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

Abstract

Phomopsis 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. 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.