This study explored the mitochondrial genome of the mollusk Donax variabilis using undergraduate researchers. The researchers successfully sequenced portions of three mitochondrial genes (CO1, COB, 16S) and used these to propose a mitochondrial genome map and analyze phylogenetic relationships. Future work will focus on developing an undergraduate research course to fully sequence the mitochondrial genome and add the data to public databases. This pedagogical approach provides hands-on research experience while advancing scientific knowledge of mollusk genomics.

1. Exploring the Mitochondrial Genome of Donax variabilis: A Pedagogical Approach
Clark, A., Reidy J. and Chung H.-M.
Dept. Biology, University of West Florida, Pensacola, United States
Acknowledgements
This work is supported by a faculty catalyst award to Dr. Chung, as well as an undergraduate research award from the Hal Marcus College of
Science Engineering and Health at the University of West Florida. We also thank Dr. Cavnar’s lab for providing Donax variabilis mitochondrial
isolate.
References
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Background
Donax variabilis, commonly named Coquina, is a phenotypically diverse mollusk
found along the intertidal zone of the Southeastern Coast of the United States.
Despite their prevalence, and the ability of Donax variabilis act as a bioindicator of
various pollutants such as hydrocarbons [1,2] , very little genomic inquiry has been
performed on this species to date. This provides a unique opportunity to incorporate
wet-lab and bioinformatics techniques into a course based undergraduate research
project that results in novel gene discoveries and, advances scientific understanding
of mollusk phylogenetics.
Goals
The long-term goal of this research is to
pedagogically explore the mitochondrial
genomics of Donax variabilis, identify
phylogenetic relationships, create a suitable
course based protocol to facilitate future
undergraduate research in biological sciences,
and construct an annotated mitochondrial
genome map.
Discussion
Successful amplification and sequencing of novel Coquina sequences (fig. A) of
cytochrome oxidase C subunit 1 (CO1), cytochrome oxidase B (COB), and 16S
ribosomal RNA (16S), has provided the basis for construction of a proposed
mitochondrial genome map (fig. B) as well as a phylogenetic analysis (fig. C). Future
resources into this project will be focused on developing and executing a course
based undergraduate research project to include all experimental design, wet-lab,
and bioinfomatics practices described . All course based results will be assimilated
into reference databases including Bar Code of Life and National Center for
Biotechnology Information.
Experimental Design Results
Figure A: Expanded view of genes with sequences obtained (dark orange), along with designed primers for each gene. A
total of 6 gene sequences were obtained out of 30 designed primer sets. Primers designed using NCBI primer blast [3].
Figure B: Hypothesized gene synteny based on obtained sequences depicted in Figure A. Synteny was determined with
Synthetic Biology Open Language Platforms (SBOL) [4], such as Benchling 2016 platform, and Serial Cloner 2.1.
Figure C: Phylogenetic relationships based on CO1 gene sequences of closely related species. Distance represents number
amino acid changes per number of CO1 amino acids. Phylogenetic tree designed using Mega 7 [5,6].
Sanger Sequencing
Process the exact order of the
nucleoKdes within a gene
fragment
Mul.-Alignment
Primer pairs are
designed to match to the
desired segment of DNA
Sample Collec.on
Coquina were
collected at
Pensacola Beach
DNA Extrac.on
Lysis of Cells and
mitochondria to
release DNA.
Mitochondrial Isola.on
SeparaKng mtDNA from
gDNA.
Polymerase Chain Reac.on
Amplify a target gene several
orders of magnitude by primers
Data Analysis
Sequences went through NucleoKde BLAST
to check alignment with other sequences within Databases
Primer Design
Primer pairs are
designed to match to the
desired segment of DNA
Build Consensus
Use base pair most
called
CO1
ND4
ND3
ND4L
ND1
ND5
COB
CO2
12S
NCR
ND6
16S
ATP6
CO3
ND2
Figure A
Figure B
Figure C