Date

Fall 2022

Document Type

Master's Thesis (Open Access)

Degree Name

Master of Science (M.S.)

Department

Applied Environmental Science

Abstract

The collection of feces from awake or anesthetized individuals is often less invasive, less time-consuming for samplers, requires fewer sampling supplies, and is more scalable than many traditional techniques when studying living organisms. To improve best practices, we investigate DNA metabarcoding as a technique for producing diet information from juvenile rainbow trout/steelhead (Oncorhynchus mykiss) feces. DNA metabarcoding may be more sensitive for prey detection and return higher taxonomic resolution for characterizing the diverse diet of this special status species. We tested universal primer pairs targeting short segments of the cytochrome c oxidase I region of the mitochondrial genome. Non-barcoded candidate primers were tested on DNA from individual prey through PCR trials before being applied in an amplicon sequencing pipeline to analyze artificially constructed mock communities of prey as well as juvenile O. mykiss GI tract samples. A primer-like DNA blocking oligo was designed and tested for its functionality as a PCR-additive to improve assay efficiency by limiting non-specific binding of universal primers to predator DNA. The best performing primer set amplified all 16 prey orders (100%) and 39 of 40 prey families (97%) in PCR trials, indicating high universality among expected prey taxa. Metabarcode sequencing of the mock community recovered 14 of 15 prey orders (93%) and 25 of 37 families (68%). Blocking oligo treatment resulted in near complete inhibition of predator DNA amplification but reduced total amplicon yield. O. mykiss stomach and intestinal contents

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