Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping

Abstract

SummaryMultiplexed inter-simple sequence repeats genotyping by sequencing (MIG-seq) is an next-generation sequencing library construction method developed for the analysis of DNA in ecology. Although MIG-seq can generate libraries from low-quality DNA, few polymorphisms can be obtained in species with small genomes. In this study, we developed degenerate oligonucleotide primer MIG-seq (dpMIG-seq) as an effective polymorphism discovery method that allows for variation in the number of polymorphisms while retaining the advantages of MIG-seq, including independence from DNA quality. In dpMIG-seq, a proportion of the simple sequence repeats in the primer sequence of the first PCR in MIG-seq was changed to degenerate oligonucleotides to enable annealing to a wider range of sequences. In tests of several crop species other than wheat, the number of loci that could be sequenced using dpMIG-seq with a data volume of 0.3 gigabases (Gb) was increased compared with that sequenced using MIG-seq. In wheat, the number of polymorphisms obtained via dpMIG-seq was higher than that obtained via MIG-seq when a data volume of about ≥2 Gb was obtained. In dpMIG-seq, different loci could be sequenced by changing the positions of the degenerate oligonucleotides. By applying dpMIG-seq, we constructed a linkage map consisting of 5,142 markers for the rice inter-subspecies F2 population, and we detected quantitative trait loci for heading date in the regions where known heading-related genes were located. Overall, our results show that dpMIG-seq is a useful tool for the genetic analysis of crop species.