DNA TESTS USED TO VERIFY PARENTAGE AND ANIMAL I.D.

by: Heather Smith Thomas

Breed registries today rely on DNA testing to verify parentage in an animal's pedigree. Every animal has a genetic code in every cell of the body. Several billion chemical sub-units of a material called deoxyribonucleic acid (DNA) constitute the long, threadlike chromosomes (and the genes on those chromosomes) that exist in the nucleus of every cell. The genetic code for every aspect of that individual can thus be found in any body cell, whether blood, bone, hair or muscle.

In recent years, researchers have mapped the genomes of many livestock species to discover their complete genetic make-up. These genomic maps have shown that all mammalian livestock species have 3 billion base pairs of DNA that are organized into approximately 50,000 to 100,000 genes—contained in different numbers of chromosomes that occur in pairs. Horses have 64 chromosomes (32 pairs) and cattle have 60 (30 pairs). Genes are the pieces of the DNA that make up the various codes for proteins and enzymes. Each gene is a specific segment of DNA on one of those chromosomes.

A DNA or gene marker is an identifiable piece of DNA that can be mapped to a unique physical location on a chromosome. A marker is any measurable inheritable difference that shows a variation between individual animals, such as coat color, body type, various aspects of conformation, various traits such as marbling and tenderness, etc.

Since the genetic code of an individual animal is present in every body cell, a tiny portion of that animal can be utilized to verify its identity. Samples of blood or hair (containing the hair roots) are the most commonly used portions that are sent to a lab to analyze for identifying that animal. DNA testing is now the standard tool for parentage verification and/or individual identity testing for all species. Some of the human applications include forensic science and criminal cases, paternity questions, or individual ID for everyone in the military (in the unfortunate event that someone is killed and all you have are body parts to be identified).

Every individual animal or human has a unique set of DNA. As explained by Brandt Cassidy, PhD (Director of Operations, DNA Solutions, Inc.—one of the companies that does animal DNA testing), if you're just comparing an individual to itself, the DNA will always match. “We often do this when working with USDA, wildlife enforcement, cattle associations to resolve rustling cases, etc. in which we need to show that a certain animal is what we think it is. DNA can thus identify a stolen animal, or show that the beef in someone's freezer came from an animal that matches the blood in the back of their truck. This type of DNA test is accurate 100 percent of the time,” says Cassidy.

Tom Holm, of MMI Genomics (the company that does DNA testing for a large number of cattle breed registries including Angus) says that if look at one animal compared with another, essentially their DNA is very similar—greater than 99 percent identical. All animals have similar body functions, for respiration, digestion, etc. There are many things that all mammals share, for instance.

But there are also differences in DNA that make one individual different and unique from every other individual. “For parentage verification and individual identity testing, we take advantages of these small differences at the DNA marker level and use them to ascertain correct parentage or identify one animal versus another,” says Holm. DNA technology for parent identification has replaced any other technology used in the past, such as blood typing.

Parent Verification -- Differences in DNA markers can be determined using various detection techniques. “Those differences are inherited from parent to offspring. For instance, every mammal, whether it's a horse or a cow has two copies of every chromosome—one from each parent. If you have a way to mark those chromosomes, you can follow their transmission from parent to offspring. For each marker, a particular offspring (if it truly is the offspring) has to get one copy of the marker from its father and one copy from the mother,” says Holm.

Cassidy explains that by tracing the pieces of DNA from the cow to the calf and from the bull to the calf, you can establish that together those two animals could have produced that offspring. “But lineage checking, when you start running genetic tests to determine parentage, is not always simple,” says Cassidy. “It is definitely most accurate when you have both the sire and the dam to check. It can become more murky if you only have one of the parents to test.”

Sometimes cattle breeders send in samples and ask the lab to check their entire herd to try to identify the parents of a certain animal. “We may find two bulls that could possibly be the sire,” says Cassidy. “The breeder then tells us that one of them is the father of the other. So just by itself, the DNA may not be enough. We must have additional information from the breeder, telling us who the actual possible sires might be.” Otherwise you might assume that the grandfather is the sire, especially in linebreeding situations where the animals have close genetics.

In nature, there are random mating selections, with a relatively large gene pool to draw from. In cattle or horse breeding, by contrast, the owner generally determines the matings within a small segment of the species population. “You lose the genetic variation that makes it easy to distinguish the correct sire and dam of each offspring,” says Cassidy.

In domestic animals you often end up with genetically similar animals because most matings take place within a certain breed or even within certain family lines within that breed. With all the linebreeding that occurs within a breed, a rancher might have several bulls with similar, though not identical, genetics. “Sometimes the mothers are also related to the fathers of the calves. Due to this lack of variation, the passage of genetic material from parent to offspring can be difficult to determine whether some of it came from the mother's side or the father's side, if you only have one of the parents tested,” explains Cassidy.

Any single marker may not carry much weight in resolving questions of parentage or individual identification. “But by combining the power of many markers, we increase the probability that an alleged parent is actually the true parent,” says Holm.

Cassidy says it is important to understand the limits of DNA testing. “The DNA test can tell you whether a certain animal biologically could not be the father. This exclusion is 100 percent accurate.” If it doesn't match, there is no way that animal could be the sire.

“Or, the test might tell you there is a possibility he could be the sire—not that he IS, but that it's possible. You can't say with 100 percent certainty, in human or animal genetics, that this is the only one that could be the father, using only the DNA information. But if there is a match at all the markers tested, then there is a possibility he could be,” explains Cassidy.

“In human situations, with random matings, you can do a statistical evaluation and give a probability, because there's not as much inbreeding and linebreeding in humans. It's a little harder in cattle because there's not as high a predictability in an inbred system as in random matings,” says Cassidy.

“But in most cases, after running a certain set of markers, if no marker excludes either parent, then the probability of parentage can be calculated,” says Holm. The technology of determining parentage, particularly in livestock, is always based on exclusion, however.

“If we are looking at the parents of a particular individual and find that the alleged father could not have contributed a certain marker to that offspring, then we call that an exclusion. In this case, we say that animal is excluded from being the true sire. Essentially, we look at an exclusion as being 100 percent proof that a certain animal is not the parent of the offspring being tested. In our lab, we always want to see at least two exclusions, to make sure we can call it that,” says Holm.

A mismatch can sometimes be caused by a mutation. Cassidy says that when DNA is passed to offspring (whether human or animal), a small percentage of the time there is an error produced (mutation) that makes the DNA of the offspring different from that of the parents. “This happens occasionally—when you have one marker that doesn't match, even though it is the correct parents. In humans this occurrence is about 1 in 300. It's probably similar in most animal systems. You can use more markers, to try to sort out the tough cases, but the problem with using more markers is that you'll also increase the probability in which you might find a mutation.”

Each lab has it's own standards regarding possible mutations. Cassidy says that he doesn't know how other labs deal with these situations—whether they automatically exclude that sample as being a possible match or say it's inconclusive. “Or they may go back to the breeder to get more information to establish that this is or isn't the true parent (with no other possibility) and then put a note on the file that it's a match but we accepted a mutation at this one site. We do this, for instance, in our whitetail deer breeding system, where the owner has enough information to provide us that this is the only possible scenario. Then we will on occasion go ahead and issue a certificate that says this is the possible father of this animal, with the caveat that there's a mutation at this particular marker. But this is very rare,” says Cassidy.

The rancher may say he saw a certain bull breed that cow, and he thinks the calf must have been sired by that bull. “But if the test comes back with an exclusion, we have to say we are 100 percent sure that it is not the sire,” says Holm. “Assuming that there are no sampling problems, exclusions are 100 percent accurate,” he says. Perhaps there was another bull in the pasture, or a neighbor's bull jumped the fence and bred the cow as well.

Sometimes breeders rely too much on DNA tests and technology, and want the lab to determine the sire of a calf using the entire database of bulls in that breed that are on file. While DNA-based parent verification is the most accurate tool for determining correct parentage, stockmen need to realize that DNA tests are not a substitute for accurate record-keeping, or close management (planned matings, accurate AI records, or knowing which bulls are with which cows, and so on). DNA testing, as done in the livestock industry today, can easily verify whether a certain individual is not the sire or dam of a certain calf, or can give a probability that it is—but is not an adequate way to try to “find” the sire or dam if the breeder doesn't have a clue about the actual identity of that sire or dam. It can be done, but this would take the use of a lot more markers and would be very expensive, according to Holm.

Parent verification in livestock uses only a certain number of markers, due to cost in running the tests. A DNA test on humans is more expensive. “In the livestock industry we are asked to do the exact same thing, for about $20 per sample, because stockmen can't afford high cost,” says Holm.

“If money were no concern, however, we could run more markers. The power of the probability of parentage is determined by the power of each of the individual markers you use, and the total number of markers you use. To keep costs down, we must run a finite number of markers. If someone wanted to test all the bulls on the planet as potential candidate fathers for any particular offspring, with the finite number of markers we use today, this would be impossible. Many, many sires would potentially qualify, just by chance. But if it was important to know, and cost was not a factor, we could run enough DNA markers to resolve parentage on every animal,” he explains.

“Running more markers means more cost. There has to be a balance between costs that breeders want to pay for this technology and how good they are in their record keeping—to need to push the envelope on this, in the first place,” says Holm. If very many more markers were used in the testing, the costs of parent verification for registering an animal would be too high for breeders to want to pay.

“But in some situations, such as when a rancher has 3 bulls that are closely related, you could run more markers on those three bulls and on the other animals that are related to them, and sort this out,” says Cassidy. This could be an option if a breeder really needed to narrow it down, and some labs will do this.

“They have up to 21 markers they could use, but they save this for a few situations where it's the last chance option,” explains Cassidy. But there is also more risk for finding one or two mutations, which would show places that didn't match—and possible exclusion of that animal even though it is the correct parent.

“DNA technology is the most powerful tool known for resolving questions of parentage and individual identity,” says Holm. “It provides a great benefit to breeders. A DNA test increases the validity and value of the pedigree,” he says. When the owner sells that animal, he/she is not just selling the animal, but also the guarantee of pedigree—with DNA testing behind it. The breeder can guarantee the bloodline represented in that pedigree. In any registry, the validity of pedigree and bloodline is what's most important. The known ancestry is what makes any animal valuable.

Standardization Of DNA Testing -- “Companies like ours have been doing parentage verification in cattle now for almost 20 years,” says Holm. “We were some of the pioneers in doing this. It took time for DNA testing to replace the blood-typing technology that was used in the past, in all breeds.” There also had to be some international standardization for the DNA testing, just as there had been for blood typing.

An international organization was involved in standardizing the earlier blood typing. This International Society of Animal Genetics (ISAG) became involved in DNA testing during the early 1990's and was responsible for coordinating selection of DNA markers to be used as standards across the industry. This technology has been accepted internationally, and is monitored and validated internationally so that there are uniform standards.

Types Of DNA Markers -- “The kind of markers used for parent verification in cattle are what we call microsatellites,” says Holm. “They are also called STR (short tandem repeats). We run 11 markers in our standard microsatellite panel. In most breed types, this provides probabilities of parentage at greater than 99 percent. In a situation where you are using closely related bulls in a multi-sire breeding group, however, sometimes they fall into that 1 percent of being inconclusive. In order to deal with those, we have a secondary panel of microsatellite markers that we run, to help resolve these cases,” he explains.

“Our lab and our competitors are now looking to switch over to a new type of marker called a single nucleotide polymorphism (SNP marker). With SNP markers, each marker is less informative, so you have to run a lot more of them. You'd have to run 80 to 100 SNP markers to have the same power you'd have with 10 microsatellite markers. But the cost of running SNP markers is becoming much lower, so it is now feasible to run 100 SNP markers for a cost equal to or less than the cost to run existing panels of microsatellite markers,” explains Holm.

“The current technology is very powerful, but the new technology with the SNP markers will probably replace it within five years for the seedstock industry, if not sooner. Part of the problem with changing to SNP markers is similar to the situation we had when we went from blood typing to microsatellite DNA markers. The microsatellite genotypes on a parent cannot be compared against SNP genotypes on progeny. They have to be tied to the same marker system. When we went away from blood typing, all the parents that were in the data base had to be DNA typed,” he says.

This will be the same kind of transition. All the parents that were genotyped with microsatellite markers would have to be genotyped with the SNP panels. “So we have to convert the data base again. Even though the SNP technology is very low cost, there is a very high cost to convert the data base. But we are in the process of doing that in the most efficient way possible,” he says.

For example, you don't need to genotype all parents in the database. “You'd wait until new progeny are submitted, with a request to test a certain old sire and dam. Then we can go back and pull their DNA out of our archive and genotype them with the new markers. So we only do it as needed,” says Holm.

“In our business, we archive all samples we receive. You can have those forever, and if you need to convert the database to a new marker system, we still have a sample on file for that animal. Even if it is alive, you don't have to go through the hassle of collecting a new sample. We just retrieve it from archives.”







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