The commercially available marker panels are predominately comprised of markers that do not code for a change in an amino acid, protein, or gene function but are chosen because they are high enough in minor allele frequency to be of value in association studies. Most are simply no more than marker locations down the chromosome much like mile markers along the interstate. They tell you where you are close to, but they themselves offer little to no real value as to giving you a service. These markers panels are not designed from looking at sequences from all of our breeds and picking markers that are truly unique to the breeds as the markers are picked to be informative across a lot of breeds for association testing (ie is marker x significantly related to meat quality), something far different than to identify what breed an animal is composed of. I do believe with a large enough test population, we can lump animals into broad groups (breeds) based on the allele frequencies, but as was warned in the Michigan State work, animals in underrepresented subpopulations of a breed don’t fit and have failed the set thresholds while in fact they do meet breed standards when more animals are included in the reference panel.
These tests are not for purity but an estimation of composition and that estimation is based 100% on the animals chosen to represent the breed. These tests simply compare the allele frequency of the test animal back to the reference animal for all 10,000 markers as in the case of what the NSR is using. Most of the time, the test that the NSR is using is comparing the test animal to the average allele frequency of the breed, between 0 and 1, (with very few markers being at 0 or 1 for any breed, ex 0.55 for breed A while breed B is 0.75) as there are very few markers, especially on these types of genetic marker panels that truly differentiate breeds being all homozygous for one allele. Think about it, each of these marker tests is to identify which of the 4 DNA bases (A,C,T,G) that an animal has with most of the tests being designed to only check for two of those bases at a time, such as for stress where the test identifies if the pig has a C or a T at the 1843rd coding base for the RYR gene. So the majority of the markers are not testing any definitive breed marker, but are testing against average allele frequencies between the breeds. If the wrong animals are chosen or not enough animals are chosen to represent the breed, then you can easily get into a situation just like the Hampshires. Choosing the “correct” animals, if less than all the parents of the last X generations are chosen, will have anyone on the wrong side of “pure” having sever doubts as to the completeness of the reference panel or even the possibility of a personal vendetta against them if ancestors that their animals go back to are not on that panel. Keeping the panel secret as in the case of the NSR, only brings both of those doubts to the forefront and do neither party any good.
There are 3 rather important things that cause allele frequencies to change with time. The first is genetic drift which is important in small populations such as North American swine breeds. The second is from genetic bottlenecks. This can be thought of as where there is a hot sire that is used heavily in a breed. His genetics are now heavily distributed through the population. The third is through genetic selection. When we select on a phenotype, hopefully that phenotype is due more to genetics than a feed bucket. Therefore when we change the phenotypes we desire, we are in fact changing allele frequencies. Why am I bringing up allele frequencies and change when discussing this breed “purity” test you ask? I am bringing it up simply to point out that we are constantly changing the allele frequencies of these markers by selecting animals to meet our desired phenotypes. These breed composition tests are simply testing markers and as I stated earlier the markers in these panels are typically not directly responsible for a single genotype.
As for the costs and testing time, I have called and personally received quotes far less than what the NSR is having to pay for their exact test. It might be simply because I know more of the people running the testing agencies personally, or because time has marched on and the costs have come down. It does not help that the NSR is one of the few groups using a smaller panel when most all the commercial companies are using the larger testing platforms (10k markers vs 50k+) at a cheaper cost. Part of the cost dilemma is simply from the number of samples that are being run by a single entity with the more animals you genotype the cheaper it is per animal to genotype. Even many of the commercial companies (companies that are competing with each other) are teaming up together to put in larger orders so they can bring the cost down. The commercial swine genetics companies are running so many samples that they receive a far cheaper price than what I can get at a university. One such company, noted to me they can get genotyping completed on a much larger panel (50k markers) for under $20 each animal and have a much faster turnaround time. There is currently the opportunity presenting itself to potentially team up with some of the companies for an order which would help all entities lower their cost and ensure there are enough samples in the pipeline to keep the turnaround time down (far less than 6 weeks). These tests are designed for high throughput genotyping with larger volume helping to lower the cost. The test that the NSR is using needs 24 animals to be run at a time while other genotyping platforms use 384 animals at a time. Don’t balk at the larger numbers, because the genotyping companies can put samples from multiple farms on the same plate to be run at the same time. This grouping will save costs. Essentially all of these genotyping platforms also have the ability to add in the genetic test for stress. The companies simply work with the organization that holds the patent and will charge the producer the cost of those tests only if the producer requests it. Essentially allowing for all the markers that need to tested to be tested at once saving time and energy. The commercial companies are also getting better genotyping costs by using the AllFlex Tissue Sampling Unit. This DNA capturing system gives far better DNA quality than semen samples, hair samples or blood cards and is easier to use. More importantly, because everyone is using the same system for DNA capture, this allowed the genotyping lab to streamline the DNA extraction process as well. Anything that makes the process simpler for the DNA lab will help lower costs.
As a geneticist, I want the pedigree integrity to be maintained above all else. If the pedigree is correct, then by extension we know the breed is correct. EBVs and breed improvement are based on taking information from the pedigree and using it to predict the animal’s value. Just knowing an animal is a certain breed offers no value to make within breed improvements with. However, knowing the pedigree and that the pedigree is correct offers a lot of valuable information to make selection from. One of my geneticist colleagues asked me what the value of a pedigree from a breed organization was if we simply needed a quick DNA test for breed identity. To me, there is a lot of weight to this statement when it seems like the breed organizations don’t want to test the dam side of the pedigree, which is where the majority of folks seem to “slip in” unpure genetics simply because for so long the dam side has never been tested. A breed “purity” platform such as what the NSR has set up still does not address the dam’s side of the pedigree thereby allowing folks that truly still want to slip in different genetics to do so if they want to take the time. Using the Hampshire 90% rule, one can take a cross dam and within 4 generations breed up and get animals that will pass. I am 100% behind catching those that cheat the system, but am simply not sure if this method is the most cost effective.
If the CPS wants to venture into DNA testing, my suggestion would be to test ALL the sires of the last few generations. Jack can pull the numbers and let us know how many sires and dams had litters recorded out of them the last several years. From a genetics standpoint, this isn’t an overkill. Before someone mentions it costing too much and want to start looking for cheap alternatives, I simply ask you to look at the Hampshire issue and ask if you want that to occur in your breed? Spend the money to do it right or do not do it at all. Since we do not have dams DNA banked, this would need to be started. Dams of all progeny recorded for say 1 year after a vote to include DNA testing should also be included in the reference panel. There may well be animals that do not fit the typical breed composition profile that are identified at this point. It is then that the breed board will need to make a call as to what the board is willing to keep the pedigree to or to pull it. However this larger all-inclusive testing will ensure all subpopulations are represented and get a fair shake. It is at that point that I would recommend putting the breed panel to bed and only using it for historical sake. Animals that are verified by DNA to be from passing parents are pedigreed. To keep costs in check, testing can be performed on all sires, class winning animals at shows (type conference and junior shows), and possibly expand to all dams in the future if the genotyping costs come down more. At least by having the DNA of the dams on file, there is the ability to test for verification of parentage on both sides of the pedigree. There is no need to test back against a threshold on an ongoing basis. As evidenced in several instances, having a threshold opens the very distinct possibility of having failing progeny out of parents that pass as well as littermates on both side of the threshold. Breeding two animals that are each 90% can result with animals falling anywhere from 80% to 100% where we expect half of the animals to be at or below 90%. Most importantly, this continuous threshold offers no value for genetic improvement and in fact hinders it as it penalizes animals that deviate from historic allele frequency. Progress always involves change and in this case the change is in allele frequency.
We all know that we inherit half our genes from each parent, but what is not set in stone is does the sire pass on his sire’s or dam’s chromosome to his offspring for each of the 18 chromosomes (plus X and Y but we all know where those come from in sires). Each chromosome is inherited independently from the rest and there are crossover events where the chromosome that is passed on to the progeny has both the paternal grandsire and paternal granddam segments. The point here is that while on average the genetics of the offspring are the average of the parents, on an individual pig level using an average is dangerous. As an example, if both parent animals are 85% pure, it is distinctly possible that a majority of those “non pure” alleles are passed on to an offspring. This would be the case if the majority of the “non pure” alleles originated from one side of the pedigree or simply by chance. You could theoretically have a progeny that is as low as 70% pure while a littermate is 100% pure. That is my concern with setting an arbitrary threshold in relation to the percentage of pure alleles an animal must have to be pure. This would have a never ending cycle of animals kicked out at shows with both breeders and kids getting black eyes for being below a set threshold even if both parents were legitimately above the threshold and the animal genotypes as being a progeny of them. We could go on testing animals forever and never reach the point where animals don’t fall below a threshold percentage. Genetic selection for any trait or phenotype will cause the alleles closest to the genetic base(s) that control that phenotype to be selected for as well due to what is termed linkage disequilibrium (LD). LD, simply put, can be thought of as the markers closest to the genetic change affecting the trait also being selected for due to how close they are to the causative change and the low probability of a chromosomal crossover between the two points. The question then becomes are they the markers that are classified as being native to the breed or not?
Let’s not forget the breed heritage of Spots and Polands. It is far easier to come up with allele frequency differences between the distinct NSR breeds, but will be far more complicated with a lot more room for error with Spots and Polands.
One thing to keep in mind is that in the court of law, DNA evidence is seldom if ever used to prove something is true (ie DNA match) but more of a more likely than not something else. It is far easier to use DNA to prove that something is not a match, ie multiple alleles are not a possibility as the defendant is something else.
My suggestions would be:
For the breed associations to genotype all breeding stock now and use that as the measuring stick for what is pure and what is in the complete breed pool vs small reference populations. There may still be the need to eliminate boars or sows that obviously do not fit, but it is doubtful it will be as many. With an increase in the number of animals tested, the cost for the test should drop and the turnaround time would drop as well as there would be more animals to test allowing the genotyping company to reach the desired numbers of animals per run much faster. Using the new genetic profile and testing sows would eliminate the possibility of people bringing in dirty genetics on the sow side. At the very least, require a DNA sample on the sows to be checked when animals are flagged. Technically you wouldn’t need to test for breed purity going forward once you decide what is “pure” and you perform progeny DNA checks against the parents. This would eliminate the percentage threshold test for purity that is not a long term solution. Once it is settled that the animals that are in the breed are “pure”, then only parentage checks (of both sire and dam) would be all that is needed to prove an animal is pure. This proposed method would be the gold standard for maintaining breed and pedigree integrity going forward.
There is far more that I would like to say, but since this letter is already long, I will stop for now. This isn’t a simply subject and before anyone acts there needs to be a thought out plan of action. Feel free to ask me any questions that you might have on the subject. If the board would like someone to offer additional insight at their board meeting when discussing this topic, I would be more than happy to travel to be there in person or present via webinar. My email is benny.mote@unl.edu and my work phone is 402-472-6033.
Regards,
Benny Mote
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