Mitochondrial metagenomics (MtMG) as a critical step towards accurate and effective diagnostics of plant-parasitic nematode communities

The main goal of this project is to establish a well-validated high-throughput protocol for nematode diagnostics that provides accurate species and subspecies resolution, is broad in its taxonomic coverage, is expedient relative to current low-throughput approaches, and reflects the entire genotypic composition of the entire plant-parasitic nematode community.We propose to address this goal via 3 objectives:Improve diagnostic capacity for plant-parasitic nematode species identification by developing, testing, and implementing the nematode mitochondrial metagenomics (Nema-mtMG) protocol.Develop a curated in-house nematode mitochondrial genomes database (Nema-mtDB) that includes all mitochondrial protein coding genes necessary to identify species that cannot be resolved with a single gene.Develop educational tools and workshops to facilitate training and adoption of the Nema-mtMG for plant-parasitic nematode diagnostics in a wide array of settings including academic research, biosecurity monitoring, and extension work.Objective 1. To ensure the accuracy and efficiency of the Nema-mtMG protocol, our analyses will proceed from known assemblages of nematode pest species (by assembling artificial plant-parasitic nematode communities from species maintained in cultures) to communities of increasing complexity in their species composition but involving known and unknown taxa of agronomically important nematode clades such as Heteroderidae, Longidoridae, Meloidogynidae, and Pratylenchidae. To properly test and validate the use of mtMG with Illumina HiSeq 2x250 bp pair-end sequencing for species-level diagnostics, we will compare it to the traditional approach that relies on the mitochondrial barcodes targeting the COI gene region. Data analyses will result in the development of new bioinformatics tools with error detection capability. Objective 2. Because mtMG sequences are only as good as reference databases that allow for annotation, we will develop a curated in-house Nema-mtDB that is publicly accessible. First, we will collate sequencing information from officially existing resources (e.g., BOLD, QBOL, Miduri, and NR-NCBI reference databases) and private collections of taxonomist colleagues and collaborators into a COI reference database compatible with HTS data processing and analysis. Importantly, we will extend beyond the COI database to include full mitochondrial genomes emphasizing all protein coding genes. To achieve this, we will apply HTS shotgun sequencing of mitochondrial genomes from individual plant-parasitic nematode species. Initially, their identity will be confirmed by traditional DNA tools and morphological analysis. The genomes will be assembled, with all protein coding genes annotated and assembled in the format necessary for HTS data analyses.Objective 3. To encourage the implementation of the Nema-mtMG we will produce protocol documents detailing all the steps of data handling, sample processing, HTS data generation, bioinformatic processing and reporting of results. We will produce presentations and webinars for online teaching, present seminars, and organize face-to-face workshops. These educational and outreach activities will be available to research laboratories, governmental agencies, and the broader scientific community. A linkage with similar efforts promoted by the BIOSCAN project of the International Barcode of Life members will hasten the development of the Nema-mtMG-based diagnostics.