Chase Paulson and Dr. R. Paul Evans- Microbiology and Molecular Biology

Project Purpose
The purpose of this project is to analyze biomarkers present in cutthroat and cutbow (a mixture of
cutthroat and rainbow trout) trout of the Payson Hatchery compared to typical cutthroat and rainbow
markers to better understand if cutthroat trout are being preserved locally and in the Western United
States.
Project Importance
Fish have been a principal aspect in human’s lives for centuries. They have been hunted, eaten, studied,
and stocked outside their native habitat. These factors along with river convergence have caused
movement of different species of fish. Some fish species can interbreed. We focus on the species
Oncorhynchus clarkii, commonly known as cutthroat trout. Cutthroat trout native to Utah include three
recognized subspecies: the Bonneville (O. c. utah), Yellowstone (O. c. bouvieri), and Colorado River (O.
c. pleuriticus) cutthroat trout (Behnke, 1992). Cutthroats are rare and have suffered a decrease in
numbers in recent years. It is very important for resource management to differentiate between different
subspecies and species of trout to identify, protect and preserve them. By comparing the set biomarkers
we have for cutthroat trout to the samples we collected, we will help stop genetic loss of these trout.
Project Profile Body
Many attempts have been made to preserve these rare cutthroats. Molecular biologist Dr. Paul R Evans
and biologist Dr. Dennis Shiozawa recently produced a phylogeny chart showing the wide distribution
cutthroat trout subspecies. By doing this, they can identify different species habitats, and provide this
information to state conservationists to help preserve the rare cutthroat trout in these areas. In 2012-3,
Evans and Shiozawa presented in cutthroat trout subspecies with next generation sequencing
technology. We are continuing this research aimed at the preservation of these subspecies.
We have decided to further this work of cutthroat trout preservation by collecting fin samples of Bonneville
cutthroat trout and a crossbreed between cutthroat and rainbow trout (Oncorhynchus mykiss) called
“cutbow.” We visited a Fish Hatchery in Payson, UT that claimed to be selling “pure” cutthroats. Upon
visiting the hatchery we collected 20 samples of cutthroat, 20 of cutbow, and 10 unknowns labeled X1-5
and Z1-5. The Hatchery owner’s sons collected the unknown samples (X1-5 and Z1-5) of both trout
species we are studying as to remove any bias. We took the DNA samples to the lab and started DNA
isolation and PCR amplification of the NADH dehydrogenase subunit 2 (ND2) mitochondrial gene. After
DNA isolation, we ran PCR of all DNA samples with the Ala13R and Glu59F primers. Preliminary results
of gel electrophoresis indicated enough DNA is present to continue the experiment. Polymerase chain
reaction was performed using a 38 cycle protocol with 40 seconds at 95° C, one minute at 50° C, and 2
minutes at 72° C (with a three second extension on each cycle). Primers for the mtDNA NADH subunit 2
(ND2) gene were determined in the lab.
Nuclear gene regions will be amplified with appropriate primer pairs to test for heterozygotes. The
nuclear genes are: transferrin (TF), rRNA intervening transcribed spacer II region (ITS2), gonadotropin
(GTH), growth hormone (GH), ikaros (IK), and the insulin-like growth factor (IGF).
Both strands of the PCR product were sequenced using BigDye Termination Cycle sequencing chemistry
as identified on an ABI 377 automated sequencer. Sequence alignment and editing were performed
using Sequencher – 4.8. Heterozygotes (cutbows) will be manually confirmed after initial identification by
algorithm. Diagnostic sequence for Bonneville, Yellowstone, and Colorado River cutthroat trout as well
as Rainbow trout will be scored using the aligned sequences compared to the Oncorhynchus nuclear and
mitochondrial DNA sequence database developed in the lab.
The lab has developed diagnostic markers with mitochondrial genes and nuclear genes (Evans and
Shiozawa, unpublished; Metcalf et al., 2007). These diagnostic mitochondrial and nuclear markers for
rainbow trout, Yellowstone cutthroat trout, Bonneville cutthroat trout in the Bear River drainage, Southern
Yellowstone (Snake River) cutthroat trout, Colorado River cutthroat trout, Greenback cutthroat trout, and
Bonneville cutthroat trout will be employed to assess the genetic status of the hatchery samples. From
these results, we will draw a conclusion stating if the trout we tested are a pure Bonneville subspecies of
cutthroat trout common to this region or merely cutbows.

Anticipated Academic Outcome
Dr. Evans and I are planning on giving all results to the owner of Hatchery and the State of Utah. We will
present at the Annual Meeting Western Division of the American Fishery Society in Colorado Springs
March 2017.
Qualifications
I am qualified for this project because of my educational background and my interest in phylogeny of
subspecies of cutthroat trout. I have been able to learn necessary techniques in my molecular biology
classes and labs to carry out this experiment. Furthermore, I have learned to be very tedious to avoid
redoing portions of the experiment due to major errors. I have sufficient background in knowledge of
cutthroat trout from fishing in my childhood and studying them. Also, Dr. Evans has helped me become
trained to work on samples of DNA isolation, using the PCR machine and constructing an agarose gel.
He has worked on Sub speciation of cutthroat trout for over a decade, published multiple papers on this
subject and presented at conferences. Dr. Evans is more than qualified to mentor me on this project
because of his multiple degrees and experience here at BYU and at other universities along with
consistent lab work on this particular subject. Dr. Evans meets with me whenever I need and wants me
to succeed in this research, while learning to apply what I have learned to the lab. Dr. Evans is great at
not giving me an answer, but he makes me think and analyze the problem and how to solve it.
Project Timetable
The project will be completed December 18th, 2016, because we want to relay the results to the owner of
the Hatchery and the State Fish Conservation Department as soon as possible. We have recorded all
methods and pictures of results thus far and will continue to do so until we complete our project.
Scholarly Sources
Metcalf JW, Pritchard VL, Silvestri SM, Jenkins JB, Wood JS, Cowley DE, Evans RP, Shiozawa DK,
Martin AP. Across the great divide: Genetic forensics reveals misidentification of endangered cutthroat
trout populations. Molecular Ecology. 2007 Nov;16(21):4445-4454.
Allendorf, Fred W., and Robb F. Leary. “Conservation and Distribution of Genetic Variation in a Polytypic
Species, the Cutthroat Trout.” Conservation Biology 2.2 (1988): 170-84. Web.