheat and can be used as genetic markers to characterize these individuals. However, the SNPs in wheat have not been effectively used in marker-assisted breeding because the SNP assays used for marker-assisted breeding usually use only one marker per assay. Multiplex assays that simultaneously analyze many markers in one assay will reduce marker assay cost and time, and increase breeding efficiency. In this study, we investigated 33 SNP markers that were linked to 20 different genes of agronomic importance in wheat using 24 selected wheat accessions. Among them, 27 SNPs were successfully multiplexed and 23 markers completely matched with the results from corresponding single marker analyses. The multiplexed assay developed based on next-generation sequencing technology in this study is suitable for rapid and high-throughput screening of SNPs in wheat.
Technical 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 rate. 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 SNP and some indel-based markers in wheat advanced breeding lines.