This study analyzed the genetic diversity of two spotted salamander populations that breed in different vernal pools near Gordon College. DNA was extracted from egg masses and adult toe clippings using phenol-chloroform purification and ethanol precipitation. An optimized amplified fragment length polymorphism (AFLP) analysis revealed higher genetic diversity and three times as many egg masses in one population compared to the other, suggesting a correlation between genetic diversity and breeding success. This molecular approach provides insights into factors influencing salamander breeding patterns.

1. Molecular Analysis of Spotted Salamander Populations: Amplified
Fragment Length Polymorphism and Genetic Diversity’s Correlation to
Breeding Success
Ken Hallenbeck, Rachel Keller, Kimberly Spaulding, Seth Gerard and Dorothy Boorse
http://www.cortland.edu/herp/keys/images/salamand/amacullg.jpg
Gordon College, Wenham, MA http://www.pbase.com/tmurray74/image/96446643
2010
Results Wilson Pond Pine Street
Abstract Methods 11 individuals 11 individuals
Spotted Salamanders (Ambystoma Field Work Amplified Fragment Length Our gel electrophoresis
maculatum) breed in vernal pools during results (Figure 4) were
Polymorphism converted into binary
- 200 BP
the spring months. Females lay fertilized Egg masses were counted in two vernal pools
egg masses on structures within their near Gordon College campus. Each was AFLP is a four-part protocol that ends in gel data groups (0 for band
breeding pool. We have counted these counted twice, and the higher of the two electrophoresis. Figure 2 displays this absence and 1 for band
egg masses in two vernal pools as an values was accepted. process visually. presence) and analyzed
- 145 BP
index of a population’s breeding success with software program
Procedure Lab Time
rate. We have developed protocol for At each pool, eggs from four masses were PopGene. We found
DNA Extraction ~2 hours
molecular analysis of these populations, taken and placed in 70% ethanol for storage that the Wilson pond
DNA Restriction ~2 hours
and found data that is highly suggestive until DNA could be extracted. population had higher - 100 BP
Fragment Ligation ~2 hours
of a correlation between breeding genetic diversity in every
success and intra-population genetic Four toe samples were taken from adult Pre-amplification ~ 2 hours measure.
- 50 BP
diversity. DNA extraction was salamanders on 3/22/2010 during mating Primer Pair Addition ~ 30 min Wilson pond also had 3x
successfully performed on developed migration. Selective Amplification ~ 3 hours the number of egg
Figure 4: AFLP gel electrophoresis
embryos and adult toe clippings. This masses. Data is laid out results. Molecular weight ladders
Figure 1: Phenol/Chloroform for comparison below: flank the sample rows.
DNA was used to measure genetic DNA purification step. Aqueous
Gel Electrophoresis ~4 hours
variability of two breeding populations DNA is decanted and TOTAL: ~16 hours
precipitated with absolute Genetic Diversity Egg Masses
with an Amplified Fragment Length
ethanol. Ho Is %P
Polymorphism (AFLP) analysis protocol Figure 2: AFLP protocol from DNA
sample to gel analysis Wilson Pond 0.2141 0.3381 76.18 73
which we optimized for the species. http://www.philsciletters.org/May%202,%202009/Genetic%20fingerprinting.htm
Pine Street 0.1341 0.2048 40.48 23
In the final amplification step two primers
Introduction http://openwetware.org/wiki/Image:DNA_extraction_w_phenol_chloroform.jpg
are attached (see * in Figure 2). These Conclusions
Spotted Salamanders and their breeding
DNA Extraction primers vary in effectiveness between
species. We experimented with eight -Our preliminary results are highly suggestive
patterns have been an object of study at Prior to egg sample extraction we cut away different primer pair combinations and of a correlation between breeding success and
Gordon College for over five years. and discarded outer egg layers. The now- determined Mse-CTA and Eco-AGG were genetic diversity. Understanding this
These studies have attempted to exposed developing embryo was digested the best fit for our sample DNA. relationship could help future efforts to curb
correlate egg mass abundance to many with Proteinase K. Toes were placed Ladder - global decline of amphibians.
- M-CTT
ecological factors. However, the driving directly into solution containing the enzyme. M-CTG -
M-CAT -
-M-CTC
factors behind salamander breeding M-CAG -
- M-CTA
- M-CAC
-We have extracted genomic DNA from eggs.
success remain unknown, and here we We performed a Phenol/Chloroform M-CAA -
This is unprecedented in literature and if
propose a molecular approach to the purification followed by an ethanol Maize
DNA
thoroughly tested could prove to greatly
problem. precipitation. This resulted in a white pellet Control
simplify the field work required for genetic
of pure DNA. Figure 3: Primer pair optimization results. Each row is a unique analysis of salamander populations.
AFLP was introduced in 1995 (Vos et al.) Mse-xxx primer paired with Eco-AGG. To determine the best pair
we counted the number of bands each pair produced. M-CTA had
and has become a standard genetic Early in our experimentation we used 21 distinct bands. -We have found an optimum primer pair for
analysis technique. It is primarily used to samples resuspended in both 20 µL and 50 use in analysis of salamander populations on
measure genetic diversity, fine-scale µL of TE buffer and found that 50 µL After optimizing the primer pair, we ran the North Shore.
population structure, and to test for resulted in the clearest electrophoresis our full experiment with 22 samples, 11
species hybridization (Bonin et al. 2007). bands. from Wilson Pond and 11 from Pine References:
Bonin, A. D. Ehrich and S. Manel. 2007. Statistical analysis of amplified fragment
Street. Gel electrophoresis was done on length polymorphism data: a toolbox for molecular ecologists and evolutionists.
Molecular Ecology 16:3737-3758.
We would like to thank Craig Story for his help with lab protocol details, and the BIO310 class for help in counting egg masses during the spring of 2010.
a 25 cm 6.5% polyacrylamide gel in a Li- Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. Lee, M. Hornes, A. Frijters, J. Pot, J.
Peleman, M. Kuiper and M. Zabeau. 1995. AFLP: A new technique for DNA
COR 4300 DNA Analyzer. fingerprinting. Nuleic Acids Research 23:4407-4414