POSTERS: Molecular plant-microbe interactions
The rps10 gene as a new barcode locus for oomycetes and its utility in metagenomics studies of environmental samples
Frank Martin - USDA ARS. Felipe Albornoz- Oregon State University, Valerie Fieland- Oregon State University, Niklaus Grunwald- USDA ARS, Hai Nguyen- Agriculture & Agri-Food Canada, Zachary Foster- Oregon State University, Jo Anne Crouch- USDA ARS, Brett Tyler- Oregon State Univer
The current barcode loci for identification of oomycetes are a portion of the mitochondrially encoded cox1 gene and the nuclear encoded ITS region of the rDNA. Both loci have the advantage of being high copy number, thereby providing a greater sensitivity when generating templates from environmental samples. However, both loci also have the disadvantage that amplification primers are not specific, leading to background amplification. Comparative genomics of mitochondrial genomes from a wide range of oomycetes has identified a unique gene order of conserved tRNA genes flanking the mitochondrially encoded rps10 gene that is not present in plants or the Eumycota. Highly specific rps10 primers for oomycetes were developed that are useful for template amplification from environmental samples and obligate pathogens in host tissue. Studies to date have confirmed the ability of this locus to correctly identify organisms to a species level with evaluation of additional taxa in progress. Primers were modified to generate an amplicon suitable for MiSeq sequencing. Amplification of a mock library of DNA from known cultures and diverse environmental samples provided proof-of-concept that only oomycetes were amplified, improved identification over ITS and enabled the description of complex microbial communities. A draft reference database and website to query and curate information for metabarcoding projects are available for feedback from the oomycete community.