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ARS Home » Plains Area » Manhattan, Kansas » Center for Grain and Animal Health Research » Hard Winter Wheat Genetics Research » Research » Publications at this Location » Publication #365822
Research Project: Genetic Improvement of Biotic and Abiotic Stress Tolerance and Nutritional Quality in Hard Winter Wheat

Location: Hard Winter Wheat Genetics Research

Title: Multiplex Restriction Amplicon Sequencing (MRASeq), a novel next generation sequencing based marker platform for high-throughput genotyping

Author
item BERNARDO, AMY - Kansas State University
item St Amand, Paul
item LE, HA QUANG - Kansas State University
item SU, ZHENQI - Kansas State University
item Bai, Guihua

Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/11/2019
Publication Date: 6/14/2019
Citation: Bernardo, A., St Amand, P.C., Le, H., Su, Z., Bai, G. 2019. Multiplex Restriction Amplicon Sequencing (MRASeq), a novel next generation sequencing based marker platform for high-throughput genotyping. Nucleic Acids Research. https://doi.org/10.1111/pbi.13192.
DOI: https://doi.org/10.1111/pbi.13192

Interpretive Summary: To develop simple, low cost, high-throughput DNA markers for marker-assisted breeding, we invented a Multiplex Restriction Amplicon Sequencing (MRASeq) marker system that uses polymerase chain reaction (PCR). The new marker system targets DNA fragments flanked by restriction enzyme cutting sites and uses a unique tail sequence to pool all amplified fragments in each sample. MRASeq generated thousands of markers that are randomly distributed across wheat and barley genomes. This novel, inexpensive, next-generation-sequencing-based genotyping platform can be used for various breeding applications in wheat and other species.

Technical Abstract: To enable rapid selection of traits in marker-assisted breeding, markers must be technically simple, low cost, high-throughput and randomly distributed in a genome. We developed such a technology, designated as Multiplex Restriction Amplicon Sequencing (MRASeq), which reduces genome complexity by polymerase chain reaction (PCR) amplification of amplicons flanked by restriction sites. The first PCR primers contain restriction site sequences at 3’-ends, followed by 6-10 bases of specific or degenerate nucleotide sequences, and then by a unique M13-tail sequence which serves as a binding site for a second PCR which adds sequencing primers and barcodes to allow sample multiplexing. The sequences of restriction sites and adjacent nucleotides can be altered to suit any species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum eastivum L.) showed a random distribution of SNPs across the genome. MRASeq generated thousands of SNPs from a wheat biparental population and natural populations of wheat and barley (Hordeum vulgare L.). This novel, next-generation-sequencing-based genotyping platform can be used for linkage mapping to screen quantitative trait loci (QTL), background selection in breeding, and many other genetics and breeding applications of various species.