MoNSTR-seq, a restriction site-associated DNA sequencing technique to characterize Agrobacterium-mediated transfer-DNA insertions in Phomopsis longicolla.

This study describes MoNSTR-seq (Mutation analysis via Next-generation DNA Sequencing of T-DNA Regions), an adaptation of restriction site-associated DNA sequencing (RAD-seq) to identify the position of transfer-DNA (T-DNA) insertions in the genome of Phomopsis longicolla, an important pathogen of soybean. The technique enables high-throughput characterization of mutants generated via Agrobacterium tumefaciens-mediated transformation (ATMT), thus accelerating gene discovery via forward genetics. This technique represents a significant advancement over existing approaches to characterize T-DNA insertions in fungal genomes. With minor modifications, this technique could be easily adapted to taxonomically diverse fungal pathogens and additional mutagenesis cassettes.