Abstract Little is known about the reproductive habits of paddlefish, a threatened

species in Wisconsin and Minnesota. Biologists studying paddlefish spawning grounds find it difficult to differentiate between the eggs of paddlefish and sturgeon based upon appearance alone. Both species are also threatened by poaching, with their eggs sold as caviar. A PCR based test has been developed to amplify a portion of the cytochrome B gene from a single paddlefish or sturgeon egg. The presence of distinct HincII restriction sites in the paddlefish and shovelnose sturgeon PCR products allows for the rapid determination of whether a single egg came from a paddlefish, shovelnose sturgeon or lake sturgeon. These tests will eventually allow fisheries biologists to better monitor and protect paddlefish spawning habitats. This test may also have forensic applications.

Sample Preparation

• Eggs kept on ice until freezing at -80oC

• Single egg mashed with sterile glass rod in 200 ul buffer (20 mM Tris, 20 mM KCl, 0.5% Tween 20, 5% Chelex, 0.2 mg/ml proteinase K)

• Samples incubated 1.5 hours at 55oC

• Samples incubated 8 minutes at 95oC

• Samples centrifuged at 14,000 rpm for 10 minutes

• An aliquot was removed for PCR amplification (1 ul)

Polymerase Chain Reaction (PCR)

Mix together...

• Sample DNA

• dNTPs

• Buffer

• Taq DNA Polymerase

• PCR Primers

CBL1 TGATGAAATTTTGGCTCACT

CBR1 GTGGAAGGCGAAGAATCG

5’

3’

95oC

5’

3’

55oC5’

3’

72oC5’

3’

3’

5’

CBR1

CBL1

Sample size vs. PCR product

After 30 cycles of amplification the primary DNA productwill be the region found between the two primers.

3’5’

Figure 1. PCR product concentration is affected by the amountof DNA in the sample. Volumes represent ul of egg homogenate.

10 u

11

ul

0.1

ul

0.01

ul

500 bp400 bp300 bp220 bp200 bp

Cytochrome b DNA Sequences

• PCR products from paddlefish, shovelnose sturgeon and lake sturgeon were cloned and sequenced.

Shovelnose Lake

Paddlefish

Lake

Table 1. Percent DNA Sequence Identity

86.6% 86.0%

90.8%

Restriction Mapping

Restriction enzymes cut DNA at specific sites. A single changethe sequence of that site will prevent cleavage.

Example: HincII cuts GTTAAC in paddlefish cyt b DNA but not GTAAAC in sturgeon cyt b DNA

PA

SN

LK

HincII

HincIINarI

Restriction Enzyme Digestion

Mix together :

• 1 ul PCR product

• 8 ul buffer and water

• 1 ul restriction enzyme

Incubate 30 min, 37oC.

Subject sample to agarose gel electrophoresis for 45 minutes.

Stain gel with ethidium bromide and photograph.

PA

un

cut

PA

cu

tS

N u

ncu

tS

N c

ut

LK

un

cut

LK

cu

tP

S u

ncu

tP

S c

ut

Std

s

500 bp400 bp300 bp220 bp200 bp

Figure 3. Restriction digest of PCR products with the restriction enzyme HincII. Samples were run on a 3% Methaphor agarose gel.

Figure 4. Restriction digest of PCR products with the restriction enzyme NarI . Samples were run on a 1% LE agarose gel.

PA

un

cut

PA

cu

tS

N u

ncu

tS

N c

ut

LK

un

cut

LK

cu

tP

S u

ncu

tP

S c

ut

Std

s

500 bp400 bp300 bp220 bp200 bp

Conclusions

• Enough DNA can be isolated from a single fish egg to allow for sucessful PCR amplification.

• DNA sequence analysis demosnstrates unique genetic markers in paddlefish, shovelnose sturgeon and lake sturgeon.

• This assay will allow the species identification of a single egg within one day.

• To date this assay has been tested on more than ten fish, from all over the country, with no observed discrepancies in the assay.