Determination of Extra-Pair Fertilization and Inbreeding Using Microsatellite Genotyping in a
Captive Population of Zebra Finches
Lindsay Miller, Julia Tucci, Debora Christensen (Mentor)
Dept of Biology, Drake University
Image from mindupbioresearch.com
RESULTS (cont’d)
INTRODUCTION
METHODS
• True monogamy is atypical among animals.
The zebra finch, Taeniopygia guttata, (figure 1)
is an exception, renown for forming life-long Figure 1. Zebra finch male
(left) grooming female
monogamous relationships.
•
Blood samples were collected from 14 parental zebra finches and 39 of their
adult offspring (figure 3)
•
DNA was isolated using a Qiagen DNeasy Blood & Tissue Extraction Kit
•
5 microsatellite regions were chosen for amplification and potential
genotyping (Table 1)
1
partner (right).
• Extra-pair fertilizations (EPFs) are detected when the expected
female is the mother but a different male is revealed as the father.
• Egg dumping occurs when a female deposits fertile eggs in a nest
other than her own so chicks are raised by foster parents.
Repeat Motif
Expected Allele Sizes (bp)
(CA)11
123, 127, 131, 133
(GT)13(TCAC)2 196, 198, 200
(GT)9
Ase50z
(CA)12
270, 272, 276, 287, 297, 301,
316
INDIGO41 (TAGA)11
298, 303, 315, 319, 324, 328,
332, 336, 417
Ppi2
(GTT)1(GT)
242, 274, 284, 286, 288, 295,
(TT)1(GT)9
298, 300
•
•
•
•
To extract DNA from blood samples taken from zebra finches and use
polymerase chain reaction (PCR) to amplify 5 microsatellite regions in
their genomic DNA
To use microsatellite information to determine the extent of
relatedness in our captive population of zebra finches
To explore the extent of monogamy in our colony of breeding zebra
finches by determining the rate of extra-pair fertilizations (EPFs)
To determine if egg dumping occurs in our breeding colony
Figure 3. Blood samples were
taken from the alar (wing) vein
of adult zebra finches.
Avian blood is nucleated, so a
small blood sample provides a
considerable amount of genetic
material.
4
5
6 7 8
Figure 5. Example of gel showing
multiple-sized PCR products from
parental DNA with INDIGO41 primers
in lanes 4 through 8. Lanes 2 and 3
show no DNA amplification with Ppi2
primers. A 50 bp ladder is in lane 1.
Locus
Ase12
Ase32
Figure 2. Example of
microsatellites differing in
the number of CTT
repeats. Image from
www.brassica.info
•
3
Table 1. Microsatellites selected for amplification (from Dawson, et al, 2005)
• Microsatellites are short tandem nucleotide repeats of 2-5 base pairs
used to determine relatedness among individuals. This can be done
by comparing the size of the microsatellites (figure 2).
STUDY OBJECTIVES
2
•
Polymerase chain reaction (PCR) was carried out on all parental DNA using
forward and reverse primers for the 5 microsatellite regions in Table 1 (figure
4)
PCR products were then run on a 2% agarose
gel and visualized under UV light with ethidium
bromide
Figure 4. The PCR procedure starts with the
template DNA, the genetic material that has been
extracted from an animal’s cell. Template DNA is
added to primers, dNTPs, and Taq polymerase.
The successful PCR product will contain many
copies of the genetic material that can be seen as
a separate band on a gel.
FUTURE DIRECTIONS
•
Troubleshoot the 2 parental DNA extractions that did not
yield a PCR product for any of the primers tested.
•
Troubleshoot Ase50z primer that did not produce a PCR
product.
•
Quantify DNA extraction results with nanodrop
•
Once all genomic DNA has tested and provides a PCR
product, parental DNA will be sent to Iowa State
University’s Sequencing Facility for size analysis using
capillary gel electrophoresis and genotyping using the 4
working primers
•
Determine relatedness amongst parents
• If parents are related (i.e., inbred), increase primer
number
• If parents are not related, use working primers for
offspring DNA analysis
•
Microsatellite regions will then be compared between
parents and offspring to determine maternity and
paternity.
RESULTS
• 4 of the 5 primers have successfully yielded PCR products: Ase12,
Ase32, Ppi2 and INDIGO41 (figure 5).
• 11 of the 13 parental DNA extractions have yielded PCR products
with at least one of the 4 successful primer sets
Acknowledgements
Special thanks to Dr. Christensen, Dr. Sleister, and Dr. Busch for sharing materials, equipment, and expertise. Thanks also to Alisha Koxlien for assisting with blood sampling and to the
Christensen lab for assistance in caring for the animals.