Whos Your Daddy

1
Whos Your Daddy?

• Lauren Lee
• D3, Woodside Pony Club
• Midcal Region

Equine Genetic Markers in Parentage Analysis
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Purpose

• In 2007, we purchased a 14.1 grey Connemara pony
  named Kilbarry Prince, foaled in 1999.
• Prince is a registered Irish Sport Pony,
  according to his passport from the Irish
  Showjumping Association.
• Because his parentage is unknown, his parents
  must be found to register Prince as a pure or
  half-blood connemara, therefore allowing us to
  compete in breed competitions for connemaras.
• After much research, I found several ties
  through dealers and similarities linking Kilbarry
  Prince as possible offspring of Prince of
  Thieves, so I will test Kilbarry Princes DNA and
  compare the two to see if Prince of Thieves sired
  Kilbarry Prince.

Kilbarry Prince
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Hypothesis

• I think that my pony, Kilbarry Prince, was sired
  by Prince of Thieves, a well-known connemara
  stallion in Ireland.

As a first step, before DNA testing, is it even
possible for a dun stallion to sire grey
offspring? Yes, it is possible for a dun
stallion to sire grey offspring if the offspring
inherits a grey gene (always dominant,
expressed as G.) from the dam. In Kilbarry
Princes case, the dun gene from the sire in
question would dillute a base color, which would
explain Kilbarry Princes premature loss of
pigmentation before the age of 10.
Prince of Thieves
Kilbarry Prince
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Background/Research

• I am going to take DNA samples of Kilbarry Prince
  and send them into the Veterinary Genetics
  Laboratory at UC Davis.
• I will have the Irish Connemara Society release
  Prince of Thieves DNA to UC Davis for
  comparison, under agreement of the owner.
• Based on the comparison of DNA microsatellite
  loci, UC Davis should be able to exclude or
  confirm Prince of Thieves as the sire of Kilbarry
  Prince (with a probability of at least 99)

Microsatellite (aka Simple Sequence Repeats
(SSRs), are polymorphic loci present in nuclear
and organeller DNA that consist of repeating
units of 1-6 base pairs in length. They are
typically neutral, co-domininant and are used as
molecular markers (or genetic markers) which have
wide-ranging applications in the field of
genetics, including kinship and population
studies (Microsatelite, Wikipedia, the free
encyclopedia).
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Procedure

• To test my hypothesis, I took hair follicle
  samples from Kilbarry Prince and sent them into
  the Veterinary Genetics Laboratory at UC Davis.
• Chemical treatment is used to break DNA up into
  small fragments. The fragments are then sequenced
  using automated equipment and usually read by
  using automated lasers. The automated equipment
  can translate the data into known genetic
  markers. One such translation is shown to the
  right.
• Kilbarry Princes genetic test resulted in 14
  different genetic markers. (See other page,
  Results to see all 14 tested markers). The
  geneticists compared these markers with the ones
  of Prince of Thieves, and using the method as
  described on the page Genetic Markers, they
  could either exclude or confirm Prince of Thieves
  as the sire of Kilbarry Prince.

Sequence ladder by radioactive sequencing
compared to florescent peaks (DNA Sequencing,
Wikipedia, the free encyclopedia). Image also
from Wikipedia
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Genetic Markers

• A genetic marker is a DNA sequence with a known
  location on a chromosome (aka a locus) and
  related with a particular trait or
  characteristic. They can either be long or short
  DNA sequences.
• Differences in Locus types are inherited from the
  genetic makeup of the parents of the offspring in
  question. (This is the same concept as the basic
  rules of Mendelian Genetics)
• For example, for an offspring of a LO type on
  the HMS6 locus, they may have had a dam with a
  type LR on the HMS6 locus and a sire with a type
  OQ on the HMS6 locus. For the offspring, the dam
  would contribute an L allele, and the sire
  would contribute the O allele, yielding a LO
  type for the offspring on the HMS6 locus.
• On the other hand, if a mare had the type AB and
  a stallion had a type CF on a particular locus,
  the stallion could not sire offspring with a type
  BO on the same locus, because the sire has no
  matching allele to result in a type BO for this
  specific locus. However, the mare could be the
  mother, because she may have contributed the B
  allele to the BO type in this example.

Mendels Genetic Theory analysis, first used on
pea plants
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Mendelian Genetics
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Results

• According to the labs at UC Davis, because of
  five (5) genetic markers, Prince of Thieves is
  excluded as a possible sire of Kilbarry Prince,
  regardless of the dam.

Typically, for parentage testing, 12-15 genetic
markers (aka microsatellite markers) are used,
although some genetic tests are performed with
only 5 genetic markers. The larger number of
genetic markers means that the certainty of the
results is much higher.
Complete results from UC Davis of parentage
analysis for Kilbarry Prince Note Only UC Davis
was permitted to review the genetic markers of
Prince of Thieves due to restrictions by the
owner.
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Conclusion

• Although my purpose was not met and I did not
  find Kilbarry Princes father in this test, I
  remain positive because I know I will find
  Princes father soon. I can use Princes current
  markers to compare against other sires, and not
  need to have Prince tested again.

I also learned a great deal in the process about
not only equine genetic markers, but about
genetic testing in the greater realm of science.