THE MOST EFFECTIVE STRATEGY TO MANAGEMENT OF ANY PEST IS THE KNOWLEDGE OF PEST IDENTITY. IN NEMATODE DIAGNOSTICS, TWO CRITICAL GAPS INCLUDE: 1. THE INABILITY TO ADOPT SPECIES-RESOLVING MITOCHONDRIAL GENE MARKERS FOR HIGH-THROUGHPUT SEQUENCING (HTS) APPROACHES (ALL SPECIES EN MASS RATHER THAN SINGLE INDIVIDUALS ONE AT A TIME), AND 2. THE INABILITY TO ID SEQUENCED INDIVIDUALS DUE TO LIMITED MITOCHONDRIAL DATABASES. THE MAIN GOAL OF THIS PROJECT IS TO ESTABLISH AN HTS PROTOCOL FOR NEMATODE DIAGNOSTICS THAT PROVIDES ACCURATE SPECIES AND SUBSPECIES RESOLUTION, IS BROAD IN ITS TAXONOMIC COVERAGE, IS EXPEDIENT RELATIVE TO LOW-THROUGHPUT APPROACHES, AND REFLECTS THE ENTIRE GENOTYPIC COMPOSITION OF THE PLANT-PARASITIC NEMATODE COMMUNITY.IN THIS PROJECT WE PROPOSE TO: 1. IMPROVE DIAGNOSTIC CAPACITY FOR PLANT-PARASITIC NEMATODE SPECIES IDENTIFICATION BY DEVELOPING, TESTING, AND IMPLEMENTING A MULTI-LOCUS SYSTEM VIA SHOTGUN SEQUENCING OF FULL MITOCHONDRIAL GENOMES (NEMA-MTMG); 2. DEVELOP A CURATED FULL MITOCHONDRIAL GENOMES DATABSE (NEMA-MTDB) WITH ALL MITOCHONDRIAL PROTEIN CODING GENES NECESSARY TO IDENTIFY SPECIES THAT CANNOT BE RESOLVED WITH A SINGLE GENE; 3. DEVELOP EDUCATIONAL TOOLS/WORKSHOPS TO FACILITATE TRAINING AND ADOPTION OF THE NEMA-MTMG FOR PLANT-PARASITIC NEMATODE DIAGNOSTICS.TO VALIDATE NEMA-MTMG, OUR ANALYSES WILL PROGRESS FROM KNOWN ASSEMBLAGES OF NEMATODE PEST SPECIES TO INCREASINGLY UNKNOWN IN THEIR SPECIES COMPOSITION BUT INVOLVING TAXA OF AGRONOMICALLY IMPORTANT NEMATODE CLADES. FOR NEMA-MTDB CONSTRUCTION, WE WILL START WITH ~300 NEMATODE SPECIES FROM CULTURES AND FOLLOW UP WITH SPECIES FROM SOIL SAMPLES. FOR OBJECTIVES 1 AND 2, WE WILL APPLY OUR RECENT PROTOCOLS FOR MITOCHONDRIAL DNA ENRICHMENT, ILLUMINA HISEQ2500 SEQUENCING, AND PHYLOGENETIC-BASED BIOINFORMATICS.WITH THE COMMITMENT OF THE USDA APHIS TO SAFEGUARD AMERICAN AGRICULTURE (GOAL 2 OF THE STRATEGIC PLAN 2019-2023), OUR PROJECT IS DIRECTLY RELEVANT TO OBJECTIVES 2.1, 2.2 , AND 2.6 THAT STRESS THE ROLE OF INNOVATIVE MOLECULAR DIAGNOSTICS.