A Simple and Versatile SNP Genotyping Method By Multiplex PCR Amplicon Sequence in Brachypodium distachyon

Next-generation sequencing technologies have enabled us to re-sequence genome to explore polymorphisms which confer various biological differences among individuals. To facilitate high-throughput polymorphism SNP genotyping, we developed a simple and versatile method based on multiplex PCR and amplicon sequencing, and applied it to determine genotypes of natural accessions in a model grass Brachypodium distachyon. As an example to assess our developed method, we carried out genome-wide genotyping of SNPs which were confidently identified in between genome re-sequencing data of natural accessions (Bd1-1, Bd3-1, Bd30-1, and Koz-3) and reference genome sequence of Bd21. Then, we examined the accuracy of genotyping with 443 SNPs of the natural accessions and a F₁ progeny (Bd3-1 x Bd21). Roughly, around 95% of SNPs are successfully called accurate genotype by VarScan program. This high-throughput genotyping method is applicable not only to B. distachyon but also to all other organisms if sequence information around SNPs is available. The method described here enables easily to design custom target re-sequencing panel in various organisms and facilitates identification of genetic association between polymorphisms and biological properties.