DNA Fingerprinting of Saginaw Bay Walleye and Spawning Populations Jennifer Richter, Gracie Lefler, Taylor Shivers, Taylor Anderson, Rachel Laskowski, Ben Cooper, Craig Coopersmith, and Dr. David Stanton Department of , Saginaw Valley State University, 7400 Bay Road, University Center, MI, 48710. 4: DNA Fingerprinting
1: Introduction •
There is a large population of walleye in Saginaw Bay.
• Walleye serve as top predators and are critical to the ecosystem. • Walleye are also economically important both for sport and commercial fishing. • The population is heavily managed and has been extensively stocked.
5: Results
• A technique used by forensic scientists to identify individuals based on highly polymorphic loci • Specific primers are used to amplify these regions by PCR (Polymerase Chain Reaction) • Fragments are sized by capillary electrophoresis in order to determine a multi locus genotype • Results can then be compared between two individuals or among a large database
1. For Svi L6, eight to eleven alleles were observed in each population (average 9.4). 2. Heterozygosities were generally high (average .79) and usually near expected values (average .76). 3. One allele was found in the Rifle River and Saginaw Bay and was not found in any other spawning population.
• We use DNA fingerprinting in order to access genetic diversity, population substructure and spawning site fidelity.
7: Estimating Amounts of Antimicrobial Compounds Present in LRRFF
Alleles and Heterozygosity (Svi L6)
Alleles
A
B1
B2
C
D
E
F
10
10
8
11
10
9
8
2: Materials and Methods • Extract total genomic DNA (DNeasy kit, Qiagen). • Amplify by PCR using dye labeled primers to VNTR regions (IDT). • Perform capillary electrophoresis to determine fragment sizes (CEQ 8000, BC). • Determine genotypes, calculate allele frequencies, heterozygosity, genetic distances (D) and population substructure (FST).
PCR machine DNeasy Kit Micro centrifuge Vortex machine 1.5 mL Eppendorf tubes Micro pipettes CEQ 8000 PCR tubes Polypropylene pestles
Collection Sites
F D C
B
A
Ho
.80
.83
.65
.90
.82
.74 .76
He
.73
.81
.73
.76
.78
.77 .75
4. Most populations were not in Hardy Alleles
E
Range (bp)
Svi 2
188 – 222
13
except the Saginaw Bay summer
Svi 4
106 – 122
8
population.
Svi 6
126 – 168
16
Svi 7
140 – 178
13
(average.06). Bay populations were most
Svi 18
114 – 126
7
similar to the Kawkawlin population.
Svi 33
82 – 106
13
6. Population substructure was low (FST = 0.01).
Svi L6
104 – 140
16
Weinberg equilibrium (P < 0.05),
DNA Fingerprint (Svi L6) 112
Date
A Tittabawassee River
4/4/13
N 48
B Saginaw Bay
7/15/15
72
C Kawkawlin River
4/3/15
48
D Saginaw Bay
1/14/14
33
E Shiawassee River
4/10/14
48
F Rifle River
3/18/16
48
140
B1 B2
.06
.06
C
.02
.05
B2
C
D
Batch 1 (8 oz) Batch 2 (1 kg)
Batch 3 (8 oz)
Isolated and purified salicylic acid (g/50 mL)
0.31
0.12
0.19
Didecyldimethylammonium salts as chloride (mg/50 mL)
8
16
10
• Samples of salicylic acid and didecyldimethylammonium salt isolated from LRRFF were submitted for carbon dating • Based on the amount of 14C present, the compounds were dated to 52000 ± 2900 and 21140 ± 100 years old, respectively • This strongly suggests that both compounds are derived from petroleum based precursors and that neither compound is a product of a recent fermentation of the plant material
9: Summary
Genetic Distances (Svi L6) B1
2
8: Determining Origins of Antimicrobial Compounds
5. Genetic distances were low
A .10
Batch #
1
Table 3: Amount of salicylic acid and didecyldimethylammonium salts obtained from commercial LRRFF from two companies
Locus
Molecular Ecology 12:1689-1702 (2003)
Location
• 50 mL aliquots were drawn from three samples of commercial LRRFF to estimate the amounts of salicylic acid and didecyldimethyl ammonium salt present • Results are summarized in Table 3 Sample
Stizostedion vitreum
• • • • • • • • •
• The solid residue obtained from concentrating the base-washed EtOAc layer was examined by 1H NMR and was consistent with didecyldimethylammonium bromide with a salicylate impurity • The salicylate impurity was removed by dissolving the solid in water and passing it thorough an anion exchange resin AG 1-X8 (Cl- form) • The solid residue obtained from concentrating this mixture gave 1H 13C and NMR spectra that were consistent with didecyldimethylammonium bromide without any trace of salicylate (Figure 5) • Didecyldimethylammonium bromide has been observed to inhibit growth of Gram-positive bacteria6 and this was confirmed on S. aureus with the solid residue and a commercially-available sample
E
• LRRFF and its extracts inhibit the growth of both Gram-negative and Gram-positive bacteria • Salicylic acid and didecyldimethylammonium salt are responsible for this activity, respectively • No antimicrobial peptides were detected in any sample
10: Acknowledgements
.04
D
.04
.09
.07
.03
E
.07
.07
.12
.08
.05
F
.05
.05
.06
.06
.06
.03
• University of Alberta • NSERC • Alberta Innovates Health Solutions • CIHR (postdoctoral fellowship) • Canada Research Chair in • Saginaw Valley State University Bioorganic and Medicinal (travel grant) Chemistry • Dr. Randy Whittal (U of A)
11: References 1. Li et al., J, Agric. Food Chem. 2015, 63, 3053-3058 and references within 2. Kim et al. J. Food Prot. 2008, 71, 325-332 2. Lee et al., US Patent 2010/0129305 A1, May 27. 2010 3. Duthie and Wood. Food Funct. 2011, 2, 515-520 4. Gershon and Parmegiani, Appl. Microbiol., 1962, 10, 348-353 5. Ioannou et al. Antimicrob. Agents Chemother. 2007, 51, 296-306