Multiplex restriction amplicon sequencing: a novel next-generation sequencing-based marker platform for high-throughput genotyping.

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, preceded 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 that adds sequencing primers and barcodes to allow sample multiplexing for sequencing. The sequences of restriction sites and adjacent nucleotides can be altered to suit different species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum aestivum 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.