Investigation of Genes Controlling Pigmentation in Alpacas

ARF Investigator Profile by Ingrid Wood, Stormwind Alpacas

Principal investigator:

David L. Kooyman, PhD

During my long teaching career, I once taught a class of fifth graders about the physiological similarities shared by human and pigs. Body parts from pigs can be used to replace, for example, defective heart valves in humans. “She's crazy,” one of my students whispered, sotto voce. Several heads nodded in silent agreement. Too bad my students did not have the opportunity to talk to David L.Kooyman, PhD, a scientist presently working in the Department of Physiology and Developmental Biology at Brigham Young University in Utah. His biography profile includes employment at Baxter International in New Jersey. As a senior scientist, Dr. Kooyman’s worked on xenotransplantation, a project where he helped to clone genes and genetically engineer pigs to be used as organ donors for humans.

Pigs were not Dr. Kooyman’s first research interest as far as species go. While working on a Masters Degree at California State Polytechnic University, his attention was focused on the reproductive physiology of goats. After completing a PhD in molecular and cellular biology at Ohio University in Athens and a post doc at DNX in Princeton, Dr. Kooyman eventually worked for previously mentioned Baxter International. His academic achievements and interests finally led to his present position at Brigham Young University. “I served several years as Associate Dean in the College of Biology and Agriculture,” Dr. Kooyman mentioned. “Prior to that, I was Chair of the Animal Science Department. I resigned my position as Associate Dean because I wanted to return to my first love… teaching and research.”

“How does the alpaca project fit into your schedule?” I wondered, assuming that camelid research played only a very minor role among Dr. Kooyman’s multitude of academic duties. His answer surprised and delighted me. “My research focus now is identifying and characterizing economically important genetic traits in South American Camelids,” he replied. His lab at Brigham Young University is the first and possibly the only lab in the world that produces llama bacterial artificial chromosome (BAC) “libraries”. In a  BAC library, large chunks of llama DNA are cloned and placed within a man-made, artificial chromosome in bacteria. “You are continuing the research begun by Dr. Emily Campbell?” I asked. “Yes, we are in the process of identifying and describing the genes associated with color in llamas and alpacas,” Dr. Kooyman confirmed. I could hear the  happy satisfaction in his voice when he added, “We’ve made quite a bit of progress in our research.”

The interview and subsequent communication with Dr. Kooyman reminded me once again how alpaca breeders have been fortunate to "piggy-back" on the results of research conducted on other species. Dr. Kooyman wrote, “We have found that identifying candidate genes for studying alpaca fleece color is possible because of the high conservation of these genes in other domestic animals previously studied and characterized.” He specifically mentioned pigs, cattle, and horses.

Alpaca breeders need to appreciate the fact that the inheritance of color/ patterns is the result of bio-chemical processes programmed by genes which code for pigment or loss thereof. Some breeding results seem bizarre to those who have never studied this science. For example, two black alpacas may produce a white cria while, on a neighboring farm, two white alpacas are the parents of a black infant. Does this make sense? It does! An animal’s final color phenotype is the result of many genes. The white alpacas in our examples are, genetically speaking, black alpacas. Their pigment was “stripped” by various genes coding for lack of pigment. The mating of sire and dam caused a series of genes to be “re-shuffled,” with obviously eye-popping results.

Let’s remember that only two pigments exist in mammals: black (eumelanin) and red (pheomelanin — sometimes spelled phaeomelanin). Two major genetic “players” determine which pigment (s) are produced by the pigment producing cells, called melanocytes. These genes are found at two loci (genetic “addresses”): the Agouti locus and the Extension locus. A large supporting cast of other genes code for shading/no shading, dilution/no dilution, and pattern/no pattern. Regardless of these modifications, we must think of all alpacas as either red or black. To use several examples: a light fawn alpaca is red, a silver grey is black, a medium brown is red, a rose grey is red, a fawn pinto is red and so on. In terms of Dr. Kooyman’s present research project, we need to primarily concern ourselves with those genes found at the Extension locus. However, we can’t get around discussing genetic mechanisms found at the Agouti locus. Shortly, you will see why that is necessary.

I mentioned that the function of genes at the Agouti and Extension loci has been firmly established in other species. Dr. Kooyman, along with other scientists such as Dr. Phillip Sponenberg, hypothesize that the Melancortin 1 Receptor (MC1R) gene at the Extension locus and the Agouti Signaling Inhibitor Protein (ASIP) gene at the Agouti locus play important roles in the differentiation of red versus black phenotypes in alpacas as well. The close bio-chemical interaction between the products of these two genes (MC1R and ASIP) can be very complicated.

Dr. Kooyman patiently explained how the melanocyte (the body cell that produces pigment) constantly produces pheomelanin (red) if it doesn’t get the directive to produce eumelanin (black). “Tell me what happens, bio-chemically speaking, to make mammals black?” I asked. “In that case, a hormone called Melanocyte Stimulating Hormone (MSH) is involved,” Dr. Kooyman answered. He emphasized, “If MSH is bound to its receptor MC1R, the pigment cell will be stimulated to convert pheomelanin into eumelanin and the animal will be black.”

Looking over my notes several weeks after the interview, I thought, “Now what exactly does 'bound to' mean?” Minutes later, I was on the phone to Dr. Patricia Craven, a fellow alpaca breeder (Cherry Ridge Alpacas), ARF board member, and a friend. She has the patience of a saint when I call on her knowledge as a research scientist to explain scientific concepts to me. “Yes, I can fill you in a little more,” Pat responded to my plea for help. “MSH is a peptide hormone and is secreted into the blood stream from the pituitary gland. MC1R is a receptor that is located on the cell membrane, the outer layer of the cell. MSH doesn’t enter the cell.” “Then how does this hormone do its job?” I interrupted. “MSH interacts with its receptor, MC1R, in the cell membrane,” Pat continued, unruffled. “The receptor recognizes the hormone and transmits a signal from the hormone to the cell. The signal causes the enzymatic conversion of pheomelanin to eumelanin.” Well, who would have thought that hormones play a part in color genetics?

During the interview, I had questioned Dr. Kooyman at this point, “But not all mammals are black, so MSH (the hormone) either isn’t produced or doesn’t work all the time?” That’s correct”, he replied. “When ASIP is bound to MC1R, MSH is unable to bind as well and only pheomelanin (red) will be produced. The actual color will depend on the other color determining genes of the animal. In order to understand how this occurs, it’s necessary to learn a somewhat complicated genetic concept. Some genes have mutated variations of the original form. We call those alleles.” Dr. Kooyman sounded almost apologetic as he added diplomatically, “That’s very hard to understand. I can’t think of an easy way to explain it.”

“You can think of alleles as ice-cream flavors…,” I started my somewhat goofy definition of an allele in layman’s terms. Dr. Kooyman laughed. “Yes, that’s a good analogy,” he acknowledged.

So, let’s talk ice cream. It comes in flavors such as vanilla and “mutated” versions such as chocolate, strawberry, lemon, and mocca. A person has only two hands and can only carry one cone (one flavor) per hand. They are all ice-cream (gene) but different flavors (alleles). Likewise, at each locus, an animal carries only two genes — one inherited from the sire, the other one from the dam. If the genetic material on that locus is the same for the entire population (ice-cream analogy: vanilla-vanilla or mocca-mocca to use two examples), scientists use the word gene. If the flavors (alleles) vary within a population (examples: vanilla-strawberry, chocolate-vanilla, mocca-mocca, strawberry-mocca), the variations are referred to as alleles. Combinations can vary considerably within a specific population, but alleles can also be easily lost forever due to natural or artificial (human) selection pressure. (A friendly warning to alpaca breeders: think long and hard before you call for specific colors/patterns to be eliminated from the entire North-American alpaca population!)

In any case, scientists established the existence of multiple alleles of the MC1R gene in humans, dogs, cattle, pigs, horses, goats, and probably many others. In many mammals, multiple allelism also occurs in the gene coding for Agouti Signaling Inhibitor Protein (ASIP). So how does the potential for multiple alleles at the Extension or Agouti locus play a role in determining whether an animal is black or red? Let me give four examples based on studies in other species.

1. The animal could have inherited an allele at the Extension locus that codes for a form of the MC1R that does not bind MSH and is therefore not functional. Dr. Kooyman explained the bio-chemical process, “The non-functional MC1R allele produces a red phenotype because the hormone (MSH) is not able to bind to its receptor, MC1R.” He further mentioned that the mutated MC1R allele coding for loss of function can produce a wide range of “red” phenotypes — from a rich, dark red to fawn so light that it appears white. He pointed to the Black Bear as an example of this phenomenon.
2. The animal could have inherited a functional allele of the MC1R gene at the Extension locus plus a functional allele of ASIP at the Agouti locus. A functioning ASIP allele would lead to production of ASIP, an inhibitor protein that prevents MSH binding to its receptor. This animal would also be red.
3. The animal could have inherited a functional allele of the MC1R gene at the Extension locus plus two non functioning alleles at the Agouti locus. In the latter case ASIP would not be produced and therefore could not interfere with the binding of MSH to its receptor, MC1R. This animal would be black.
4. As with just about everything in science (or so it seems to me), there are exceptions to the rule. Dr. Kooyman made it clear that, in some animals, a dominant black MC1R allele produces a receptor that allows the hormone MSH to bind even if the ASIP allele is functional and ASIP is produced.

It is quite possible that alpacas, like dogs, have more than two Agouti locus alleles (flavors) to “choose” from. Once again, let’s remember that the individual alpaca carries only two alleles at each locus. After examining breeding records, Dr. Sponenberg in fact proposed a series of Agouti alleles, and their identification certainly lends itself to another project funded by the Alpaca Research Foundation.

At this time, let’s clearly define Dr. Kooyman’s research objective: in his laboratory at Brigham Young University, he will identify all the alleles of the MC1R gene in alpacas within the available population. “Comparing genotypes to phenotypes will help to determine if multiple alleles exist at the MC1R gene in alpacas,” Dr. Kooyman further clarified his mission.

The interview drifted back to more private matters. Dr. Kooyman’s interest in camelids developed in 2002. His oldest daughter Kristy owned llamas and alpacas at that time.

She urged her father to apply his considerable knowledge and expertise to the animals she loved and Dr. Kooyman described as “delightful.” In 2004, he made contact with South American scientists involved in camelid research. This led to his election as lead scientist of the “Camelid Focus Group”. The group’s goal is a comprehensive, integrated project in camelid production with the initial emphasis on fleece characteristics including color and elimination of guard hair. Dr. Kooyman expressed a keen interest in helping the people of the Alto Plano. “Many live in poverty,” he stated, “my contribution to their welfare can be in the area of genetics.”

Dr. Kooyman shared with me that he and his wife have four children, one of which died a little over a year ago. When, at some time during the conversation, I referred to his three children, he quietly but firmly corrected me, “No, we have four children.” This loving inclusion of the deceased child in such a natural, unaffected manner made a deep impression on me.

We talked about Dr. Kooyman’s upbringing on the edge of a rural environment in Iowa. Summers were spent on his uncle’s farm helping to raise crops, cattle, and pigs. Ah yes, those pigs of organ donor fame! It seems like quite a journey from feeding slop to pigs to researching color genetics in camelids. It is an equal stretch for many alpaca breeders, most of whom are not scientists, to comprehend the complexity of color inheritance. The knowledge of bio-chemical processes is crucial for a truly deeper understanding of the subject area. However, it is not, in my opinion, a prerequisite for learning what I call the “system” of color genetics.

Long before the first alpaca stepped on North American soil, scientists and breeders developed a system of letters to express the mechanisms responsible for mammalian color phenotypes. It also enhances communication among breeders. An article describing the various color loci and genotypes of fancy mice is entirely comprehensible to a breeder of dogs and horses. When Dr. Kooyman is ready to share his research results, I will present them in this “breeders’ language”. The words Melanocortin 1 Receptor gene need not even be mentioned. Breeders will be able to take Dr. Kooyman’s findings and apply them to their breeding programs. This is indeed an exciting project and only the mere beginning of what a scientist with Dr. Kooyman’s knowledge can accomplish for the benefit of the alpaca community.

Ingrid Wood is the owner of Stormwind Alpacas. Her small farm is located in a rural community in New Jersey. She offers a PowerPoint presentation An Introduction to Camelid Color Genes to interested groups and private parties. Her website, www.StormwindAlpacas.com includes all contact information.