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

SUMMARY Next‐generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR‐based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG‐seq primer set (MIG‐seq being a PCR method enabling library construction with low‐quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG‐seq (dpMIG‐seq), enabled a streamlined protocol for constructing dpMIG‐seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG‐seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG‐seq protocol for advancing genetic analyses across diverse plant species. Significance Statement Given that plants contain impurities in their leaves, DNA purification is required to construct NGS libraries with reduced genomic complexity using restriction enzymes, which poses a hinderance to rapid genotyping. We developed degenerate oligonucleotide primer MIG‐seq and its accompanying protocol, a method for constructing NGS libraries with varying degrees of complexity reduction from unpurified plant DNA using PCR, and demonstrated the feasibility of expeditious genetic analysis across various crop species.