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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Mycotoxin Prevention and Applied Microbiology Research » Research » Publications at this Location » Publication #350438
Title: Resolving the Mortierellaceae phylogeny: a test of Multi-Locus Sequence Typing (MLST) and phylogenomic approaches

Author
item VANDE POL, NATALIE - Michigan State University
item STAJICH, JASON - University Of California
item O Donnell, Kerry
item DESIRO, ALESSANDRO - Michigan State University
item BONITO, GREGORY - Michigan State University

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 7/21/2018
Publication Date: 7/21/2018
Citation: Vande Pol, N., Stajich, J., O'Donnell, K., Desiro, A., Bonito, G. 2018. Resolving the Mortierellaceae phylogeny: a test of Multi-Locus Sequence Typing (MLST) and phylogenomic approaches [abstract].

Interpretive Summary:

Technical Abstract: The Mortierellaceae are an ecologically and industrially relevant lineage of early diverging fungi comprised of six genera classified within what is now recognized as phylum Mucoromycota. This polyphyletic family is estimated to contain at least 100 species, the phylogeny of which cannot be fully resolved with ribosomal markers. The ITS region is too divergent to align across the entire lineage and while the 28S region can be aligned across the family, there is poor backbone support for the resulting phylogenetic tree. In this research, we tested the use of high-throughput targeted amplicon sequencing for generating multi-locus sequence data to improve phylogenetic resolution of diverse lineages within the Mortierellaceae. First, we analyzed three de novo sequenced Mortierella genomes with a non-biased bioinformatic pipeline to identify potential non-ribosomal markers and designed PCR primers for multiplexed PCR amplification. We identified 13 loci that performed well across a diverse test panel of Mortierella isolates. We amplified these loci across 330 Mortierellaceae isolates, prepared libraries with an Illumina Nextera kit, and sequenced them on an Illumina MiSeq platform. Our non-biased locus selection successfully identified established phylogenetic markers (i.e., TEF1 and RPB1); however, 7 loci were members of gene families or under selective pressure and therefore inappropriate to use as phylogenetic markers. This multilocus sequence typing (MLST) approach is very robust in extracting phylogenetic information from isolates contaminated by non-target organisms (e.g., Fusarium or Mucorales), an advantage over Sanger sequencing of universal barcodes or genome sequencing. However, we were less successful in resolving cross-contamination by other Mortierellaceae. Low-coverage genomes that were available for 60 of our isolates were analyzed to identify 400 informative markers. These markers were used to build a strongly supported genome-based phylogeny. We are identifying MLST loci within the low coverage genomes to serve as a backbone constraint for MLST phylogenetic analyses. This combination of approaches leverages both the sequencing depth of genomics and the sampling depth of amplicon-based sequencing. A resolved phylogeny and identification of non-ribosomal markers will improve identification and placement of novel species and genotypes as they are isolated, and inform higher taxonomy of this family. We will discuss the resulting phylogeny, informative non-ribosomal loci, our evaluation of the MLST approach, and implications for other lineages and organisms.