The Punnett Square | Preservation Breeders Can Utilize Population Genetics – What are some of the craziest things you have ever heard someone say about the heritability of traits where it concerns breeding dogs? Are there still some “old tales” you hear from time to time? Some recent ones that I have heard include “crossing colors dilutes the richness of black” and “you’ll never be able to breed away from the reproductive problems—they’re just part of the breed.”
In spite of what people may think, believe, or feel, we are at a remarkable crossroads in time, where scientific theories of the 19th Century have paved the way for revolutionary 21st Century technology. What our predecessors, 40-60 years ago, were merely observing as correlations of heritability in the whelping box can now be analytically predicted with DNA obtained from blood samples and cheek swabs.
Let’s take a look back at some of the history on genetics. We’ll also discuss coefficients of inbreeding and some aspects of population ecology. More importantly, what does this mean for breed preservation, and why is an understanding of “population” so important?
The Principles of Heredity
Gregor Mendel is recognized in the biological sciences for discovering the principles of heredity. Originally from Brno in the modern Czech Republic, Mendel’s scientific work during the mid-nineteenth century focused on cross-pollination of pea plants, wherein he was able to describe the resultant expression of dominant and recessive traits. At the time, this work was ground-breaking and revolutionary, but it took another 35 years before it was widely acknowledged and accepted.
Going hand-in-hand with Mendel’s inheritance of traits is the Punnett Square. Most of us who breed dogs are already familiar with this. A surprising number of us, I have learned, have never heard of it. Named for Reginald Punnett, a British geneticist of the late nineteenth and early 20th centuries, the Punnett Square is a tool used by biologists to predict the probability of genotype outcomes. That is, what will be inherited in the offspring of two parents. For example, a color trait in dogs, like black, is dominant, the K locus, and when it is recessive, it allows for the expression of colors other than those controlled by the E locus. Where “Kb” is dominant and “ky” is recessive, and two parents each with a genotype of “Kbky” are bred together, then we would be interested to see a Punnett Square revealing a 25% probability of dominant black offspring, a 50% chance of black offspring carrying recessive black, and 25% probability of offspring that are not black. Therefore, it would also be interesting to know which other color alleles the parents are carrying in order to predict the potential genotype and phenotype of that other 25% that will not be black.
THE PROBABILITY OF INHERITANCE
The Punnett Square can also be applied to other traits, such as hereditary diseases, like Degenerative Myelopathy, a degenerative neurological disorder. While it may be tempting to toss carriers from gene pools, it is not necessary. A Punnett Square can be employed by breeders to illustrate the probability of inheritance in order to avoid producing an affected offspring. For example, if we take a clear dog designated “NN”, and a carrier designated “NC”, and draw a Punnett Square, we would see that the odds of producing an affected dog are 0%, with the odds of producing a carrier at 25%. Keeping carriers in a breeding program has the beneficial effect of increasing the size of a breeding population, and keeping genetic variation high in order to avoid other pitfalls that can occur, such as bottlenecking.
“Population” is defined in Ecology as a group of individuals of the same species living and interbreeding within a given area. This is an important concept to understand where it concerns purebred dogs. If we replace “species” with “breed” then we immediately understand that we have reduced our “breeding population” from all dogs to just the dogs in that breed. If we understand further that, “…within a given area…” can mean the population of our kennel, of our peer group, of our breed club, of our State, Region, Country… we can see how this population can begin to expand to include more individuals in the population of consideration. When we have the mechanisms at hand, why stop at Country-level? Why not think globally?
Bottlenecking generally occurs in a population due to natural events such as droughts, diseases, famines, etc., which act dramatically to reduce a population size in rapid proportion. One theory known as the Toba Catastrophe was presented in the 1990s, suggesting that our own human species crashed to between just 3,000 to 10,000 individuals worldwide (compared to nearly 8 billion today thanks to the Industrial Revolution). The consequence of a genetic bottleneck, as you may have already concluded, is that it significantly reduces the size of a population, and subsequently, the genetic variation within the population. Only through mechanics of population genetics can bottlenecks be resolved. These include gene mutation, gene drift, and gene flow. While the Toba Catastrophe theory is not without controversy (as with most human evolution theory), it is illustrative of the problems which can occur in species—or breeds of dogs—when genetic bottlenecks do occur. This includes inbreeding depression, which can be particularly detrimental to small populations. Consider a small population of a rare breed in North America if it is cut off, isolated, and highly inbred to maintain “type.” Think back to what was mentioned earlier about “in a given area.” The breeder of rare breeds MUST collaborate, and MUST collaborate far and wide. It is grossly inefficient to say “I must protect my breed and trust no one,” or the fatal flaws of inbreeding depression will be perpetuated.
Coefficient of Inbreeding
Dog breeders may be more familiar with concepts of inbreeding depression through another term, such as coefficient of inbreeding, or COI. As we see in some breeds like Doberman Pinschers, a population does not even have to be small in number to have a great frequency of dogs with high COI. What does a high COI mean? From a preservationist’s point of view, a high COI means there is low genetic variation and more problems caused by inbreeding depression. For a fancier who likes to win ribbons, a higher COI means that there is more consistency of type across offspring. Which is more important? Can there be a balance? According to the Institute of Canine Biology, a COI of 5% or lower is best to maintain type while preventing going into what they term as “the extinction vortex,” or the inbreeding depression problem.
The Institute describes the “extinction vortex” as a negative feedback loop. It is a level of inbreeding which produces smaller litters, higher levels of mortality, and expression of defects, with a negative effect on the size of the population, resulting in more inbreeding and more problems, eventually driving the population into extinction. Sadly, we see this happening in our purebred dogs each year. Recently, I was told by one well-respected breeder-judge, “You’re never going to fix these problems; it’s just part of the breed.” The same person told me that I have to breed type, type, type. This only perpetuates the negative feedback loop.
Before moving on, let’s consider that population growth has an equation: N = (b-d)+(i-e), where “N” is the number of individuals in a population, “b” is the number of births, “d” is the number of deaths, “i” is the number of immigrants, and “e” is the number of emigrants. AKC parent clubs typically keep statistics on “b” and “i” but not on “d” or “e.” While these numbers can be extrapolated, particularly in the United States where “e” is typically low, birth rate and death rate can be controlled and influenced by biologic factors. These include the inbreeding depression mentioned earlier which affect fecundity (offspring produced per female), sperm motility, congenital defects, heritable predispositions to disease, etc. Death rate can also be influenced by infectious diseases like canine influenza, heartworm, parvo, or other preventable agents. The end result is decreased “fitness” within the population.
What color will the puppies be?
One question that I always get when my Estonian, Red Affenpinscher male produces a litter is “what color are the puppies?”
While he was born as a surprise to his breeders because they did not do genetic trait testing, we have that benefit now. And so, it is possible to predict the outcomes for a litter.
In the hierarchy of color inheritance, the E locus controls for white, cream, “apricot” in Poodles, “yellow” in Labradors, and “red” in Affenpinschers. When it is double recessive, it overrides the K locus which controls for Black.
In the graphic below, which is a Punnett Square, “E” represents the dominate allele for White/Yellow/Red, and Kb represents the dominant allele for Black, with “e” representing recessive and “ky” representing recessive black.
We can see that the offspring produced from the mating below, where one parent (on the Y axis) is phenotypically Black, and the other on the X axis is phenotypically Red, the offspring outcomes are as follows:
75% phenotypically Black, and 25% phenotypically neither Red nor Black. The color of the minority offspring will have their color determined by another locus. In Affenpinschers this will most likely be the A locus which will produce a Belge or Black and Tan, though other colors are not out of the question.
Fitness for Reproduction
You may have already heard this term “fitness” before. It does not, as some think, refer to musculature or athleticism. Instead, it refers to the ability of an organism to survive and reproduce, thus passing on its set of “successful” genetics to the next generation. In a population—there is that word again—replacement rate, that is, the number of offspring required to replace each parent, is approximately 2.1 for each female’s reproductive lifetime. To grow a population, such as a rare breed on the verge of extinction, the birth rate (plus the immigration rate) will need to be greater than 2.1, and the COIs will need to be kept low. It will do no good to have a high population with high COIs as this will only serve to exacerbate the vulnerability of a breed and hasten the pace into the extinction vortex.
My own goals for breed preservation have been to focus on genetic variation, and to keep COIs low. This is not entirely possible by limiting my programs to US-bred dogs alone. We have already, in this country, high levels of inbreeding and a vanity for type that is detrimental to preservation in too many of our breeds. How do we quickly achieve greater genetic variation in our US dogs then? The solution is simple: import.
Why import, though, when there are so many dogs available here? While this may be true in more popular breeds, it is not true of every breed that the AKC recognizes. Even in already established breeds with solid populations, COIs can be (and are) relatively high as a consequence of maintaining type. Because our canines live in our man-made environment, a genotype that may not be adaptive or able to thrive can be kept alive and able to reproduce more progeny with more maladaptive traits. Take, for example, breeds that are only successful because litters are consistently born via cesarian section. Could these dogs do what they were originally bred to do if they are otherwise sick, broken, or unable to provide some level of care for themselves? We also see a lash back from puppy buyers who blame breeders for deaths like epilepsy. Deleterious recessives or “health problems,” or other undesirable traits, can be bred away from first, if breeders know about them, and second, if breeders understand the mechanics for breeding away from deleterious recessives using genetic diversity tools.
A ‘Giant’ Example
I originally started importing to expand my line of Pepper & Salt Giant Schnauzers. My foundation bitch, born and bred here in the United States, was heavily linebred to maintain her kennel’s “type.” If I had limited her reproductive future only to dogs in the US that I knew about at the time, I would have repeated the pattern and perpetuated the problems that she came with. Logic dictated that if I was going to correct those problems I needed to look abroad. As a consequence of my bitch’s COI, some level of inbreeding depression was already apparent. Aside from her irregular heat cycles, she missed her first two matings, and when she did finally get pregnant from a surgical AI, she only produced one singleton. The sire was a 100% outcross from Italy. That offspring is a 0% COI puppy with no common ancestors, and he is now an AKC Champion.
After a considerable wait, I finally have a mate for him, but the process of reproducing this rare variety of Giant Schnauzer is very slow, methodical, and a concentrated effort. While working to maintain a semblance of type is important, preservation of the Pepper & Salt Giant—which is nearly extirpated from North America—takes priority. In fact, the Pepper and Salt already went through a genetic bottleneck event after World War II and had to be recovered by crossing back to the Standard Schnauzer (as the German Pinscher recently did as well). We also have evidence, thanks to the Veterinary Genetics Laboratory at the University of California-Davis, that our Pepper & Salt Giants are genetically distinct from Black Giants. These really are two separate breeds. While the color is described and stated in the breed standard, we also have fully approved judges who ask, “Is (Pepper & Salt) an allowed color?” “Are you in the right ring?” and state further, “I’m sorry, I’m going to have to withhold because I don’t know how to judge this.”
Many of our breeds are in danger of extinction. The Kennel Club (United Kingdom) lists 32 endangered native breeds and seven more classified as “at watch.” While the AKC does not have a similar list, it does have an article listing “11 rare breeds you never knew existed.” While “most popular breeds” is frequently available, it is of little value. It would be helpful for those truly interested in preserving our purebred dogs, and for purposes of raising awareness, if AKC published a list each year similar to the Kennel Club. It is possible that some breeds are in greater danger, and that some will disappear in our generation while others are in no danger at all.
Regardless of the breed you have committed your heart to, breeding practices which enhance genetic variation by including the introduction of new genetics into a breeding program—whether they come from another country or from another breeder you met at a show—will ensure future breed vitality and “fitness.” We can all achieve positive outcomes for purebred dogs by supporting the importation and free movement of dogs between countries, collaborating wisely with international partners, keeping our contractual promises intact, maintaining good relationships, and by showing these dogs both here and abroad whenever and wherever possible.