oneinamillion.pdf

An incredible genetic

circumstance creates a

unique DNA puzzle to solve.

By Christine Hamilton

One In A

Dunbars Gold, a 1996 brindle stallion by Two D Nine and out of Outa

Chiggers by Outa Utopia. Genetic testing has shown the horse to be

an extremely rare chimera, an individual with two DNA types.

DON TROUT

Million

52 FEBRUARY 2006 THE AMERICAN QUARTER HORSE JOURNAL

“ EXCLUDED .”

For the second time Denise Charpilloz had sent in hair from

her 2004 foal out of her mare Sharp One for DNA testing and

parentage verification. And for the second time, the

Veterinary Genetics Laboratory at the University of

California, Davis, had excluded not only the stallion Dunbars

Gold as the foal’s sire, but Sharp One as the foal’s dam.

Every now and then people make mistakes when they submit

mane (or tail) hair samples for DNA testing on a foal. It usually

happens when people accidentally mix hair if they’re collecting

samples from more than one horse. Although very rare, sometimes

mares can actually switch foals or the wrong stallion’s semen is

unintentionally shipped and used to breed a mare.

All those scenarios result in a DNA test that “excludes” a

mare or stallion as a foal’s parent. Usually it doesn’t take long

to figure out what went wrong.

But Charpilloz knew there were no mistakes here.

“I saw it being born!” she said. “I didn’t mix it up with any

other foals; there were no other foals!” And Dunbars Gold’s

owner, Carole Dunbar, had only one stallion to ship semen from.

AQHA’s registration department turned back to the lab’s

geneticists: could they please give the case another, much

closer, look?

But in her review, something else caught Penedo’s attention.

“The oddest thing about the stallion’s blood sample results

was that the DNA types for sex-linked markers were typical

of a female and not a male,” Penedo said. “There was no evidence

of a Y chromosome.”

Penedo decided to retest Dunbars Gold.

“We went back to the original hair samples and used a single

hair for the DNA test,” she said. “And we performed several

of these single hair tests.

“Some of the tests yielded a perfectly good type for a male

individual and some a good type for a female individual.”

Some of the results also showed two DNA types within a

single hair root.

“At that point I thought, ‘this horse is chimeric*,’”

Penedo said.

*Pronounced “ky-mare-ik”

Chimerism

IN GENETICS , A CHIMERA IS AN INDIVIDUAL FORMED FROM TWO

different cell lines. Scientists believe that it happens when

two nonidentical twin embryos (fertilized eggs) fuse into one

embryo very early in their development.

The embryo develops into a normal, complete individual

that has two different DNA types. He or she might have

kidneys that developed from one DNA type and a heart or

skin cells from the other type.

Extremely rare, chimerism has been documented in other

species, including cats and humans.

“Dunbars Gold has one cell line that is female and one cell

line that is male,” Penedo explained. “The cell lines have

slightly different DNA types, but both qualify to his sire, so

there’s not a parentage issue involved. It’s almost clear that

there were two embryos produced, and they fused.”

Although chimeric in his skin and hair, Dunbars Gold’s

reproductive organs were apparently formed by his male

DNA type.

“It’s an interesting biological developmental event. He

went on to develop as a completely normal male,” Penedo

said.

Puzzling Pattern

THE CASE LANDED ON THE DESK OF CECILIA PENEDO , PH . D ., THE

lab’s associate director of service and genomic research and

development.

Penedo immediately noticed that Dunbars Gold and Sharp

One are brindles.

“It’s a very rare coat pattern in horses,” she said (though

common in dogs and cattle). “People have not been very suc-

cessful reproducing this pattern through breeding, and we’ve

never really had much information on the genetics of it

because it is so rare.”

In fact, of the more than 4.7 million American Quarter Horses

that have been registered with AQHA (excluding appendix

horses), the registration department has a list of only 15 horses

that have exhibited some form of the brindle coat pattern.

Her interest sparked, Penedo began reviewing the

lab’s testing on the horses.

“When I looked up the stallion, Dunbars Gold, I

found that we had tested him three times before we

established his (DNA) type,” Penedo said.

The first two tests the lab ran were on mane/tail

hair samples.

“At that time, there was something odd about his

type,” she continued. “It looked like the hair samples

came from two different animals, like the hair had

been mixed together. We requested a second sample

and had the same problem.”

Convinced the hair samples were getting con-

taminated, for the third test the lab requested a

blood sample from the horse.

“When the blood sample came in we got a perfectly

good type that would be consistent with one animal,”

she said.

The blood test results also had some things in

common with the hair results.

The lab used the DNA type obtained from blood

for the parentage verification on Sharp One’s 2004

foal (the first of the stallion’s foals to be tested for

parentage verification).

I , E O

N O

, W

The brindle mare Sharp One, owned by Denise Charpilloz of Vancouver, Washington. The

parentage verification of her 2004 foal revealed an incredible coincidence and gave a

once-in-a-lifetime research opportunity to geneticists at the University of California, Davis.

THE AMERICAN QUARTER HORSE JOURNAL FEBRUARY 2006 53

If Dunbars Gold was

chimeric in his skin and

hair, could there be a

link between the equine

brindle coat pattern and

chimerism? What if the

problem the lab was having

qualifying Sharp One as

the foal’s dam was also

due to chimerism?

Like Dunbars Gold,

Sharp One’s DNA type

had also been initially

established from mane/

tail hair samples. However,

according to Penedo,

there was no evidence of

chimerism in those hair

samples.

“I wondered if she

could be the opposite of

Dunbars Gold,” Penedo,

said. “If she might show

chimerism in her blood

but not her hair.”

When the lab tested

Sharp One’s blood, that’s

exactly what they found:

two different cell lines (and

DNA types) in her blood. But both of her cell lines were female.

The lab then tested Sharp One’s body hair taken from the

darker and lighter areas of her brindle pattern.

“We were able to identify the two cell lines in the different

(body hair) patches,” Penedo said.

When the lab compared the 2004 foal’s DNA type to Sharp

One’s 2003 foal (her only other living foal so far), they found

that each came from her two different female lines.

Y O

F D

E A

E C

Y F E

Sharp One’s two coat colors in her brindle pattern

radiate from the central swirl on her forehead.

Sharp Barcoder is the 2004 son of two rare chimeric

brindle horses. He’s a sorrel with no brindling.

The lab has now tested several of his foals, and they were

all produced by his male cell line.

When Penedo turned her attention to Sharp One, she again

made note of the rare brindle coat pattern the mare had in

common with Dunbars Gold.

ABOUT THE HORSES

Dunbars Gold

Dunbars Gold, aka “Aurie,” is a product of Delos Dunbar’s 40-plus

years breeding American Quarter Horses. Foaled in 1996 in Buchanan,

Michigan, Aurie is by Dunbar’s homebred red dun stallion Two D Nine,

and out of the palomino mare Outa Chiggers by Outa Utopia.

Delos’ daughter, Carole (Aurie’s owner), remembers other

brindles in Aurie’s family. Delos raised Two D Nine’s dam, a dun mare

named Miss Two Larue by Two Larue, and out of the dun mare Latigo

Kiowa (the family’s very first Quarter Horse).

Dunbars Gold might have both factors working in him, both

chimerism and the genetic predisposition for brindling, Carole said,

because there is brindling in his family.

“Two Larue had a sister that was brindle,” Carole said.

“And Aurie has an older full brother that we sold as a yearling,”

she added, (registered as Audacious One). “When the new owners

saw Aurie they said, ‘You know, the full brother that we have? You

can see brindle stripes on him when he’s wet.”

A longtime judge with the American Paint Horse Association,

Carole is proud of the stallion and the family of primarily dun horses

her father raised, but she didn’t show Aurie.

“I said to myself, ‘if I show this guy, someone’s going to offer my

dad a lot of money and he’ll sell him!’ ” Carole remembered with a

laugh. “He’s incredible. He’s as sweet as he is beautiful.”

Sharp One

“I couldn’t believe that nobody else had bought her!” said Denise

Charpilloz of the unique 1999 brindle filly she found on the Internet,

way out in Wisconsin. That was back when Sharp One, aka “Salsa,”

was a yearling.

Since then, Charpilloz has bred Salsa four times trying to repeat her

unique brindle pattern; something that she’s not sure she can do now that

she knows the mare is a chimera. Salsa has had two sorrel colts, one mis-

carriage and is in foal to the AQHA stallion Major Hearts Desire for 2006.

“The fact that there are so few brindle horses is why there have

not been any studies done at all until this one (case),” Charpilloz said.

“I don’t think the genetics surrounding the occurrence of brindling

in horses has been figured out entirely.”

Regardless, Charpilloz loves her unique mare by Force One and

out of the red dun mare Sonnys Coppertone by Sonny Supermann.

“I love the beautiful, swirling, marbled effect of the brindle pattern,”

Charpilloz said. “People are attracted to it; it stands out in a crowd.

It is different; it is rare; it is special.

“My question now is,” she added, “does she have an ovary on one

side that produces one of the (DNA types)? Or are there eggs (from

both types) in each ovary?”

Charpilloz might be the only horse owner on the planet who can

wonder that about her mare.

54 FEBRUARY 2006 THE AMERICAN QUARTER HORSE JOURNAL

“Unlike Dunbars Gold, she is chimeric in her (reproductive

organs),” Penedo explained. “She is producing two different

types of eggs, they can be from one or the other cell line,

which is a very interesting situation.

“Once we put the puzzle together, we were able to qualify

the foal without any problem.”

Penedo is quick to point out that there is evidence of a type

of brindling pattern in horses that appears to be inherited,

linked to a coat pattern gene, as it is in dogs and cattle.

“We can’t say that it is always embryo fusion that leads to

the brindling pattern in horses,” she said.

“But for the very classic, clear brindling pattern like we see

in Dunbars Gold and Sharp One, I wouldn’t be surprised if

that was caused by chimerism,” she continued.

“If it results from embryo development, then there is no

genetic control, and you can’t really breed for it. Unless there

is a gene controlling something that makes it more likely for

the fusion to occur, such as something that increases the

likelihood of a twin pregnancy.”

Penedo thinks that you would only see outward evidence of

chimerism in the coat pattern if the two embryos that fused

had genes coding for contrasting coat colors.

“For example, if two bay embryos fused, or two chestnuts,

you wouldn’t see anything,” she explained. “I think you’d see

the brindle pattern if you had a combination of a base color

with one dilution gene. In this case it was the dun gene. Both

(Dunbars Gold and Sharp One) have dun in their background.

“It’s been a great learning experience,” Penedo added. “It is

shedding a little bit of light on the brindling pattern in my

mind.”

Denise Charpilloz named her newly registered foal Sharp

Barcoder, aka “Deuce,” and he’ll soon head to his new owners,

Stacy and Alex Payne in McMinneville, Oregon.

Of course, Deuce is a solid sorrel and there’s nothing

chimeric about him. But he is the occasion for an incredible

genetic coincidence: If Charpilloz hadn’t been trying to breed

for the rare brindle coat pattern, these even rarer chimeric

individuals would never have met.

And geneticists worldwide would not have had this once-

in-a-lifetime chance of studying their DNA. What are the

odds of that?

The Pieces Fit

“ WE NOW HAVE EVIDENCE OF CHIMERISM FROM TWO DIFFERENT

brindle horses,” Penedo said. “We think that the brindle

pattern in some horses could be explained by this very rare

event, where two embryos fuse early on in their development

and go on to make just one, single individual with different

cell lines.”

CONFUSING COLOR

“The best way to think about it is, most chimeras are brindles,

but not all brindles are chimeras,” said Phillip Sponenberg,

D.V.M, Ph.D., noted author and equine color geneticist from

Virginia Tech’s Virginia-Maryland Regional College of Veterinary

Medicine in Blacksburg, Virginia.

“Chimeras are mixtures of two individual animals,” he continued.

“Those animals usually end up being striped, like a brindle horse.

“However, there does seem to be a coat color pattern (in

horses) that is brindle that is not related to chimerism,” he

continued. “There is a gene floating around that can bring

about the brindling coat pattern.”

Sponenberg thinks that it might have to do with the gene that

causes the “sooty” coat color.

“I suspect that the brindling gene reorganizes the sootiness into

stripes,” he said. “For example, if you look at horses that are bay

brindles, they still have the black points, so the brindling doesn’t

affect the lower legs. It just seems to reorganize the body color.

“I think that for the (brindling to be expressed), the horse

has to have both the gene for the brindling, as well as the right

background color to demonstrate it on. That’s why I think

they’re so hard to breed for.”

The point is you can’t tell that a horse is chimeric just by

looking at it.

“That’s where (genetic) testing is very useful,” he pointed out.

Christine Hamilton is a field editor for The American Quarter

Horse Journal. To comment, write to chamilton@aqha.org.

Learn More

Here are some Internet sources on equine color genetics:

• Equine Color Genetics information page, compiled by Daralyn

Wallace: http://hometown.aol.com/raunfalcon/colrpage.htm.

• Horse Colors: http://greenfield.fortunecity.com/dreams/

799/hc/horsecolors.htm.

• HorseColor.com: http://www.horsecolor.com/.

• Cremello & Perlino Educational Association Web site:

http://www.doubledilute.com/.

• The Veterinary Genetics Laboratory at the University of California,

Davis, performs a variety of horse coat color tests and offer

information on the genetics on their Web site at:

http://www.vgl.ucdavis.edu.

• For more on brindle horses specifically, look at J. Sharon

Batteate’s Web site at: http://members.aol.com/brindleho.

Check out these books as well, most available online through

Quarter Horse Outfitters at www.quarterhorseoutfitters.net.

• “Equine Color Genetics,” by D. Phillip Sponenberg

• “Horse Genetics,” by Ann T. Bowling

• “Horse Color Explained,” by Jeanette Gower

, .

P P

F P

A horse exhibiting the sooty coat pattern, where black hairs countershade a base

color, giving the coat a sooty appearance. Sponenberg thinks that the inheritable

brindle coat pattern in horses works to reorganize the sootiness into stripes.

THE AMERICAN QUARTER HORSE JOURNAL FEBRUARY 2006 55

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