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

Abstract

Significance and Impact of the Study: 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. AbstractPhomopsis longicolla (Hobbs) causes Phomopsis seed decay and stem lesions in soybean (Glycine max). In this study, a novel, high-throughput adaptation of RAD-seq termed MoNSTR-seq (Mutation analysis via Next-generation DNA Sequencing of T-DNA Regions) was developed to determine the genomic location of T-DNA insertions in P. longicolla mutants. Insertional mutants were created via Agrobacterium tumefaciens-mediated transformation, and one mutant, strain PL343, was further investigated due to impaired stem lesion formation. Mutation analysis via Next-generation DNA Sequencing of T-DNA Regions, in which DNA libraries are created with two distinct restriction enzymes and customized adapters to simultaneously enrich both T-DNA insertion borders, was developed to characterize the genomic lesion in strain PL343. MoNSTR-seq successfully identified a T-DNA insertion in the predicted promoter region of a gene encoding a cellobiose dehydrogenase (CDH1), and the position of the T-DNA insertion in strain PL343 was confirmed by Sanger sequencing. Thus, MoNSTR-seq represents an effective tool for molecular genetics in P. longicolla, and is readily adaptable for use in diverse fungal species.