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THE UNDERGRADUATE RESEARCH ACADEMY
OFFICE OF UNDERGRADUATE ASSESSMENT & PROGRAM REVIEW
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STUDENT
Dustin Hancks____________________________________________________
MENTOR
Dr. David Duvernell_______________________________________________
PROJECT TITLE
Single Nucleotide Polymorphisms within Fundulus notatus and
Fundulus olivaceus
Abstract: The abstract is a brief but comprehensive summary of the contents of the
proposal in plain language, approximately 150 words. Readers receive their
first impression of the flavor of the topic from this abstract. The information in
the abstract needs to be concise, well organized, self contained, and
understandable to persons outside the discipline.
Fundulus olivaceus and Fundulus notatus are two species of killifish that can be found
widely distributed in overlapping regions within the Midwest and the Gulf Coast. They
are rarely found together in their habitats, but sites have been discovered where both
species are present. Controlled laboratory crosses have produced fertile, viable hybrids of
the two species although it is thought that hybridization is rare in nature. Discrimination
of hybrid and non-hybrid individuals based on physical features has proven difficult. To
study hybridization between these species molecular genetics techniques are needed to
identify diagnostic differences at the DNA level. The objective of this project is to find
DNA sequences that contain variation, specifically single nucleotide polymorphisms in F.
olivaceus and F. notatatus genomes, which will be used to distinguish them
diagnostically and identify hybrids.
Upon submitting this proposal, I verify that this writing is my own and pledge to fulfill all of the
expectations of the Undergraduate Research Academy to the best of my abilities. I understand that failure
to do so may result in return of fellowship money to the University and forfeiture of academic credit and
honors recognition.
Signature of the Student
I am able, willing, and committed to providing the necessary facilities and to take the time to mentor this
student during this project. I verify that this student is capable of undertaking this proposed project.
Signature of the Faculty Mentor
This project is within the mission and scope of this department, and the department fully supports the
faculty mentor and student during this venture.
Signature of the Department Chairperson
I testify that all necessary research protocols (human, animal, toxic waste) have been fulfilled, and I
support this proposed faculty-student scholarly activity as within the mission of the College/School.
Signature of the Dean of the College/School
* See Appendix A for glossary, page 5.
Research Proposal
I. Introduction and Significance
A biological species is defined as an “actually or potentially interbreeding group of
populations”; any taxa that can produce viable and fertile F2 generations or backcrosses
are regarded as being of the same species (Mayr, 1942, Barton and Hewitt, 1989). An
important question in ecology and evolutionary biology is what sorts of characteristics
define species and what mechanisms exist to keep closely related species distinct?
Hybrid zones are places where the reproductive barriers between species have broken
down. Hybrid zones give us an opportunity to study the mechanisms of speciation as it
occurs within nature. Analyses of these zones give us insight to how reproductive
isolation, selection, and gene flow between divergent species occurs. By studying
hybrids within nature we are able to acquire a better understanding of how these
mechanisms work.
Within the drainage of the Mississippi River and the coastal drainages of the Gulf of
Mexico, live two species of killifish; Fundulus olivaceus and Fundulus notatus. These
fish are virtually identical to the eye, distinguishable only on the presence or absence of
spots (Thomerson, 1966). On the other hand, their genomes exhibit distinctive
differences, with F. notatus having 40 chromosomes and F. olivaceus having 48 (Setzer,
1970). These fish have been known to hybridize with one another under controlled
breeding experiments in the lab. Furthermore, hybrid zones have been found within
nature, though they are uncommon. These fish are unique species, but are able to
interbreed with each other.
Genetic analysis can be performed to find distinguishing features within the genomes of
F. olivaceus’ and F. notatus’, respectively, in order to effectively distinguish the two
species from one another. Divergent sequences within the DNA can be used to
distinguish the two species and identify hybrid individuals. We want to look for genetic
markers or landmarks of the genome that are unique to each species. Using
representative samples of each species, from isolated populations, we will sequence the
DNA in order to look for these markers, diagnostic species differences identifiable at the
DNA sequence level, at several nuclear gene loci.
II. Literature Review
The species of Fundulus olivaceus and Fundulus notatus are two of the most widely
distributed species of the Cyprinodontid genus of Fundulus. They inhabit distinctly
different habitats; F. notatus can be found to be predominant in areas that are flood plain
sloughs and low-gradient creeks, while F. olivaceus more often occupies fast-flowing
clear, gravelly upland streams (Braasch and Smith, 1965). Each species has been found
present in the other’s habitat when the other species is absent, though it is rare to find the
two species occupying the same habitat together (a condition called syntopy)
(Thomerson, 1966).
The reproductive isolation of these species appears to be due to slight differences within
their ecological and species mating preferences (Thomerson, 1966). F. olivaceus and F.
notatus are very closely related phylogentically and are able to produce viable, fertile
hybrid offspring despite their chromosome number differences (Setzer,1970). The
reproductive isolation in nature could be due to lack of fitness of the hybrid individuals.
This appears not to be the case, because the hybrids found within nature and in vitro are
fully functional. These hybrids are able to produce offspring, along with being able to
backcross with individuals belonging to either of the parental species (Thomerson, 1967).
Hybrid zones exist where the two species occur together in parts of Illinois and Missouri,
in some cases with documentation of up to sixty years, yet they retain their specific
identity (Braasch and Smith, 1965), (Thomerson, 1967). Mitochondrial DNA and
microsatellite analysis has been performed recently in order to determine the frequency of
past hybridizations within nature (Duvernell, Schaefer and Ravanelli, In Review). While
these genetic markers can be used to distinguish the two species, their use in identifying
hybrid offspring is not completely satisfactory. Additional, diagnostic genetic markers
are needed for a thorough genetic dissection of hybrid zones
III. Objectives and Hypothesis
The hypothesis to be tested is single-nucleotide polymorphisms that differentiate F.
olivaceus from F. notatus can be identified and used to distinguish each of these species
and their hybrid offspring. These polymorphisms, when found, will allow the
construction of diagnostic tests that will be able to identify specifically F. olivaceus, F.
notatus, or F1 hybrid individuals. The testing of the populations for hybridization events
will allow us to better understand mechanisms of reproductive isolation, species-specific
mating, and the nature of speciation. These diagnostic tests will be important for future
studies that deal with these species.
IV. Materials, Methods, and Timeline
The sampling design in this study will include populations of each species that are
isolated from the other species as well as populations from sites where both species are
present. These will include: Gasconade River, a known habitat for F. olivaceus in central
Missouri; Piasa Creek, a known F. notatus habitat in western Illinois; and Brushy Creek,
a known population consisting of both species in southern Illinois. The samples were
previously collected (Duvernell, Schaefer and Ravanelli, In Review) and DNA samples
are available in the lab.
We would like to find specific sites where the nucleotide present is unique to the species.
DNA consists of four nucleotides: Adenine (A), Guanine (G), Cytosine (C), and
Thymine (T) that make up the genetic code. For example, F. olivaceus may have a T,
while at the same spot, F. notatus has a G present. These polymorphisms, variations
within the DNA sequence, will allow us to create diagnostic tests to identify each species
and hybrids at any point within their life cycle.
Gene regions of interest within the DNA will be isolated and amplified by a Polymerase
Chain Reaction (PCR) and then sequenced on the ABI 310 Genetic Analyzer. The DNA
sequences will be aligned and analyzed to identify differences between species using
GeneTools vers. 2.0 (BioTools Inc.). Genes that have been identified as candidate loci
for analysis include: TPI, Surf 3 (Douglas Crawford, pers. comm.), and S7 (Jason Allen,
pers. comm.). We will identify additional gene loci to analyze by searching sequenced
fish genomes available online via GenBank, and designing PCR protocols for isolation of
sequences from F. notatus and F. olivaceus.
Once the sites of polymorphism are found, one of two approaches will be taken in order
to develop diagnostic tests to identify each species and hybrid individuals. The first
method utilizes restriction enzyme digests. A restriction enzyme can be used to
distinguish specific sequences of DNA if sites occur within the recognition site of the
enzyme. The enzyme will cut the DNA if it contains the specific sequence within its
recognition site. An example of a restriction enzyme is EcoR I, which recognizes DNA
sites containing the sequence GAATTC. If the sample sequence is ACGAATTCCG the
site will be recognized and the enzyme will cut the DNA between the G and A sites. On
the other hand, if the DNA sequence is TGGTATTCGC, the enzyme will not recognize
the sequence, and will not cut the DNA. Once digested, the samples will be analyzed by
performing gel electrophoresis on a 2% agarose gel in 1xTAE buffer. The principle
behind electrophoresis is that a molecule with a net electrical charge will move in an
electric field. The sample is placed in the agarose gel that contains a matrix through
which the sample is pulled by the electric field. The rate of migration of the sample is
inversely proportional to its molecular weight. The samples in the gel will be visualized
under UV light. Ethidium bromide, a molecule present in the gel, intercalates between the
DNA base pairs. The UV light excites the ethidium bromide, which fluoresces the pieces
of DNA that may or may not have been cut by the restriction enzyme. Based upon
whether or not the cut occurs, we will be able to identify whether the DNA sequence is
characteristic of one species or the other, or whether both alternative DNA sequences are
present, as expected for hybrid individuals that would contain one sequence from each of
the parent species.
The second method uses PCR to discriminate between sequence variants. This consists
of designing primers, short strands of DNA that recognize specific sequences for DNA
replication. The primers will be designed based upon the DNA sequence analysis of each
species and will target nucleotides that differ between the species. Each sample DNA
will be PCR amplified with two sets of primers, which will be specific for each of the two
species, respectively. The PCR products will be electrophoresed through an agarose gel
and visualized under UV light. Amplification of a DNA product using species-specific
primers will be diagnostic for the presence of DNA diagnostic for that species. Finally,
all of the samples within the collected populations will be screened utilizing the
diagnostic tests constructed in order to validate techniques.
V. TIMELINE
Event
Estimated Time
Identification of gene loci
1 month- September
Amplification of genes of interest (PCR) 1 month- October
Sequencing of genes
1 month- November
Identifications of sites unique to each
species
Development of diagnostic assays
1 month- December
Testing of diagnostic assays
1 month- February
Screening of individuals
1 month- March
Analysis of results
1 month- April
Total time for project
8 months
1 month- January
Bibliography
Barton, N.H and G.M. Hewitt. 1989. Adaptation, speciation and hybrid zones. Nature,
341: 497-502.
Braasch, M.E. and P.W. Smith. 1965. Relationships of the topminnows Fundulus notatus
and Fundulus olivaceus in the upper Mississippi River valley. Copeia 1965:46-53
Duvernell, D.D., J. Schaefer and A.M. Ravanelli. Hybridization and introgression of
nuclear and mitochondrial genes between Fundulus notatus and F. olivaceus in
Southern Illinois. In Review.
Mayr, E. 1942. Systematics and the origin of Species. Columbia University Press: New
York.
Russell, Peter J. 2002. iGENETICS. Benjamin Cummings: Boston.
Setzer, P.Y. 1970. An analysis of a natural hybrid swarm by means of chromosome
morphology. Trans. Amer. Fish. Soc. 1970: 139-146.
Smith, Philip W. 1979. The Fishes of Illinois. University of Illinois Press: Urbana.
Thomerson, J.E. 1966. A comparative biosystematic study of Fundulus notatus and
Fundulus olivaceus (Pisces: Cyprinodontidae). Tulane Stud. Zoology 13:29-47
Thomerson, Jaime E. 1967. Hybrids Between the Cyprodontid Fishes, Fundulus Notatus
and Fundulus Olivaceus in Southern Illinois. Transactions Illinois Academy of
Science. 1967: 375-379.
Appendix A- Glossary
assay- A diagnostic test.
F2 generation- Offspring produced by crossing F1 individuals. Crossing two parental
individuals produces F1 generation.
gel electrophoresis- A process which separates proteins and DNA by pulling the samples
through a gelatinous matrix by using an electric field, separating samples based on
molecular weight.
genome- The sum of an individual’s genetic material within their chromosomes.
hybrid- Offspring produced by the reproductive cross between two species.
in vitro- Latin for in lab.
karyotype- A complete set of an organism’s chromosomes, species-specific. i.e. Home
Sapien 2N=46 chromosomes.
loci- Location of a gene on a chromosome.
microsatellites- Highly repetitive sequences of DNA found that exhibit high variability.
mitochondrial DNA- DNA from the mitochondria organelle (powerhouse of cell),
inherited maternally.
nucleotide- One of the four building blocks of the genetic code, either A,C,T,G.
phylogeny- Evolutionary relationship between species, i.e. humans are closely related to
chimpanzees phylogenetically.
polymerase chain reaction- A method used to replicate defined DNA sequences
selectively and repeatedly from a DNA mixture (Russell, 2002).
polymorphism- A variation within the sequence of DNA, between individuals, and/or
species.
primer- A small segment of DNA with a specific sequence which is used to initiate DNA
replication at a site with the complimentary sequence.
restriction enzyme- Enzymes which identify unique sequences within DNA and cut it at
the recognition site.
syntopic populations- Populations where both species are present.
transcription- The transfer of information from a double-stranded DNA molecule to a
single-stranded RNA molecule.
Budget Justification
The molecular procedures described in the proposed study require the following supplies:
Item
Quantity
Supplier
Price
Ex-Taq DNA Polymerase
250 units
Fisher
$150.00
Custom Primers
4 primers
Fisher
$63.84
Agarose
100 grams
Fisher
$143.92
Tris-Acetate-EDTA buffer, 50X
solution (TAE 50X)
Pipette Tips, 0.5-10 ul
500 mL
Fisher
$32.10
960
Fisher
$49.95
Pipette Tips, 1-200 ul
960
Fisher
$48.85
Pipette Tips, 100-1000 ul
$49.60
Fisher
$49.60
Eppendorf Tubes, 1.5 mL Tubes
1000
Fisher
$35.00
PCR tubes, 0.5 mL
1000
Fisher
$50.00
Restriction Enzymes
2
Fisher
$110.00
Shipping Handling
$66.74
Total
$800.00
Ex-Taq Polymerase is an enzyme that synthesizes the DNA in the PCR reaction.
The agarose and TAE 50X is utilized in the gel electrophoresis procedure. The primers
are needed in order to design and perform the diagnostic tests. The different size pipette
tips will be used to transfer solutions while performing the restriction digests and PCR
reactions. The various size tubes are used in the restriction digests and PCR reactions as
well. The restriction enzymes will be used in the diagnostic testing of the samples. Other
supplies are provided by the mentor’s lab, which include: centrifuge, electrophoresis
apparatus, various size mechanical pipettes, PCR machines, incubator, ABI 310 Genetic
Analyzer, and software for genetic analysis.