Optimization of DNA Extraction Methods for Sequencing of the NADH Dehydrogenase Subunit 2 (ND2) Gene in Rainbow Trout (\u3cem\u3eOncorhynchus mykiss\u3c/em\u3e)

Morphological, behavioral, and genetic variation due to broad geographic ranges is common in salmonid fishes. This variation can be attributed to the isolation of populations from natural phenomenon or human alteration to habitats, which affect gene flow and genetic drift. These changes may play a role in intraspecific genetic variation, potentially isolating them into separate subspecies or locally adapted populations. Rainbow Trout (Oncorhynchus mykiss) are one of the most widely distributed fish species in western North America but no study has identified the main evolutionary lineages from across its range. Identifying and comparing distinct evolutionary lineages can be achieved by comparing sequence data from mitochondrial genes but requires extraction of archived tissue samples of rainbow trout and amplification of selected genes. In this study, we compare different DNA extraction methods for rainbow trout tissue samples, some of which are more than 25 years old. DNA was extracted from tissue samples following a 5 or 15% Chelex method, or by following Zymo Research extraction kit instructions. Following extractions, polymerase chain reaction (PCR) for the highly conserved NADH dehydrogenase subunit 2 mtDNA gene (ND2) was completed using a BIO RAD thermal cycler and a gel electrophoresis is done to determine whether the ND2 gene was successfully amplified. Photographs of the agarose gels were taken using a ChemiDoc imaging system, and samples showing a band at 1050 base pairs are sent to the Molecular Research Core Facility at Idaho State University for sequencing. The genetic sequences are then compared and used to create a phylogeny and distribution for Rainbow trout subspecies. Sequencing of individual specimen and comparison across the species range will aid in the development of management and conservation strategies for rainbow trout across the species range