Optimization of DNA Extraction and Pcr Methods for Pacific Lamprey Gut Content Analysis

摘要

The Pacific lamprey (Entosphenus tridentatus) is an anadromous, eel-like, vertebrate endemic to the North Pacific. The species is ecologically, culturally and economically important in the Pacific Northwest region of the United States. Despite their intrinsic value however, they are threatened by significant population declines coupled with a paucity of information regarding their ocean phase and basic biology. The goal of this study was to optimize the efficiency of DNA extraction and polymerase chain reaction protocols for Pacific lamprey diet analysis. This will ultimately provide insight into the prey species of pacific lamprey and be used to inform conservation practices in the NE Pacific region. DNA was extracted from 18 Pacific lamprey individuals that were collected as bycatch onboard Pacific hake (Merluccius productus) and pink shrimp fishery (Pandalus jordani) vessels. Whole genomic DNA was extracted using standard Qiagen DNeasy Blood & Tissue Kit protocols; and verified using spectrophotometry. Customized primer design was guided by the MiFish project for detailed metabarcoding of marine fishes and other vertebrates. Our findings revealed an intriguing region of lacking DNA that is unique to lampreys (likely an insertion in bony fishes), and adjacent to the 12S rRNA gene. We leveraged this information to design specialized primers that will aid in distinguishing lamprey DNA from dietary items in future analysis by DNA barcoding and next-generation metabarcoding platforms that scale up to include additional individuals and collection localities.