Crassostrea virginica DNA Sequencing and Analysis

Natalie Warner ,
1 Adjoa Cofie ,
Idongesit2 Joshua Uffen ,
2 Anzalone ,
2,3 Josue Mass ,
2

Tyler Carrington2, and Tara Harmer Luke1,2

1Biochemistry and Molecular Biology Program, 2Biology Program, 3Physics Program

School of Natural Sciences and Mathematics, Stockton University
Galloway, NJ

Abstract Methods
Crassostrea virginica, the Eastern Oyster is both an economically and
● Samples were collected in the Mullica River Great
ecologically significant inhabitant of our local coastal ecosystem. Once
widespread, this species has declined over the years, and local oyster fisheries Bay Estuary in New Jersey in September of 2017.
restoration projects are currently underway in this region. Using genetic
material collected from oysters of the Mullica River Great Bay Estuary in New
Jersey, the research experiment aimed to characterize genetic variability
among various oyster populations. DNA was isolated from oyster samples,
amplified via polymerase chain reaction (PCR) and sequenced. The DNA
● Extracted genomic DNA from oyster samples using
Figure 4: DNA sequence alignment of oysters samples sequenced and related species.
sequences collected from samples were compared to known sequence via
BLAST, DNA sequence alignments were constructed, and a phylogenetic tree Qiagen DNeasy Blood and Tissue Kit (Qiagen)
was inferred. This confirmed the identity of the species, as the DNA matched
known Crassostrea virginica sequences. The experiment provides preliminary The DNA sequence alignment shows 12 oyster samples
DNA sequencing data that will allow this project to expand in the future to sequenced with related organisms that were compared
include genetically identifying subpopulations of oysters by looking at more
specific variable regions of DNA.
● Conducted PCR reactions with NS1 primer, Euk516r with one additional out group. The 12 oyster samples
sequenced match that of Crassostrea virginica. Most of
primer, master mix, template, and free water
Introduction the related species match or are very close to matching
nuclease. Crassostrea virginica. The out group: Lingula reevii has
Crassostrea virginica is a salt-water bivalve that lives in many more differences than Crassostrea virginica.
marine and brackish habitats. Locally it is known as the
Eastern oyster, but it also known as the Atlantic oyster,
Virginia oyster, American oyster, or Wellfleet oyster. It is ● SV Gel and PCR Clean-up reaction with gel run.
native to the east coast of North America, and has been
(Qiagen)
harvested for at least 3,000 years. The oyster fisheries
in New Jersey were economically important for many
years, but stocks have declined significantly, due to
harmful fishing techniques, over harvesting, water ● Sequenced PCR products using BigDye Terminator
pollution, and disease.
v3.1 Cycle Sequencing Kit (Invitrogen) and run on an

Applied Biosystems 3500 Series Genetic Analyzer

(Thermo Fisher)

● Once sequence was obtained, BLAST search was Figure 5: Phylogenetic tree of oysters samples sequenced and related species.

performed. The phylogenetic tree shows the relatedness between

the oyster samples sequenced, the related species, and
the out group. The oyster samples sequenced are
directly related to Crassostrea virginica, which they
indeed are Crassostrea virginica. Ostrea chilensis and
Figure 1: Crassostrea virginica
Ostrea circumpicta are closer in relativity than are
Stockton University has been working on a local Ostrea denselamellosa, Saccostrea glomerata, and
oyster restoration project, focusing in part on the Mullica Saccostrea kegaki. Lingula reevii, the outgroup, is less
River Great Bay region, which is currently the only self- closely related than the other species.
sustaining fishable oyster population in New Jersey’s
coastal bays. This is part of a larger project that aims to Conclusion
rebuild and restore local oyster populations. More than All of the DNA samples that were sequenced matched
150,000 juvenile oysters have been seeded atTuckerton known Crassostrea virginica sequences. This is the
Reef, the first-ever bottom oyster restoration site in the initial step of a project that, when refined, will be able to
Figure 3: BLAST search results and identity to Crassostrea virginica.
Southern Barnegat watershed. aid Stockton’s oyster restoration project by allowing
researchers to determine local variation within oyster
Results populations. This experiment has helped to gathered
preliminary data which will be used in the coming
The oysters from the Mullica River Great Bay Estuary
semesters to hone in on more specific genes with
that we sequenced matched the Crassostrea virginica.
greater genetic variability.
Sequence with 99% to 100% identity. Other organisms
used in analysis includeCrassostrea angulata,
●
References
http://www.seacoreseafood.com/product/chebooktoosk-oysters
Crassostrea ariakensis, Crassostrea gigas, Crassostrea ● Qiagen DNeasy Blood and Tissue Kit https://www.qiagen.com/us/shop/sample-technologies/dna/genomic-
dna/dneasy-blood-and-tissue-kit/#resources
nippona, Ostrea chilensis, Ostrea circumpicta, Ostrea ● https://www.google.com/maps/place/Stockton+University+Marine+Field+Station/@39.5420331,-
74.446066,14337m/data=!3m1!1e3!4m5!3m4!1s0x89c0e3afcff14b13:0x4e94318eb6e4d818!8m2!3d39.5349108!4d-
74.4635658!5m1!1e4
denselamellosa, Saccostrea glomerata, Saccostrea ● BigDye Terminator v3.1 Cycle Sequencing Kit
https://www.thermofisher.com/order/catalog/product/4337455
kegaki, and Lingula reevii. ● Applied Biosystems 3500 Series Genetic Analyzer (Thermo Fisher)
Figure 2: Mullica River Great Bay Estuary, site of oyster collection https://www.thermofisher.com/us/en/home/industrial/forensics/human-identification/forensic-dna-analysis/dna-analysis/3500-
series-genetic-analyzer.html