If you're a dog breeder and you believe in genetics (of course you believe in genetics!), you know that there is a certain predictability in breeding that is the result of genes being passed from parent to offspring. You take advantage of your understanding of the process of inheritance when you breed for certain colors, or you use a taller sire to put a bit more leg under your next litter. 

On the other hand, as most breeders will tell you, "breeding is a crap shoot". You are trying to breed for one a particular trait in your puppies and get instead a litter of puppies with something else or, even worse, a mish-mash of variety that beggars your belief in genetics.

Of course, the problem here isn't that understanding genetics is worthless. It's that you are trying to select a breeding pair based on the genes you would like them to pass on to their offspring. You are trying to select for particular genes, but you are assessing the genetic merit of a dog for a particular trait based on its phenotype - how those genes are expressed in a dog. This method of selection - evaluation of phenotype - only works well if phenotype is a good indication of genotype. As anybody who has ever bred a litter of dogs knows, for some traits phenotype is a good indication of genotype, and for other traits it isn't.

The most important concept in selective breeding is "heritability". If you ask 10 breeders to define heritability, I can almost guarantee that most will get it wrong. Most people will define heritability as the inheritance of a particular trait by a dog.

​But actually, heritability is a concept from population genetics. Heritability tells you how much of the variation in a trait in a group of dogs is the result of differences among them in their genes.

Now, let's say we could create an exact clone of each of those puppies and raise that duplicate litter separately. Each puppy goes to a different home, is fed differently, exercised differently, and raised under conditions that are unique to that puppy. Now when you measure the weight of each puppy at 1 month, you will probably get different numbers compared to each pup's clone, and there will probably be more variation in the litter. The pups have exactly the same genes, but ended up at 1 month with different phenotypes. We know that genes are important to weight, but in this case there are clearly effects of environment.

Here is where most people get tripped up on the definition of heritability. In the first example, the heritability of weight will be equal to 1. In the second example, the heritability of weight will be less than one, perhaps something like 0.3. Same trait, different heritabilities. Why? Because heritability is not about a particular trait in an individual, but rather tells us about the trait in a population of dogs. Here's why.

The phenotype of most traits depends on both genes and environment. We could write this in shorthand as

So for our example of weight in puppies above, the puppies in first litter were raised in identical environments, so the "E" term in our little equation is 0; there is no effect of environment on puppy weight, and all the variation you see among the puppies is due to genetics. So for these puppies, the heritability of weight is 100%.

I don't want to get into the details of heritability and how it is determined (these are things we discuss in the ICB courses). Rather, I want to make a simple but extremely important point. 

When you assess particular breeding combinations, you use whatever information you have about phenotype of the parents or relatives to make assumptions (or guesses, really) about the genes each parent is likely to pass on to each puppy. If the heritability of weight at 1 one month is 1.0, as for the first litter, you know that 100% of the variation in weight from pup to pup is due entirely to genetics. If you wanted smaller size in your next litter, you should keep one of the smaller puppies to breed and you should also get the genes for smaller size.

But what about the second litter, for which heritability of weight was only 30% because there was lots of variation in how they were raised? If you want smaller pups in your next litter, you can't assume that you are correctly selecting the genes you want for size just by the weight of the puppies. Some pups might have had more to eat, some might have been couch potatoes and put on some puppy fat, some might have been slow eaters and never got as much to eat as the others. In this case, phenotype is not a very good indication of genotype; there is lots of variation in phenotype because the "E" term in that simple equation above is not zero.

Okay, here's the rub. For most of the traits you want to select for (or against), there will be multiple genes involved as well as non-genetic factors that will affect phenotype. If you select the pick of the litter based on phenotype alone, you will not necessarily be selecting the puppy with the best genes for that trait. For puppy weight with a heritability (in our example) of 0.3 (30%), most of the variation in phenotype (70%) is because of things that have nothing to do with genetics. If your strategy for selection relies heavily on the phenotype of the dog, you are more likely to be wrong than right. 

If you don't know the heritability of a trait you are trying to select for or against, you are likely to make a lot of incorrect decisions based on phenotype alone. If you're assuming the heritability of a trait is 1.0 - i.e., what you see is what you get - you are going to have a lot of those "breeding-is-a-crap-shoot" experiences, selecting for one thing and getting something else. On the other hand, if you know that the heritability of a trait in dogs raised in uncontrolled conditions (different households, various diets, variable amounts of exercise) is 0.15, then you should understand that phenotype is not a very good reflection of the genes for that trait in an individual puppy and that you will not be very good at selecting the puppies with the genes you want. On the other hand, if the heritability of trait is typically 0.8, then phenotype will be a decent reflection of the genes for that trait inherited by that dog.

The critical point here is that heritability tells you how much of the variation in a trait is due to variation in genetics. If heritability is low, efficient selection will be difficult because phenotype is a poor reflection of genotype. If heritability is high, more of the variation in the trait among individuals is a result of genetics, so selection based on phenotype will be more effective.

There are ways to get around the limitations of selection when heritability is low. One is to use information about the relatives of a particular dog. If the heritability of hip dysplasia in a breed is usually about 0.2, and you found a sire you like but his hip score is mediocre, have a look at the hip scores of his litter mates, his parents, and, if he has already been bred, his own offspring. All of this information will provide clues about the genotype of that sire you like. If his relatives have good hips and he is the outlier, you can have more confidence that he probably has good genes, but perhaps he had an unfortunate experience as a puppy or was overindulged by an owner who fattened him up by rewarding everything he did with a cookie. You can do this assessment statistically using something called "estimated breeding values" (EBVs), which does some fancy math and comes up with a number that tells you about the quality of genes in a dog for a particular trait. (Some kennel clubs now provide EBVs for hip dysplasia.)

Hate that "crap shoot" part of breeding? You can reduce the guesswork in breeding by understanding heritability. Know the typical heritability of the trait you are selecting for, and take that into consideration when you evaluate phenotype. And also make use of information available from the ancestors, siblings, and descendants of a dog, because those animals share many genes and can help you decide how much weight to put on your assessment of phenotype when you are trying to select for particular genes.

Breeding will always have an element of chance, because that's how genes are inherited. But it doesn't have to be a crap shoot. Understanding how to use some simple tools like heritability can help you make better decisions and result in more predictability and consistency in your puppies.

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Breeding strategies should not be ideologies. They are the means to achieve a genetic end, and the savvy breeder will use them appropriately. Breeds with small gene pools that can no longer produce healthy dogs have to use breeding strategies to fix that problem, and in many cases cross-breeding is be necessary. This is the case with the Norwegian Lundehund, a breed on the brink of extinction that is bouncing back thanks to a scientifically managed cross-breeding program. There are other genetic rehabilitation projects ongoing that involve cross-breeding, all under the guidance of scientists with appropriate expertise. Cross-breeding is not a philosophy. It is a breeding strategy, and one I would point out was used routinely by "purebred" dog breeders 100 years ago because they understood that a strictly closed gene pool was unsustainable. They didn't do it because they were "pro" crossbreeding; they did it because it prevented the deterioration in health of their animals that resulted from too much inbreeding. Modern dog breeders have forgotten that lesson, and the long list of genetic disorders in dogs is the predictable result.

I do not have a "personal stance" about any aspect of dog breeding. I am not a dog breeder; I have no opinion about how to do it. I understand the science of breeding and the role of population genetics in determining the health of the gene pool and the population of animals. Everything I say is based on facts.

This isn't the first time somebody has rushed around fear-mongering about me. I am happy to tell you completely, clearly, exactly who I am and what I am doing. My "alignment" is to science, and to the professional scientists whose expertise and research is essential to understand how we can improve the breeding of dogs. I communicate with them, I collaborate with them, and I respect them. I get my information from them and the army of other scientists that are working towards improving the health of dogs, not from people on Facebook with opinions and no expertise. I suggest you do the same.

As a final note:
The International Partnership For Dogs (IPFD) is an organization of kennel clubs, canine health organizations, scientists, genetic testing organizations, and others involved in improving the health of dogs. They met just a few weeks ago in the UK to discuss issues affecting the health of purebred dogs. The producer of Pedigree Dogs Exposed was there, as she was for all of the previous meetings of IPFD. A committee was formed to address issues of genetic diversity in dogs, and she was placed on that committee. She has the respect of the people at that meeting, and if they respect her, so do I.

So stop the fear-mongering. Do your homework. Read the science. Learn something. Align yourself with the people that have the passion and expertise to do right by dogs and are working towards better health based on science.

​A paper has just been published that is being grossly misinterpreted by the press, and even by the Institute where the study was done (Marchant et al. 2019). This is the title of the study:

​These are the critical points of the study:

A) Norwich Terrier

• This is a study about breathing problems in the Norwich Terrier.
• The study found a gene that causes swelling of the airway tissues of Norwich Terriers, which iinterferes with breathing.
• The Norwich Terrier is not a brachycephalic breed.

B) Other breeds

• They also found the mutation in most Bulldogs.
• They found the mutation in only a few French Bulldogs.
• They did not find the mutation in Pugs.
  
  

C) Brachycephaly

• The Norwich Terrier is not brachycephalic, so the effect of this mutation is not linked to brachycephaly.
• Most French Bulldogs do NOT have the mutation.
• The mutation is absent in Pugs.
• Therefore, this gene is NOT the main cause of breathing problems in Bulldogs, Frenchies, and Pugs..

From these points, it is obvious that the press release issued by the Roslin Institute, where the study was done, is incorrect. This is what they said:

​"Dog DNA find could aid breathing problems
Scientists have discovered a DNA mutation linked to breathing problems in popular dog breeds.

Breathing difficulties are most often associated with flat-faced breeds, such as French bull dogs and pugs, but scientists have found the mutation is also carried by Norwich terriers, which have proportional noses.
​
The finding could inform future genetic tests that could help vets identify animals at risk, and help breeders avoid producing affected pups."

Read the points above. This study did not find a mutation that causes breathing problems in brachycephalic breeds. Only a few Frenchies had the mutation, and it is completely absent in Pugs. The mutation was found in Norwich Terriers, where it has NOTHING to do with brachycephaly because the Norwich Terrier is not brachycephalic.


This article is also wildly incorrect. The gene was not found in Pugs. So no, this is not the gene that makes Pugs wheeze. The gene does occur in Bulldogs, but this study did not examine whether it is a cause of breathing problems in this breed, for which there is much evidence that the brachycephalic head shape is a major risk factor for breathing issues. This piece doesn't even mention the Norwich Terrier. In fact, this headline should have said something like "Mutation that causes breathing problems in Norwich Terriers is not the cause of problems in brachycephalic breeds."
​

I repeat AGAIN:

​No, we have NOT found the mutation that causes breathing problems in brachycephalic dogs.

This is NOT a breakthrough for health issues in brachycephalic dogs. In fact, it essentially rules out this particular gene being a significant cause of the problem because it is absent in Pugs and relatively rare in Frenchies, both brachycephalic breeds, and it is linked to the breathing problems in the Norwich Terrier, which is NOT a brachycephalic breed. 

Only a day after publication, the social media groups for owners and breeders of brachycephalic breeds are all a'twitter with news of this terrific research development which (they claim) vindicates flat faces as the cause of breathing problems in their breeds. This study does not do that.  

REFERENCES
Marchant et al. 2019. An ADAMTS3 missense variant is associated with Norwich Terrier upper airway syndrome.
PLoS Genetics 15: e1008102. https://doi.org/10.1371/journal.pgen.1008102

Hip dysplasia remains one of the most significant causes of pain and disability in dogs despite decades of research into the causes and diligent efforts by breeders to reduce risk through selective breeding. A new review article aimed at veterinarians provides a useful summary of the current state of our understanding of its causes (Witte 2019). The comments here are detailed in that review.

A number of non-genetic (environmental) factors are known to affect the risk of developing hip dysplasia, and some of these appear to be especially critical to manage properly in the puppy. Housing on a slippery floor, access to stairs, and some types of exercise can significantly increase risk in puppies. Higher weight at birth and while growing elevates risk. On the other hand, dogs raised on farms and those born in spring and summer are less likely to develop hip dysplasia. Aside from nutritional deficiencies, or overconsumption that leads to overweight, there is no evidence that the type of the diet plays a role in development of dysplasia. Although exercise in the adult might lead to clinical signs of pain or lameness, there is little evidence that the amount of exercise alters the progression of development of osteoarthritis once the dog matures. Dogs neutered before 6 months have a higher risk of developing hip dysplasia.

Managing the risk of hip dysplasia remains a challenge for breeders and dog owners. Breeders must consider both genetics in mate selection and environmental factors, especially when the puppies are young. When puppies go to their new homes, owners must be educated about the factors that elevate the risk of developing hip dysplasia, especially weight, stairs, and unsuitable exercise and activities.
​
​

REFERENCES
Witte PG, 2019. Hip dysplasia - understanding the disease. Companion Animal 24:77-81.

The adrenal cortex is the outer layer of the adrenal gland, which is a small organ near the kidneys. It secretes hormones that are part of the stress response (e.g., cortisol), and it also produces aldosterone, a hormone essential for maintaining normal levels of minerals involved in the regulation of blood pressure.

In some dogs (and in humans), the immune system fails to recognize the tissue of the adrenal cortex as "self" and destroys it. The result is Addison's disease, which can be fatal. Addision's is especially common in some breeds, including Standard Poodles, Portuguese Water Dogs, Cocker Spaniels, Nova Scotia Duck Tolling Retrievers, and Bearded Collies. The frequency in Poodles is relatively high, estimated at 5-10% (Famula et al. 2003).

Studies looking for a genetic basis for Addison's have identified SNPs that might be associated with the disease, but causative genes have not been found. Because Addison's is an auto-immune disease, we might expect to find relationships between risk and the genes of the immune system. In dogs, these genes are called the dog leukocyte antigens (DLA), and they come in combinations called haplotypes. There has been some success in identifying DLA haplotypes that confer risk of disease in several breeds (Hughes et al. 2010, Massey et al. 2013, Pedersen et al. 2015). 

A study just out takes a closer look at the association between DLA haplotypes and Addison's Disease in Poodles, using a larger sample size than previous studies and also looking for potential associations related to sex of the dog (Treeful et al. 2019). The results are both interesting and sobering. While the data are for Poodles, the implications are relevant for every breed, so you should read this even if you don't have Poodles.

​Furthermore, haplotypes 2-8 and "other" are uncommon in the breed, which means that the options for "breeding away" from haplotype 1 to reduce the incidence of Addison's in male dogs will be limited by the paucity of dogs to breed to.  Haplotype 5 is a risk for females, presenting similar problems.

The problem here is that we know very little about the DLA genes and how they work. In mammals, the immune system genes have the highest diversity because the individuals with the most diversity also have the highest rates of survival. This tells us that high diversity is important, and we can surmise that low diversity will compromise the function of the immune system. In dogs, limited diversity from a small number of founder dogs, together with inbreeding and loss of diversity through selection and genetic drift, has resulted in both reduced diversity and uneven representation of haplotypes as seen in the Poodle, where haplotype 1 is overrepresented.

Breeders are likely to be strongly tempted to use information about DLA haplotypes to inform breeding decisions, based on the perception that the frequencies of some allele should be increased and others decreased. Dr Lorna Kennedy, who has studied the canine DLA genes for many years, cautions against this. (Note that Kennedy uses the term MHC, which stands for "major histocompatibility complex,"  the broad class of immune system genes to which DLA belong.)

The one recommendation Kennedy makes is to avoid producing puppies that will be homozygous for DLA haplotypes. Otherwise, as she notes, because the interactions among genes are complex and the workings of the immune system poorly understood, breeders should not attempt to alter haplotype frequencies through selective breeding. At the very least, if there is a serious problem in a breed, breeders could consult with experts that can advise on the best course of action.
​

Perhaps the best strategy, once again, is to protect the existing genetic diversity in your breed and avoid producing homozygosity by inbreeding. 

Breed Status

1. Breed history
2. Current number of dogs and locations
3. Pedigree information
4. Health information

Threats to sustainable breeding

1. Small population size
2. High inbreeding
3. Inbreeding depression
4. Small effective population size (Ne)
5. Health issues
6. Not enough breeders
7. Inadequate guidance for breeders

Breed-specific concerns

1. Iconic Norwegian dog of high cultural importance
2. Preservation of unique anatomical and behavioral traits​

​

• Increase the size of the population
• Reduce breeding age of bitches to 15 months
• Breed at least one litter from every healthy bitch
• Develop methods to assess breed-specific traits
• Develop methods to assess temperament and breed-typical behaviors
• Use DNA genotyping to assist mate selection
• Develop a comprehensive strategy for data collection on dogs, litters, health, etc.
• Develop an efficient breeding strategy using population genetic analyses
• Assess efficiency of breeding strategies using appropriate computer models

​

From the start, the Norwegian breeders progressed step by step following a planning strategy, the major outline of which was developed for general goals (e.g., increase the size of the breeding population), with tweaks and adjustments as necessary as the plan progressed. These breed-specific breeding plans are called "Rasespesifikk Avls-Strategi" (RAS) in Norwegian.

The first step in the development of a sound breeding strategy is determine what you have to work with, and a review of history to understand how past events have shaped the current population of animals. For this, they did a comprehensive review of history, documenting the historical use of the breed for Puffin hunting, events that affected population size and breeding such as disease outbreaks, legislation, changes in culture, kennel fires, and replacement of dogs with other hunting methods. They developed a pedigree database that was as complete and error-free as possible. They documented fertility, litter size, mortality, and disease when information was available. They also summarized historical changes in the level of inbreeding, number of sires and bitches in the breeding program, offspring per individual, and imports and exports.

​Threats to sustainable breeding
After assessing these data, they identified the problems that needed to be addressed and articulated the goals to solve them. For this, breeders worked closely with animal breeding experts and population geneticists at NordGen who assisted in developing and testing the particulars of the plan.

These were summarized as:
​ 

​Breed-specific concerns
There's no point in doing a genetic rescue if the dogs produced at the end aren't the breed you started with. A critical part of any program is to have a clear picture of the critical traits of type that make the breed unique so the breeding program preserves those in the dogs that result. These traits would include physical features like size, color, coat type, proportions, shape of ears, tail, feet, and muzzle, and also instinct and behaviors, temperament, features specific to purpose, conformation including gait, shape of eye, proportions and planes of the head, and so on. A special consideration for the Lundehund is the preservation of their extreme flexibility in the shoulder and neck joints, polydactyly, ability to fold the ears, rotary front movement, and double coat that is waterproof and warm. Overall, the goal is to produce dogs with good Lundehund type that are healthy and fit for function.
​

​Development of a breed-specific breeding plan (RAS)
The specifics of the RAS planning schedule are in their document (download below), which is broken up into sections that focus on specific aspects of the breeding plan. You can review that document, but here are some of the highlights: 

​The size of the breeding population affects critical aspects of genetic management. As for the Condor, the success of the breeding population will hinge on increasing the census number of individuals as rapidly as possible. This will improve genetic stability (chance fluctuations of gene frequencies) and broaden the options for breeding. A top-level concern for the short- and long-term goals of the program was to increase the effective population size (Ne), setting a goal of Ne = 200 (90 males and 110 females) by 2020-2025, as well as increasing the total census size of Lundehunds in Norway to 1,000 by 2025.  It was stipulated that the highest recommended inbreeding coefficient is 3.25, based on a 5 generation pedigree.

You can download these two RAS documents, which lay out the information and goals for the Lundehund breeding program. Note that the date ranges are a bit different, which I suspect reflects updates in the plan as they progressed
​
Download RAS Schedule (2014-2025):  Breeding strategy for the Norwegian Lundehund
Download RAS data summary (2014-2018):  RAS – breeding strategy for the Norwegian Lundehund

Many dog breeds are struggling to manage a growing list of genetic disorders at the same time as the options for managing inbreeding are becoming more and more difficult. Avoiding problems with the known mutations and lines producing complex diseases only shifts selection away from one corner of the gene pool and towards another, producing an ever-narrowing bottleneck that ultimately only makes matters worse.

The holy grail is discovery of a population of dogs that has been isolated for generations and contains genetic diversity long lost in the main population of dogs. Failing this, however, many breeds do in fact retain enough genetic diversity to improve health and open up new options for breeding. But when a breed reaches the limits of improvement using the diversity remaining in the breed, the only other option is to restore the genetic diversity that has been lost to selection and genetic drift. ​

The goal here is to outline a basic program to rehabilitate or rescue a dog breed that is facing challenges due to inbreeding, small population size, low genetic diversity, or other factors that make sustainable breeding difficult. The path to genetic improvement will be different for every breed, but the considerations are generally the same, so this can provide a rough template for the rehabilitation or rescue of any dog breed.

​
Genetic assessment of the breed from pedigree & 
​DNA data

Central to the development of a plan for genetic rescue is information about the current
genetic status of the breed. The pedigree database is a primary source of historical genetic
information, and DNA analysis can now be used to supplement this with much detail about
actual genetic diversity, gene frequencies, inbreeding, relatedness, etc.

Historical Information From Pedigree Data

1. Number and identity of founder dogs
2. Changes in population size over time
3. Loss of genetic diversity
4. Changes in effective population size
5. Average inbreeding over time
6. Average kinship over time
7. Genetic contributions of founders
8. Addition of unrelated dogs (new founders)
9. Fraction of dogs used for breeding

Genetic Information From Pedigree Data
The pedigree database can provide a significant amount of critical data about the current genetic status of the breed, including several measures of genetic diversity, genetic composition of current dogs, size of the gene pool, relatedness of dogs in the population, etc.:

1. Level of inbreeding (coefficient of inbreeding, F, or "COI")
2. Genetic relatedness (mean kinship, mK, and average mK for the population)
3. Effective number of founders (fe)
4. Eeffective number of ancestors (fa)
5. Founder genome equivalent (fg)
6. Effective population size (Ne)
7. Origin of genetic variation

​

Genetic assessment of the breed from DNA data

Genotyping using high-density SNP chips (e.g., Illumina CanineHD SNP chip; > 170k markers) can provide a wealth of information about the genetics of the current animals in the population, and it can also be used to estimate historical trends in effective population back hundreds of generations. Information from DNA analysis will be "realized" instead of "estimated" or predicted, because it is based on DNA marker status and not on probability of allele inheritance from pedigree data. 

Genetic information from DNA genotype analyses (SNPs)

1. Genetic diversity (observed and expected heterozygosity, Ho & He)
2. Genetic structure(Fst)
3. Current and historical effective population size (Ne)
4. Genomic inbreeding (as F)
5. Genomic inbreeding relative to the population (Fis)
6. Genomic kinship coefficients and kinship matrix
7. Fraction of polymorphic loci
8. Population genetic structure (e.g., principal components analysis, cluster analysis)
9. Genealogical relationships determined from DNA and cluster analysis
10. Assessment of disease risk without knowledge of the genes involved or mode of inheritance
11. Localization of inbreeding on individual chromosomes (runs of homozygosity, ROH)
12. Genetic relatedness to other breeds
13. Across-breed comparisons of patterns of homozygosity on the chromosomes (ROH)​

​

Assessment of Potential Breeds for Crossing

The goal of a genetic rescue is ideally to restore a breed to its original genetic state. We rarely know what that is, but 

​Phenotype Information

1. Size and weight
2. Conformation (e.g., proportions, topline, tailset, head size, athleticism)
3. Specific features of type (e.g., ears, skull, coat  type)
4. Temperament and behavior
5. Purpose

Genetic Information

1. Inbreeding and mean kinship within cross breeds
2. Haplotype sharing of cross breeds with other breeds (indicating historical relatedness or crossing)
3. Genetic relatedness to rescue breed
4. Shared blocks of homozygosity with rescue breed
5. Potential to produce genetic diversity in F1 crosses

By Carol Beuchat PhD

Okay, what other things do we need to think about in the process of getting from generation 1 to 2, and from 2 to 3, and onward?

If we had a population of dogs that were healthy and terrific examples of their breed, and we wanted to replicate that in the next generation, what should we do? We should make copies of all of the genes in all of the dogs of our founding populations, mix those genes up, and package them into puppies that will become the healthy dogs of the next generation.

And for the next generation? We want to do the same thing. Gather up all the genes in that first generation, replicate every single one, give them a good mix, and distribute them in the puppies of the next generation.

Of course, we might want to change something about the dogs - make the legs longer, or produce more dogs of a particular color. We can use our skills as a breeder to preferentially breed the dogs with the traits we want, using the magic of selective breeding. We make more copies of the genes we want by breeding more of the dogs with those genes, and this changes the mix of genes in the gene pool in the direction of creating dogs with the traits we want.

In a perfect world, we could continue like this forever.

But our world is not perfect. Not every gene in the current generation will end up in a new puppy, either because we have chosen not to breed some of the animals (selecting for longer legs or whatever), or simply by chance. Over time, selection and random chance will shape the nature of the gene pool, changing the frequencies of genes little by little with each generation.

If we started with founder dogs of perfect health, we want to make sure all the critical genes for health get passed from generation to generation so we continue to produce healthy dogs. But what happens if we lose a few of those, either by selection or chance? The Dalmatian, for instance, inadvertently lost a critical gene for nitrogen metabolism, with the result that the dogs suffered from the formation of urinary stones. How did breeders fix that problem? They put the critical gene back in the gene pool. They did this by crossing a Dalmatian to a breed similar in structure (the pointer), then selected the offspring that inherited that critical gene, which is identical in all dogs. It was a brilliant and simple fix. With each backcross into the breed population, with selection of the offspring that inherited that gene, the frequency of the new gene increased, the fraction of pointer genes in the gene pool dropped exponentially, and in a few generations the dogs were genetically pure Dalmatian.

Now, we have technology that might someday allow us to go in and replace that single lost gene using something called CRISPR. But it was easily restored, not with technology, but with clever breeding in a single cross.

Back to our hypothetical breeding program. If we have been randomly (by chance) or deliberately (by selection) losing genes every generation, and all the genes in our founder dogs were essential for some function, we can expect some things are not going to not work like they're supposed to. These broken things become genetic disorders - allergies, temperament problems, low infertility, cancer, kidney disease, heart failure, and any of a very long list of canine maladies.

We understand why this happens.

If we start with a population of healthy dogs and want to keep them that way, there's one critical thing we need to do - make sure every single one of the "dog" genes - the ones necessary to build a healthy dog - is passed on to dogs in the next generation, generation after generation after generation. ​​Fiddle with the genes for type all you want, but you have to protect that original collection of "dog" genes that are necessary for building dogs that are healthy and fit to do what they were bred for.

This is the critical feature of preservation breeding. If we don't do this, we will break things.

Reality
To be fair - and realistic - it's nearly impossible to get all the genes of one generation into the next one, even if that was our sole aim as breeders. It certainly doesn't happen if we breed in a way that guarantees that some genes will be lost, and the two obvious ways this can happen is just by chance (an inescapable property of genetic inheritance) and by selection. Dogs that are bred will pass on only some of their genes; and dogs that are not bred pass on none.

If we replaced each critical "dog" gene lost like they did for the Dalmatian, we could keep a healthy population of purebred dogs forever. But if those critical genes are not replaced, there will be a deterioration in the health and function of our dogs over time. We should expect this; it's an inescapable consequence of losing genes from the gene pool each generation.

As purebred dog lovers, this is where we are. We have dogs with great genes for type - the particular traits that make each breed unique - but we have all sorts of problems with function, even to the point where breeding has become difficult in some breeds. We are hoping to cope by indentifying the critical genes that have been lost (by looking for the broken genes that have taken the normal gene's place), and we are spending millions studying the diseases that have resulted from loss of a particular critical gene. But neither of these efforts is solving the problem because we are still missing the normal copies of those critical genes.

What else could we do to solve this problem of missing genes? Why not the obvious? Why don't we just put those lost genes back, or make more copies from the dogs that have them? For some problems, it will be just a single gene as it was in the Dalmatian. In some cases, it might be multiple genes that together are necessary for some important function. But if we understand that every gene in those founder dogs played an essential role in building a healthy dog, it should be obvious that the only solution to a problem caused by missing genes is simply to put them back. In fact, this is really the only solution to the problem. Without that critical gene for nitrogen metabolism, the Dalmatian will produce urinary stones - in fact, any dog missing that gene will produce urinary stones. To solve the problem, put the normal gene back in the gene pool.

The key element of preservation breeding
If you want to have a sustainably breeding population of purebred dogs, you have to prevent the loss of the genes necessary for function from one generation to the next, or you have to replace the genes that are lost. We can't do the first if we practice selective breeding (which of course we do), so we have to solve the problem by replacement. To get back to healthy dogs, we need to restore the gene pool needed for health.

If a critical gene has been lost from one subpopulation of the breed (e.g., the dogs in the UK, or the bench lines of a retriever), it can be restored from dogs in another population that have retained that genes. In fact, there are breeding strategies that will reduce the loss of genes from a population over time by taking advantage of the ability to restore a lost gene from another population of dogs in the breed. This strategy is used by animal breeders that want to breed from a particular population of animals for many generations. It involves some clever population management and rotation of dogs among several populations that are maintained by inbreeding. In fact, this is how wild animal populations are able to persist for thousands of generations. Individuals from one population migrate to another, brining with them some of the genes that were lost over time in the new population. If populations are prevented from doing this, if they are isolated on an island for example, they eventually lose so many of the genes necessary for function that they go extinct.

For some dog breeds, critical genes might be lost from the entire gene pool, as the nitrogen metabolism gene was for Dalmatians. To replace those genes will require crossing to another breed that is selected to address the specific genetic problems most efficiently. Crossbreeding is routinely used in animal breeding to change particular  traits or to restore genetic health when there has been a loss of genetic diversity.  Cross-breeding is just one of several strategies breeders can use to achieve particular goals. Inbreeding, linebreeding, outcrossing, and crossbreeding are all used strategically by breeders to "shape" the gene pool so it best serves as the genetic pantry of ingredients you use in your breeding program.

The secret to producing healthy animals generation after generation - not just purebred dogs, but animals of any sort - is to maintain a gene pool that contains all the genes necessary for health. Protect the genes you have, and replace the ones that are lost, and you can breed healthy dogs forever.

It would be fair to say that the pioneering studies conducted by Scott and Fuller using dogs in the 1950s and 1960s gave birth to the modern scientific discipline of behavioral genetics. It has only been in the last decade, however, that dogs have again caught the interest of biologists interested in the evolution and domestication of dogs, and once again research on dogs is pushing forward the fields of animal behavior and cognition.

With the availability of affordable DNA analysis that can provide information about hundreds of thousands - or even millions - of markers, we have started to look for the genetic basis of behavior at the level of the gene. A few genes have been identified that are associated with specific behavioral traits in dogs, like xxxxxxxx (Bridgett). There is also a growing number of studies that looked for the genetic basis of behavioral differences in dog. But as I explained in an earlier post, the associations of behavior with genes have been generally weak and not especially useful for identifying genes of major effect or improving traits through genomic selection. 

A new study, however, is a game-changer (MacLean et al 2019). It has been made available before submission for publication, so it has not been subject to peer review and you should keep that in mind. But what it offers is a tantalizing first look at links between genes and behavior in dogs. 

Previous studies have looked for variation in genes that could account for differences in behavior in a group of dogs. The strength of the association between genetics and a trait is indicated by a statistic called "heritability", and in most studies of behavioral traits in dogs of a particular breed, the heritability was found to be low; i.e., variations in behavior were not associated with genetics.

Instead of focusing on a particular breed, the new study looked for major differences in behavior and genetics across many breeds. And here, they were successful.

In this study, a large set of behavioral assessments from CBARQ testing of > 17,000 dogs was paired with DNA genotyping data for > 5,600 dogs of 141 breeds  by combining information from two separate studies (Hayward et al. and Parker et al.) that used the current gold-standard analysis platform (high density SNP markers; > 100,000). They identified 14 behavioral traits of interest and looked for differences in the behavior of breeds that appeared to be associated with differences in the DNA, which would tell them the heritability of these specific traits.

​What they found was fascinating.

In this graph, the behaviors they looked at are indicated down the y-axis, and the strength of the association between genetics and the trait, i.e., heritability, is on the x-axis. The stronger the association, the higher the heritability. The grey dogs are measurements of heritability made on dogs of the same breed. The green and yellow dogs display heritability of those same traits when the associations are made looking across breeds.

You can see here that heritabilities determined from dogs of the same breed are generally low, less than about 0.3, which means that 30% or less of the variations in behavior can be attributed to variations in genotype.

But when you compare across breeds, the average heritabilities are high, from about 0.4 (40% of variation explained) to more than 0.7 (70% of variation explained.

This is very exciting, because it's the first time we have been able to detect strong links between genes and behavior. 

This is a game-changer. Our understanding of the genetic basis of behavior in dogs has gone molecular.

We now have large databases for both behavior and genotype, and these will continue to grow in size because the methodology for both is standardized (CBARQ for behavior and high-density SNP for genotype). These allow us to do analyses like the one below, which is a "heat map" that depicts the behavioral scores by breed displayed along with the dendrogram displaying their genetic relatedness. You can easily find the sporting dogs because they are high in trainability, and the small breeds like the chihuahua, rat terrier, and miniature pinscher stand out for scoring  high in traits that we might describe as reactive or "fiesty" (e.g., aggression, fear). The herding breeds score high in trainability while the hounds are low. And now we are finally able to explore the genetic basis for major breed-specific differences in behavioral traits among dog breeds. 
​

This paper will need to go through peer review, revision if necessary, then publication, so don't expect to see the final version for at least a few months. But I suspect we're about to see a flood of studies that also leverage the large datasets now available for both behavior and genetics. Buckle up!

NOTE: If you are interested in the links between genetics and behavior, now is the time to start boning up on the jargon and concepts you need to understand these studies. You can find relevant articles in the blog posts on the ICB website (just search for a topic or keyword). Even better, ICB has courses designed for dog people with no background in science that will get you up to speed. This genie is out of the bottle. A little homework now to learn the basics will pay off every time you read about an exciting new paper about the genetics of behavior in dogs!

Wow. What on earth is that?

This is not a paper about behavior. It is about measuring something called heritability (h ^2). Here's the translation of that first equation:

Heritability is the fraction of the variation in a trait that can be attributed to variation in genetics.

This study shows that there is a measurable fraction of the variation in a number of behavioral traits in dogs that is due to genetics. 

Heritability doesn't mean "inherited". It doesn't tell you how much variation there is in a trait. It doesn't tell you how likely a dog is to inherit a behavior. None of those things.

Clearly, to understand this paper, you need to be really clear on the meaning of heritability. You can start learning about it by reading some of my blogs on the ICB website, starting with this one:

                Understanding the heritability of behavior in dogs             

There are others if you do a search.

Also, please consider taking the Genetics of Behavior course. We will go through some papers like this one (including this one!) so you have a good understanding of what the information does - and doesn't - mean. There will be more studies like this one. Make sure you get the most out of them by understanding the science.

Check out those blog posts, and consider taking my course that starts (coincidentally) next week, 4 March 2019:

REFERENCES

MacLean EL, N Snyder-Mackler, BM vonHoldt, & JA Serpell. (unpub) Highly heritable and functionally relevant breed differences in dog behavior. BioRxiv preprint accessed 27 February 2019.. doi: http://dx.doi.org/10.1101/509315.

Dealing with the behavioral issues of dogs is a big business. Trainers and therapists abound, as do the professional suffixes tagged after the names of those who took some sort of course or were accredited by an organization.

I have no idea whether the majority of these people know what they're doing. I can tell you, though, that the scientific literature about behavior in dogs is a morass of tangled, insufficient, and often contradictory studies. In fact, in the field of canine behavior, science is marching forward very slowly, even as the ranks of lay behaviorists swell and practitioners claim to become ever more authoritative and confident of their expertise.

How reliable are behavioral assessments of dogs? Are they repeatable, meaningful, and verifiable? This especially matters when dogs are being assessed for working ability, as in breeding programs for service, police, and other types of working dogs. Breeding and training these dogs is time consuming and expensive, and being able to accurately assess the usefulness of a potential working animal is the differences between success and failure.

A recent study evaluated the reliability and validity of various behavioral tests that are used to assess the traits of working dogs (Brady et al 2018). What the researchers found should give pause to anybody that professes to have a deep and true understanding of canine behavior. 

For the selected studies, they evaluated the reliability, repeatability, and predictive validity of the behavioral tests,  both within and among "raters" (the individuals conducting the evaluation). They had an assortment of problems to deal with, like the use of different terminology for the same trait. More seriously, they found that data were often inadequately reported, statistical analyses were misinterpreted, and there was an overall lack of concordance in the various methods used for assessment.

​Ultimately, they concluded that there is "a widespread lack of information relating to the reliability and validity of measures to assess behaviour and inconsistencies in terminologies, study parameters and indices of success...This review indicates that we are still not addressing concerns over the lack of standardisation (sic) amongst research on dog behavioural tests". 

What this means is that we are probably not as good at assessing behavioral traits as we think we are or, at the very least, it's hard to tell how good we are because of limitations of the studies and the lack of standardization in terminology and protocol. Certainly, it should give pause to anybody relying on "experts" for advice, most of whom cannot lean on published studies to support claims about the validity and reliability of their assessments.

Buyer beware.

You can download a copy of the study below.

REFERENCES

Brady, K., N. Cracknell, H. Zulch, and D.S. Mills. 2018. A systematic review of the reliability and validity of behavioural tests used to assess behavioural characteristics important in working dogs. Frontiers in Veterinary Science 5(103);doi: 10.3389/fvets.2018.00103.

By Carol Beuchat PhD
​

Have you ever wondered how scientists figure out the genetic basis of a particular trait? This is especially difficult if the trait is polygenic and many genes plus environmental effects might be involved.

These are called "continuous" or quantitative traits; most of the traits under selection by dog breeders are of this type. For most of us, the process of identifying the markers associated with particular traits is a "black box" from which information mysteriously emerges from huge files of cryptic genotype data. How are scientists able to identify which of the many of thousands of markers play a role in the expression of a specific trait? ​​

In ICB's has a new course, The Genetics of Continuous Traits, we'll take the lid off the box and show you how scientists get from genotype to phenotype using DNA data you provide for your own dogs! For a learning example, we will study the genetics of body size, which is a trait that varies widely and continuously among breeds and mixed-breed dogs.

​We will use the raw DNA data file provided by Embark for your own dog, together with information about the size of your dog that you will provide. If we get data for enough dogs of a single breed, we can also explore the genetic basis of variation within a breed, also for a variety of mixed-breed dogs.

We will review some published studies for comparison with our own results, so you will also get to make comparisons with "real world" information.

This course is a great opportunity for you explore "under the hood" and learn how scientists go from a table of data to new information extracted from the data for your own dog!

Don't miss this great learning opportunity and the chance to analyze the data for your dog!

This unique online course is $125 and lasts 10 weeks, with students free to work along with the class or at their own pace. There are no prerequisites and a scientific background is not required - just your keen interest in dogs!

Class starts 11 February. Register now to join us!
​

Through a series of clever experiments, Mendel opened the black box that held the secret of the gene, which he concluded was responsible for the inheritance of traits in plants and animals. Knowing this, breeders could do some simple breeding experiments to reveal whether the genes for particular traits were dominant or recessive, which allowed them to design breedings that produced predictable traits.

While this took some of the mystery out of breeding for many traits, for other traits prediction remained elusive, and breeders realized that in some cases the genetics of a trait must be more complex. The problem, of course, was that some traits are determined by many genes. How could breeders do selective breeding on these so-called polygenic traits to reliably produce particular traits?

The puzzle was finally solved by a university geneticist named Jay Lush. In the 1930s, Lush realized that, for many traits, there could be lots of variation from individual to individual in a trait. That is, some traits were not "binary".

​For instance, there could be a range of size in a population of cows from small to large and everything in between, and not just two (binary) sizes of large and small. He realized that this was the case for many traits and began to think about how genetics could account for this. What Lush had stumbled upon was the key to selective breeding for traits that can vary continuously - genetic variation.

He described this in his groundbreaking book, Animal Breeding Plans.

Here, Lush is making the two observations that transformed animal breeding in the mid-1900s: 1) that genetic variation provides the raw material for selection, and 2) that some of this variation was "additive". Put another way, the effects of multiple genes acting on the same trait can be added. One large dog bred to another large dog could produce offspring larger than either parent.

This is what he is alluding to when he says that it's not necessary for a breeder to find the exact traits desired in a potential breeding animal, because genetic variation can be used to create a new mix of genes that will produce a trait not present in the current population. 

Lush's observation launched a new field of genetics, called "quantitative genetics", that was based in mathematics and statistics and applied specifically to traits that vary continuously. The advances in our understanding of quantitative genetics subsequently revolutionized animal breeding and was the basis of the spectacular improvements in production that became the modern system of commercial breeding of livestock and other animals.

One of the best examples of the gains that could be achieved by understanding the basis of additive genetic variation is for milk production in cattle. At the beginning of the 1900s, average milk yield was about 2,000 kg per year.There were no cows to be found anywhere that could produce 8,000 kg of milk per year. However, with the application of selection based on quantitative genetics in the 1940s, milk production soared and by 2000 it averaged more than 8,000 kg of milk per year - a four-fold increase in milk production over a period of about 50 years. Gains in production were made incrementally, a bit more each generation, and the basis for progress was finding advantageous combinations of genes that, added together, would push a trait in the desired direction. Breeders used the genetic variation in the animal population to create new combinations of genes in the next generation, then selected from among those animals the ones that improved on the trait over their parents.

There was one critical thing that was absolutely necessary for this to work: there needed to be genetic variation in the population. Breeders had to protect the genetic variation in their breeding stock, because without it new combinations of genes would not be possible and there would be nothing to select for.
​

The realization that selective breeding could produce animals that were superior to both parents revolutionized animal breeding. The use of additive genetic variation to produce animals with traits not present in the original breeding stock became the fundamental process of selective breeding in domestic animals.

Of course, dogs provide many examples of traits that have been shaped by selective breeding that takes advantage of additive genetic variation.

Body size is an obvious one. Researchers have identified multiple genes that can be associated with variation in body size in dogs. Some of these have a large effect, accounting for a significant fraction of the variation in size. But there is still much variation that is not yet accounted for genetically, and there might be dozens or even hundreds of other genes that have tiny effects individually but collectively account for differences among individuals.

Let's look at another example.

People probably started racing dogs thousands of years ago, and it's fair to suspect that over this time there has been selection to improve speed. 

One option would be to breed only the best to the best. Identify the sire and bitch with the fastest racing times and breed them together. Will some of the offspring be faster than either parent? Perhaps. Might some be slower? Perhaps. So what do we do from here?

We could mate the fastest male in the litter with the fastest female, and again select the fastest of their progeny. 

We might end up with dogs faster than the first two parents, but over the generations of inbreeding and strong selection (breeding only "best to best"), progress will start to level off. We will have eliminated much of the original genetic diversity in the dogs of the first generation. In fact, the puppies in each litter will share a larger and larger fraction of the same genes every generation. We are not going to get faster and faster dogs forever. Why? Apart from the deleterious effects of inbreeding, we have tossed out the genetic variation necessary to produce improvement in a continuous trait by exploiting additive genetic variation. We have bred ourselves into a dead end.

So how can you produce faster dogs from selective breeding and not run into this problem of a genetic dead end?

Redefine what you mean by "best". We do definitely want to select the best animals to breed, but we need to think of it in terms of the genetics of the population. To improve on a polygenic trait, we need genetic variation to select for. Instead of selecting a single best dog, we can select a subpopulation of dogs that are all faster than average. We will then use the genetic variation in those dogs, and the random inheritance of that variation in the offspring, to push racing performance in the direction of higher speed.

Can we keep producing faster and faster dogs forever? Probably not. If we don't run out of genetic variation, at some point we will run up against the limits of design and physiology. But we can definitely use selective breeding in this way to improve most traits that are polygenic in dogs.

So think about this. What traits do you strive for that are polygenic? Size. Temperament. Working ability. Coat quality. Ear length. Muzzle length. Shoulder angulation. Intelligence. The list is long.

Note that there is another issue here that breeding in this way addresses. The phenotype of polygenic traits is likely to be a reflection not just of genetics, but also of a roster of non-genetic factors, some of which might be known but most will not. When you select only the "best" individual to breed, you are selecting for the combination of genes and environment that produced what you perceive to be best. But you really only want to be selecting based on genes, because only that part of a trait can be inherited. Maybe the fourth "best" dog has great genes for the trait you want but was raised in a less than ideal situation. That dog's genes will get tossed if you only select the single best animal. By breeding a subpopulation of the top performing dogs, you preserve the genetic variation you need for selection and also reduce the influence of non-genetic factors in your choice.

You have no doubt noticed that this method of breeding, where the "best" is defined as a subpopulation of individuals instead of a single dog, is not the way dog breeders usually breed. We usually choose to breed only the dog we perceive to be the "best", ignoring the possible effects of environment on our assessment of the other apparently-inferior dogs. Doing this, we narrow the gene pool and increase inbreeding every generation, both of which will limit our ability to continue to improve traits. This applies to health as well as physical and behavioral traits. We can't "breed around" health issues if the necessary genetic variation no longer exists in the population of breeding animals.  

Can we breed better dogs by taking advantage of additive genetic variation? Yes, by changing the way we define "best" during selection. The improvement we can achieve might be significant - in health, in physical traits of importance, in temperament, and many other traits that we care about. 

Let's breed better, healthier dogs. Learn about the quantitative genetics of continuous traits and how you can use this knowledge to your advantage in your breeding program.
​

Since tests for specific mutations have become available in the last few years, the mantra of breeders is that their puppies are from "health tested" parents. To breed without first doing the available DNA tests is considered the height of irresponsibility in the fancy. But there is a right way to use DNA test results and also a wrong way. 

If you are reading this, you probably consider yourself to be a responsible breeder. Let's do some cyber dog breeding using some information from DNA testing and see how you do! All you need is a pen and paper along with your expertise as a responsible breeder.

Okay, we have a bitch we want to breed, and we have selected seven possible sires (S1 to S7). We have ranked these according to how well we think they fit what we're looking for in a sire in terms of conformation, temperament, health, longevity, working ability, or whatever criteria are important to you. We have given three green starts to the dogs we rank the highest and one green star to the dogs ranked lowest. However, we would be willing to breed to any of them. Assume we have done the relevant phenotype tests (e.g., hips, elbows, eyes, heart, etc.) and the results for all of those were acceptable.
​

The first thing we need to do is run the appropriate DNA tests for the recessive mutations known to be in the breed. In this case there is one, for the "red circle" trait. Our bitch is  carrier, as are two of the sires. I have identified the carriers with a red X on the chart, and the dogs that were clear have blue check marks.
​

Now we have our health-tested dogs from which we are going to select the perfect sire for our bitch. Using your rankings for overall quality, together with your information from health testing, rank the sires with 1 being your first choice and 7 your last choice. 

On your piece of paper, make columns for S1 through S7, and in the first row below that write your ranks for each of the potential sires.

​

Let's say you went to a little more trouble before making your breeding decision and you were able to get the  predicted average inbreeding coefficient of the litters that would be produced from our bitch and each of the seven sires. (You can get this information from the kinship coefficient, which is an index of the relatedness of a pair of dogs and can be determined either from pedigree or DNA information. The kinship coefficient of a pair of dogs is equal to the inbreeding coefficient of their potential offspring.)

Let's add the predicted litter COI to our table. With this information to consider as well, again rank the sires from 1 to 7 and add these data to the next row of your table. (Did this alter your first set of ranks?)

This is usually as much information as a breeder has about the dogs they want to breed. 

You did the testing for the "red circle" mutation in our potential parents and discovered that two of the sires were carriers. For both of them, there would be a 25% risk of producing puppies that are homozygous for the red circle mutation. You probably chose to eliminate those two sires from breeding consideration, which reduced the risk of producing affected pups in this litter from 25% to 0%.

You breed using your top-ranked sire and your bitch produces a beautiful litter of puppies. You advertise your pups as being from health-tested parents, keep a lovely pup for your breeding program, and send the rest off to pet homes. Sometime later one of the pet owners contacts you with the report that their puppy has a serious inherited blood disorder, and they ask how this could be if you claimed your pups were from health tested parents?

Indeed. How can this be???

We only know what we can know. We can know if a dog possesses a mutation if we have a test for it. We did our tests so we knew about the status of the red circle mutation in all of the potential parents.

But most dogs have many recessive mutations lurking in their genome for which there is no DNA test, and we have no way to know they are there if they are not expressed. Clearly, there was a mutation in your parents that you didn't know about. You feel horrible, offer to replace the puppy, and bury yourself in pedigrees trying to figure out how you could have produced a puppy with a genetic disease after being so careful.

If there is no DNA test for a recessive mutation, a disease can suddenly appear out of the blue that you have never seen in your lines if you happen to pair two carriers. This is why, if somebody claims that they only breed within their own kennel because they "know what's in their lines", you know that either they don't understand genetics, or they are being dishonest. It is not possible to know what recessive mutations are in your lines if you have no way to detect them.

So this leaves you in a pickle. All dogs have recessive mutations, and there are always going to be some that you don't know about. How do you avoid them? This is like tip-toeing across a mine field in which there are 3 mines that you can't see but are flagged, but you know that there are 28 more hiding out there somewhere. One wrong step and you will meet your maker. If these are mutations hiding in your dogs, this is a pretty hectic way to breed.

What if there were two mine-fields, but you can choose which one to cross. The first minefield has 3 mines you know about and can avoid and 28 that are hidden. The second mine field also has 3 that are flagged, but only 6 more that are hidden. If you're not ready to start your after-life, you would logically decide to cross the second mine field, where the risk of getting blown up is only 25% of the risk in the other one.

​Now we can see the problem. Our bitch has not one but three mutations that are also found in our "health tested" sires. Have a look at your last set of sire rankings. Now that you can see these other mutations, are you still happy with those ranks? If you would like to change your rankings, add another row to your table with these new assessments.

Great, you say. But in the real world, if we don't have tests for those unknown mutations, there is no way for us to avoid them, right?

Actually, there is. You just need one piece of information that in fact you already have - the predicted inbreeding coefficient of the litter.

Remember that inbreeding is defined as the probability of inheriting two copies of the same allele from an ancestor. By definition, then, inbreeding is homozygosity. If the inbreeding coefficient of a dog is 25%, then the probability of inheriting two copies of the same allele from an ancestor is 25%. If that allele happens to be a recessive mutation, then the risk of producing a genetic disorder caused by that recessive mutation is also 25%. Likewise, if the inbreeding coefficient is 10%, then the risk of a genetic disorder produced by a recessive mutation is also 10%.

Think about this. You did mutation testing to eliminate the 25% risk of producing a recessive genetic disorder from two carrier parents. But we just explained that the inbreeding coefficient tells us the risk of producing a genetic disorder from ANY recessive mutation, the ones we know about as well as the ones we don't.

So, we can be clever about this. We can control the risk of genetic disease from all recessive mutations by controlling the inbreeding coefficient of the litter. If we test for red circle and eliminated the carrier sires, it would be dumb to then breed to sire #1. We have paid to eliminate the 25% risk from the known mutation then done a breeding with a 31% risk of producing a disorder from some other mutation we don't know about.

Let's read that again.

We tested, eliminated the carrier sires so there is no chance of producing affected puppies from the known mutation, then we bred to a "clear" dog with a 31% risk of producing puppies with a disorder from some other mutation we don't know about.

You might be breeding "health tested" dogs, but you are still producing puppies with a significant risk of genetic diseases from recessive mutations.

Go back to what we know about the inbreeding coefficient. If our litter has a predicted COI of 10%, then the risk of ANY mutation causing a problem is only 10%, including the one we paid to test for. This is because parents with a kinship coefficient of 10% are less similar genetically than parents with a kinship coefficient of 25%. If the parents have fewer genes in common, there is a lower chance of a puppy getting two of the same mutation from both.

So it's simple. Reduce the risk of genetic disorders in your puppies by reducing the predicted inbreeding coefficient of the litter. 

If you've never thought about this before, this should be your light-bulb moment. All this "health testing" is a waste of time and money if we choose parents that produce a litter with an inbreeding level of 38%.

This is the WRONG WAY to use DNA testing. The breeder believes they are doing everything they can to produce puppies that will live long, healthy lives. But in fact they mark the known land mines with flags then assume the coast is clear without considering the risk produced by the many unseen explosives.

To produce healthy dogs, we need to get the risk of problems caused by ALL recessive mutations as low as possible, the ones we know about as well as the ones we don't. You can estimate this risk from the kinship coefficients of the potential parents. These numbers can be computed from a pedigree database, or (even better) they can be determined from the genotype data produced by DNA testing.

Don't waste your money on DNA tests then breed in a way that has a high risk of producing disease. Use those tests to eliminate all risk from mutations we can test for, then use kinship coefficients to make sure we have reduced as much as possible the risk from the unknown mutations hiding out in the genome of every dog.

To learn more about the genetics of dogs, check out
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There are some fun tools we can use to explore the effects on genetics of changes to some of the properties of a population. This is one that we use in my population genetics courses to help students understand how genetics can be affected by changes in various properties of a population such as size or addition of new individuals.

One of these is an online population simulator called Red Lynx. It's easy to use, and if you have access to the internet you can take it for a test drive.

Go the the Red Lynx website at ​https://cartwrig.ht/apps/redlynx/. 

On the landing page, click on the button to start the Red Lynx simulator.

This will take you to the simulator and you should see the screen below.

Red Lynx simulates the "behavior" of a single allele, "A1", in populations with various properties. There are two alleles at every locus, so actually A1 has a pair (lets call it A2). But we only need to simulate one allele, because if the frequency of allele A1 in a population is going up, the frequency of A2 must go down. If we model the behavior of A1, we know that A2 will be responding as a mirror image. So, we will run our simulations using only a single allele at a locus.

On the simulation page, you will see a graph, and below that a series of parameters that you can change using some simple sliders. We are only going to play with the first three: number of generations, population size, and initial allele frequency (green arrows). You can change these either by using the slider or just type the number you want in the box. You can ignore the rest of the sliders on the page.

​This line is the predicted behavior of the allele A1 in this population over time, assuming a 50/50 chance of inheriting the allele at each generation in a population under "ideal" conditions - no selection, no migration of A1 alleles in or out of the population, and no mutation. You can see that the initial frequency is 50%, and from there it change in frequency in the population with each generation.

Now, without clearing the graph, do a few more simulations. Each of these lines will be different because the role of chance in inheritance at each generation. If you do enough of these, you might see one of the lines go either all the way to 0% and flat-line (red arrow), or to 100% and stay there (green line). In the first case, all copies of the red allele have been lost from the population; for the green arrow, the alternative allele (A2) has been lost completely, leaving only A1, a situation we call "fixed" for that allele.
​

So what?

I hear you say "That's cool, but so what?".

Let's say the A1 allele was for something like the ability to smell a particular scent. If this isn't something you would be selecting for, this simulation suggests that there is a chance it could be lost from the population just by chance, a phenomenon called "genetic drift". Maybe this scent wasn't important to you, but if it was to the dog (e.g., a pheromone), it would have consequences that might affect some aspect of the physiology or behavior of the dog. Perhaps it is an allele that prevents fearfulness; losing it from the breed would definitely have consequences for the welfare of the dog. Or maybe it is an allele at a locus that is most beneficial when heterozygous - that is, the genotype A1A2 is more beneficial to the dog than either A1A1 or A2A2, something called "over-dominance". If one of the alleles is inadvertently lost from the population, all individuals will be homozygous for the other allele, and beneficial effects of heterozygosity will be unavailable.

So now let's do the fun stuff.

Let's make the number of generations in the simulation 100 so it will be more relevant to dog breeding. Change the population size to 50, and leave the allele frequency at 50%. Use the button to clear the graph, and run some simulations. 

You should find that the frequency of A1 goes to one extreme or the other (0% or 100%) in many of these simulations. 

Now run some simulations with different population sizes, perhaps 100 and 25. How does this affect the graph?

This simulator makes it easy to explore the effect of population size on the behavior of alleles in the population that you might not be paying any attention to. Clearly, the genetic stability of a population is sensitive to the number of individuals.

Why does this matter to your breed?

How big is your breed? What fraction of the puppies produced are used for breeding? Remember, only reproducing animals count when we're considering genetics. What are the effects of breeding contracts or spay/neuter policies on the population genetics of your breed? How stable is it genetically to the chance loss of alleles by genetic drift?

These changes in allele frequency occur in all populations. If a population is divided into closed subpopulations, the same phenomenon will occur in each of them. This will cause them to drift apart genetically over time. Breeders can take advantage of the genetic differences in these subpopulations to use them as outcrosses that can restore alleles that have been lost in another subpopulation. You can simulate this using the slider for "migration". If breeders can work together to monitor the genetic status of these subpopulations in a breed, they can reduce the loss of genetic diversity, slow the increase in inbreeding, and benefit from the hybrid vigor produced by an outcross.

​Is the population of your breed stable, or is it one of the many with registrations dropping off and fewer active breeders than there used to be? The simplest thing you can do to reduce the chance of losing important genetic diversity from your breed is to use more of the dogs produced in the breeding program. If the usual fraction is 20%, increase it to 40% or more. Don't send so many puppies off with contracts that prohibit breeding, or breed once before spaying or neutering. How much of a difference will it make? You can check it out using the Red Lynx simulator. Just remember that the simulator assumes a population with no selection, no migration, and no mutation (unless you stipulate conditions for these using the sliders). Certainly a purebred population will be under selection for the traits for type, but you can assume there is no selection for the "neutral" alleles that have no effect on type.

This simulator can also have a very practical use. Let's say your breed is discussing separating various colors in the breed into separate populations that would not interbreed. Or perhaps it is different sizes, or some feature of phenotype. If the groups are prevented from interbreeding, what will the consequences be for the genetic stability of the resulting smaller populations? There are many examples of breeds being split in the past - Norwich and Norfolk were separated based on ears, Toy and Standard Manchester were separated by size, the Belgian Shepherds are separated in the US based on color (but not in Europe), and perhaps you know of others. Splitting a breed WILL have consequences to the genetics of the separate populations, as you can see here effects of genetic drift on allele frequency. There will also be changes in other genetic properties of the populations such as the rate of inbreeding, and breeders should carefully consider these before deciding to make any changes in the breed.

In a previous post, I showed how inbreeding and strong selection increase the risk of genetic disease (via increased expression of deleterious mutations) and diminish overall health (via inbreeding depression). I also explained how we could improve health and reduce the risk of genetic disorders by making some simple changes in the way we breed. I showed why these things happen using a simple path diagram.

(If you didn't read my previous post, you should do that first and return here: Simple strategies to reduce genetic disorders in dogs)

There are other consequences of inbreeding that might be less familiar to the dog breeder but can ultimately be more important because they also feed into the same cycle of "inbreeding -----> genetic disease".

Below is  the figure from my previous blog post, to which I have added another loop (in green). (I have collapsed the loop that was to the left of health consequences and simply replaced it with the word "health".)

Once again we start with inbreeding in a population of dogs. Mating related dogs produces inbreeding, in which a puppy gets identical copies of an allele from both parents, increasing the fraction of genes that are homozygous. To here, we are following the same steps as we reviewed before.

At this point, we will add a new detail to our path diagram. The homozygosity produced by inbreeding might be scattered over the chromosomes, or it can be in blocks of adjacent genes on a chromosome. 

Blocks of homozygosity on the chromosomes are called "runs of homozygosity" (ROH), and they have two important properties.

a) the blocks of homozygous alleles tend to get longer and longer with inbreeding;

b) linkage disequilibrium increases with inbreeding.

We need to define "linkage disequilibrium". We usually think of inheritance as one of the two alleles at a loci that are passed on to a descendant by random chance. This is a simple and useful way to think about inheritance, but in reality it can be more complicated. In fact, the regions of homozygous loci that form runs of homozygosity tend to be inherited together as a block. This non-random inheritance at adjacent loci in a run of homozygosity is called "linkage disequilibrium". 

So, follow along using the chart below -

In step 4, why do mutations get trapped in blocks of homozygosity? Inbreeding removes genetic variation. To breed "away" from a deleterious allele, the alternative (i.e., normal) allele must replace it. When there is inbreeding to increase homozygosity for genes for type, as well as selection to reduce genetic variation, the normal allele can become rare. Homozygosity of the mutation within ROHs will increase. Now it becomes difficult or impossible to remove or even avoid a mutation, and over time the number of mutations that are "trapped" increases.

5) mutation trapping increases the "genetic load", the number of deleterious alleles in the gene pool

6) the expression of recessive mutations increases

This path now joins the main pathway that we discussed previously.

​You can see that the breeder is now trapped. We are trying to improve type and reduce genetic diseases at the same time as we lose the genetic variation needed for improvement. We are also making it more and more difficult or even impossible to breed away from genetic issues because of linkage disequilibrium. Of course, we continue to select strongly for type, which insures that the blocks containing those genes remain homozygous. The mutations in these blocks are trapped forever.

Now this path feeds back into the larger pathway, where we remove affected dogs or entire lines in an effort to get rid of the genes causing problems. But this reduces the size of the gene pool and throws out valuable genetic variation.

​And so it goes, round and round, the quality of the gene pool deteriorating a bit more with every generation.

This is a real pickle if you're a breeder. What all this means is that the more effort you put into breeding for improved health using the usual strategies of inbreeding and strong selection, the bigger the problem gets. This is where we are.

How do we fix this?

If we breed a very inbred dog to one that is not closely related, we can reduce homozygosity (inbreeding) in the puppies. Less inbreeding means smaller blocks of homozygosity, so linkage disequilibrium is reduced. Mutations and other unwanted genes that were trapped in runs of homozygosity are now "set free", so you can breed away from them. There is also new variation that you can make use for improvement of phenotype, so selection will be more effective.

Note that the most useful dogs for an outcross will likely be the ones that are not what you would choose to breed to on the basis of phenotype. The dogs you gravitate to with good type are likely to have all the same ROH you are battling with already, and there will be little to gain. Paradoxically, dogs with more phenotypic variation, especially as it varies away from the extremes in type, will have the most to offer you in terms of escaping from the feedback loop illustrated above. You probably won't get stunning puppies out of a breeding with a dog with mediocre type, but you will escape the loop that is strangling your breeding program and sending purebred dogs towards the cliff.

I can hear a bunch of people complaining that outcrossing will introduce new mutations to your line. Yep, it might. You would probably know about the dominant mutations in a dog you're breeding to, so the new mutations are likely to be recessive. Look again at the feedback loop and you will see that those recessive mutations are completely harmless as long as you don't breed dogs together that have the same ones. And if you keep inbreeding low, you can eliminate those mutations through selection because they aren't trapped in those blocks of homozygosity. Also put your foot down on popular sires; the last thing you want is to produce dozens of puppies that each carry half the mutations found in the favorite dog. Remember, inbreeding is how we ended up with the current problem in the first place. The solution is simple: don't put two copies of the same mutation together in a puppy.

You can fix this problem. You can eliminate health problems in a generation. You can use selection more efficiently. You can add the variation necessary to produce better dogs than you have now. Lives will get longer. Vet bills will go down. Breeding will get easier. Litter sizes will increase. The health and welfare of dogs will improve.

The secret to improving the health of dogs is not more DNA tests, but sound genetic management. We know what is causing the high burden of genetic disorders in dogs, and we know how to prevent this. Trying to eliminate problems by chasing down pesky mutations will not get us out of the feedback loop that is causing the problem. Breeding ever more selectively won't help either. These things make the problem worse.

The solution is genetic management. Understand how the problems are created. Understand the critical importance of genetic variation and reduced inbreeding for improving health. And breed in a way that will be sustainable into the future.

If you learned something useful from this article, there is much more information out there that will improve the health of dogs and make your breeding program more successful. The best way to increase your knowledge and understanding is through one of the courses offered through ICB. These courses are specifically designed for breeders and they truly are the best way for breeders to learn what they need to know to improve the health and well-being of dogs.

The next course is Managing Genetics for the Future and starts 7 January 2019. You can learn more about it and register here -

​https://www.instituteofcaninebiology.org/managing_genetics.html

I hope to see you there!

Over the last few decades, the number of genetic disorders in dogs has been increasing at an alarming rate. This is despite the diligent efforts of breeders to breed healthy dogs. Why is this happening?

I have created a very basic flow chart to illustrate how a breeding strategy to reduce genetic disorders in a population will actually have the opposite effect. I will go through each of the steps in the process, and you can follow along on the picture.

1) Let's start with a population of dogs in a closed gene pool like the one in the left circle below. Because all the dogs in the population come from a small number of founders, they are all related. Breeding two together is likely to produce offspring that are homozygous for some loci; that is, they inherit two copies of an allele that originated in a common ancestor of both parents. In the flow chart, the inbreeding step results on average in an increase in homozygosity in the offspring.
​

2) Every dog carries dozens or even hundreds of recessive mutations. For the most part, these cause no problems if there is only a single copy of the mutation and the other allele at the locus is normal. But if two copies of the mutation are inherited, there is no copy of the normal allele. This is why inbreeding, which results in homozygosity. increases the expression of these recessive mutations.

3) Homozygosity also has more general detrimental effects on function such as reduced fertility, smaller litters, higher puppy mortality, shorter lifespan, etc., which we collectively call "inbreeding depression". Inbreeding also increases the incidence of polygenic disorders such cancer, epilepsy, immune system disorders, heart and kidney issues, and others. 

4) Dogs with genetic disorders are usually removed from the breeding population.

5) Removing dogs from the breeding population reduces the size of the gene pool.

6) Smaller gene pools have less genetic diversity.

7) With less genetic variation in the population, the genetic differences among individuals are reduced and their similarity and relatedness increases.

​8) Breeding related animals is inbreeding, so once again this step results in an increase in homozygosity.

From here, we now have a negative feedback loop that goes back to the top of the list of steps. Again, the increased homozygosity increases inbreeding depression, the risk of cancer, epilepsy, and other polygenic disorders, and the expression of recessive mutations.

The result of this negative feedback loop is the steady deterioration in health of the population over the generations unless there is appropriate intervention.

Let's have a look at the cycle for the population of dogs in the circle on the right in the illustration.

a) Breeders are very selective about which dogs are used for breeding. In general, about 25% of the purebred puppies produced are bred, and typically this is only one or two puppies per litter.

b) Of course, this means that 75% of the puppies are not bred. Removing them and any unique genes they may carry from the breeding population reduces the size of the gene pool.

c) Smaller gene pools have less genetic diversity.

d) If there is less genetic diversity, the dogs in the population are more similar to each other genetically.

e) Breeding dogs that are similar genetically will produce homozygosity in the offspring.

f) Homozygosity increases the expression of deleterious mutations.

Ultimately, the path feeds into the steps we have already described that form a negative feedback loop that increases the incidence of genetic disease.

The goal of selective breeding is to produce quality dogs. The two breeding strategies we have just described, breeding of related dogs (inbreeding) and breeding "the best to the best", do not result in improvement except in the short term. In the long term, the loss of genetic diversity limits the possibility of genetic improvement because the population has lost the genetic variation needed for selection. Inbreeding depression and increased incidence of genetic disease reduce the quality of the breeding stock, and improvement - or just maintaining quality - becomes more and more difficult. Without intervention, animal populations bred this way will go extinct.

If you understand the downstream consequences of breeding decisions, which are depicted as steps in these flow charts, you can prevent this cycle of genetic deterioration. For example, the simplest action to take is to be less restrictive about which animals are bred. Breeding 50% instead of 25% of the puppies produced will reduced the depletion of the gene pool. Breeding dogs that are less closely related will reduce the risk of producing genetic disorders in the puppies, and it will also reduce inbreeding depression. Replacing genes lost from a population through outcrosses to another population (i.e., through a plan of rotation breeding) or introduction via a cross-breeding program will broaden the gene pool and mitigate the negative effects of selective breeding.

DNA testing is a valuable tool but not the holy grail. By itself, it will not result in healthy dogs. Ultimately, to improve the health of the purebred dog we need to understand basic population genetics and follow a sound strategy for genetic management. Solving the problem requires understanding the cause and how some simple changes in the way we breed can dramatically improve the quality of the dogs we produce.
​

Continue to read Part 2 of this article:
"​MORE ON "Simple strategies to reduce genetic disorders in dogs"
​

To learn more about the genetics of dogs, check out
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​

By Carol Beuchat PhD

There are more than 1,000 known breeds of dogs, only a few hundred of which are "recognized" by a kennel club. By any measure, this is a spectacular level of structural, physiological, and behavioral diversity to see in a single species, and we owe a large measure of the development of human civilization to the dogs that have helped along the way.

There is growing awareness now that many of these breeds are becoming rare and run the risk of disappearing entirely. The UK Kennel Club recognized this possibility a few years ago and created a list of native breeds that are vulnerable to extinction because of declining numbers. In some breeds, the numbers haven't moved much or continue to decline, but in others the heightened visibility has resulted in an increase in the number of puppies produced.

The Dandie Dinmont Terrier is one of the breeds on the vulnerable list, with numbers produced per year hovering between about 80 and 150 per year since 2010. So the birth of a litter of 5 adorable little pups just in time for the holidays is cause for celebration and noteworthy enough to make the news.
​

Also celebrating are the folks at the Mongolian Bankhar Dog Project. While all is bleak and frigid on the Mongolian plateau, they are welcoming the arrival of 11 new puppies that will be placed in about 2 months with one of Mongolia's nomadic herders. Each will puppy be introduced to the livestock, grow up as a hoof-less member of the herd, and serve as their protector for the next decade or more. The Bankhar Project is bringing these dogs back from the brink of extinction by replacing guns with these traditional Mongolian livestock guarding dogs, allowing the herders to coexist peacefully with the snow leopard, wolves, and other native predators. By the way, that whelping box is not in a cozy abode in the middle of the spare room. It's a hut outside and it's bitter cold. These pups are coming into the world as they have for thousands of years, ready for the weather and with no expectation of napping on the sofa. I haven't verified this yet, but I'm betting that the "fog" you see puffing in the video is the condensation of the warm breath coming from behind the video camera.

​Also in the frozen north, the Norwegian Lundehund Club is readying for the production of the first backcross generation in their breeding program to save the Lundehund from extinction. The dam is Lundhund x Buhund cross and the sire is a Lundehund. The F1 dogs were healthy and very much resembled Lundehunds, retaining almost all of the unique anatomical features of the breed (polydactyly, ability to close the ears, extreme flexibility). The progeny of the backcross should produce pups that are even closer to breed type.

We celebrate these preservation breeders! They are doing the hard work of making sure that the precious genes in the few remaining dogs of these breeds are preserved - duplicated, recombined, and packaged into a new generation of puppies that will carry the breed forward. Each tiny new nose, while appearing in the depths of winter, holds the promise of a brighter future for a breed.

To learn more about the genetics of dogs, check out
ICB's online courses

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...the latest canine news and research

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In 2015, the UK Kennel Club published a basic genetic analysis of pedigree breeds based on the data in their stud books. This analysis showed that, for many breeds, there were high levels of inbreeding and low effective population sizes. Subsequently, to address concerns that some of their native breeds might be at risk of extinction because of declining numbers, they created a listing of breeds that were vulnerable or at risk. They have subsequently monitored registrations of these breeds and annually update the statistics online. 

Although AKC would have the records for yearly registrations, they don't make those statistics available. Unless breeders or breed clubs compile these records themselves, there is no way to track breed population sizes in the US. Based on pedigree records of a few breeds that I happen to have, however, there is good reason to be concerned.

Population crashes have consequences. Genetic diversity is lost, and the rate of inbreeding necessarily goes up as effective and census population sizes go down. We can expect the incidence and number of genetic disorders to increase. Health will decline and interventions by breeders will become increasingly ineffective. It's not a good situation.

What are we doing about this? Nothing. If AKC is monitoring breed populations, they are not making that information available. Unlike kennel clubs elsewhere in the world that have geneticists on staff to provide guidance to breeders about genetic management, or organizations like the Animal Health Trust with expertise and research capabilities that are available to breeders, in the US it's every breeder for themself. Breed clubs and their health committees make decisions about breeding strategies in the absence of relevant expertise, and more often than not the general strategy is to remove dogs from the breeding population that are afflicted with disorders for which there are no tests (e.g., epilepsy, cancer, immune system disorders, etc). 

I am not aware of any plans to address these problems, by the kennel club, breed clubs, or smaller groups of breeders. In fact, breeders might not even be generally aware of their dire situation. For the breeds with data below, time for action has nearly run out. 

I have argued before that dogs have high value as a genetic resource. Dogs shaped the development of civilization. In many places in the world they are still essential for the special abilities they have for management of livestock, not to mention their roles as service animals, detection of everything from invasive plants to cancer, military and police service, and companions that improve our own health and longevity while they enrich our lives. 

There is plenty of evidence that purebred dogs are not doing well. And we also know that in many cases we are not doing what we can (as for the Doberman). Breeds are about to disappear. Are we going to just let this happen?

The first point here is that there is no way to know what specific genes were present in the founder lines more than a century ago. Each dog added to the mix would add another spoonful of genes from the large, diverse genetic stew available among dogs at that time. Certainly, it is fair to expect that the original gene pool was very diverse.

Could the Ky allele have been present in that original gene pool? That would depend on how common it was and how it was distributed among dogs, recognized breed or otherwise.

Eduard Korthals developed the breed in the Netherlands in the late 1800s, with the aim of producing a gun dog that was versatile, hardy, and devoted to its master. To do this he crossed a variety of dogs, some of unknown lineage but carrying some of the traits he wished to produce in the breed. Among those of various types were probably spaniels, retrievers, pointers, and otterhounds. The breed was created as many others have been, as "blender breeds" that borrowed a bit of this from one breed and a bit of that from another to produce a new breed of dog well-suited to a particular purpose in the eyes of its creator.

So here are the crucial bits of information for this discussion of the Ky allele as evidence of genetic contamination.

1) The Ky allele is the ancestral form of the K allele. It is found in all wolves. It is thus likely to have been in the earliest dogs.

2) How common is the Ky allele in dogs? To answer this question, I contacted two highly-regarded canine geneticists with access to large databases of dog DNA data, including alleles for color: Dr Adam Boyko (Cornell University and Embark Vet) and Dr Jonas Donner (Genoscoper and MyDogDNA). They both confirmed first that the Ky allele is the ancestral K allele that is found in wolves. They also both said that the Ky allele is very widespread among breeds and dogs in general. Boyko said "I think it's in more breeds than not...even some black breeds like Schipperke and Belgian Sheepdog." Donner said that "Ky/Ky is the fixed genotype in the modern wolves we have tested, so yes it is the ancestral allele...The frequency of the ky allele in dogs overall is about 70%".

Not only is the presence of the Ky allele not evidence of a cross-breeding event, it would be surprising to NOT find it in a modern dog breed created by crossing various breeds as well as dogs of ambiguous lineage. Ky is recessive to other K alleles, so it can be present in a population for many generations without being expressed. Having a few tan point dogs pop up in a long line of dogs where this has never been seen is proof of nothing more than the roles of chance and allele frequency in the expression of recessive alleles.

There is no scientific or even rational basis for the claim that Griffs carrying the Ky allele are not "purebred" or "true" Korthals. The policy of some groups to purge all the "contaminated" dogs carrying the Ky allele from breeding populations is both wrong-headed and detrimental to the breed because it reduces the size of an already limited gene pool and unnecessarily removes quality dogs for a cosmetic trait that won't even be expressed if a dog is just a carrier. Those claiming that the "pure" or "true" Korthals does not carry this gene and that dogs that do are "just mutts" do a true disservice to the breed for the apparent purpose of elevating their own dogs to a higher status than others. This is not to better the breed, and it doesn't. It is nothing less than a shameless scam.

The data to disprove the fallacy of the claim that dogs carrying the Ky allele are descendants of cross-breeding are abundant and readily-available. There is no evidence that it is associated with any detrimental or undesirable trait in this breed (which has also been claimed) or any other. Remember: this is the K allele found in wolves. In fact, the K allele is not a "color allele". The K gene is otherwise known as the β-defensin gene, and as the name implies it plays a role in the synthesis of peptides with antimicrobial activity in the skin, assisting in its role as a barrier to infection. (Candile et al 2007, Hedrick 2009, Leonard et al 2012).

While breeders are struggling to manage a number of emerging health issues in this wonderful breed, it should be a matter of policy that the loss of genetic diversity for any reason should be minimized to the fullest extent possible. A genetic management plan that strategically prioritizes retaining genetic diversity through the breeding of dogs carrying less common alleles should be encouraged by the custodians of the breeds, and breeders that do this should be supported.

There are some other important things breeders can do to improve the future of this breed. It would be useful to do a breed-wide survey of genetic diversity using a genotyping service like Embark. This will provide information on both breed-wide and genomic inbreeding, the size of the gene pool, distribution and frequency of alleles for known mutations and traits (including color), and the potential presence of lineages of differing genetic composition that can be managed in a way to support retention of the genetic diversity currently available in the breed. These data can produce much information that would assist breeders interested in developing breeding plans that will improve the health of the breed as well as its highly valued qualities as a working dog and a companion. You can see examples of these types of analyses for a number of breeds using the ICB Breeder Tool at http://bit.ly/2OE88by​, and ICB can assist you with this.

It would also be extremely useful to do a genetic analysis of a pedigree database. This will provide wealth of information about history that might not otherwise be known through other records. It can also reveal current breeding practices that are detrimental to the preservation of genetic diversity, allowing breeders to take steps that will provide more genetic stability and facilitate management of existing health issues. You can see an example of this type of analysis here (The amazing secrets hiding in your pedigree database). This analysis is for Afghan hounds, but it provides a good model of the wealth of information that can (and should) be extracted from a pedigree database with the appropriate software and expertise. ICB can provide this type of analysis, and it would be very worthwhile to do for this breed.

I have been asked to weigh in on the "Ky debate" many times over the last 10 years or so, and I hope this will put the final nail in the coffin of this debacle that has probably done great damage to the gene pool and therefore the future of the breed. Breeders should consult with scientists that have the appropriate expertise to address critical issues of genetic management instead of trying to do this "in house". There is first the issue of bias interfering with judgement, in the same way that a surgeon should not operate on a member of their own family. But most especially, genetic management of small populations of highly inbred dogs is very difficult; a policy decision to address one problem can have an unanticipated effect on something that was not considered. Here, what appeared to be a simple policy decision about a "color" allele probably did lasting damage to an already small gene pool; certainly it strained relationships among breeders and has fostered the development of cliques that have divided what should be a family of breeders that collectively support the best interests of the breed. I would encourage breeders to heal the wounds, get some factual information about the current genetic status of the breed, and work together to solve problems and insure a bright future for the breed.

One of the problems with strong selection in a closed gene pool is that the dogs become progressively more and more similar genetically. Although selective breeding in purebred dogs results in consistency, it also has the downside that a breed can find itself stuck in a genetic cul-de-sac from which it can't escape. If you need to change the direction of selection, for example to breed towards a different trait or away from a genetic disorder, you need to find dogs with the genetic diversity necessary to support selection in a different direction. Genetic diversity is the raw material for selection; you can't change anything without it.

How much genetic diversity is there in your breed? How can you find the dogs that might have the genetic variation that you're looking for?
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There are several different ways to determine how much genetic diversity there is in your breed, but let's look at one that uses the kinship coefficients that we have already been talking about (see Parts 1, 2, and 3 in this series; links at the bottom).

Let's say that we have a population of animals that are not closely related. If we compare the genes in each dog with those in every other dog in the population, we can compute each dog's mean kinship, which I explained in a previous post. Since we know the dogs in our population are relatively unrelated, we know that the values for mean kinship will be low (they can range from 0 to 1). If we graph the mean kinship of all of the dogs in the population, we might get a chart that looks like the first one on the left below for Labrador Retrievers, in which all of the values are less than about 0.06. On the other hand, if we have a population of closely related dogs, we might get a graph like the last one on the right below for Irish Wolfhounds, in which most of the values are greater than 0.3. If you scan the graphs for the other breeds depicted below, you will see that the distribution of values for mean kinship can vary considerably.

What do these graphs tell us about genetic diversity?

Remember that mean kinship is the average relatedness of a dog to all others in the population. The Labrador Retriever is one of the most popular breeds in the world so numbers are very high, and there are populations of show dogs, hunting dogs, personal pets that will all be a bit different from each other genetically, and populations in different countries will probably be different from each other as well. This is a breed with a LOT of genetic diversity, and the mean kinship values are mostly low. But if you were to make another graph with just the show dogs, the distribution would probably be skewed towards higher values because those dogs would be more closely related to each other than they are to field line dogs.

When you compare Labradors with Irish Wolfhounds, the graph is skewed strongly towards much higher values. This indicates in this population of over 200 dogs, the average relatedness was very high. The exception is a few dogs that fall far below the pack and are not closely related to the bulk of the population. This indicates that most of the dogs are closely related to each other.

Because the kinship coefficient is based on genes that are the same because they are inherited from the same ancestors, we can interpret these graphs in terms of relatedness. If you breed two first cousins from parents that are not inbred, the predicted inbreeding coefficient of their offspring is 0.0625, or 6.25%. Looking at the graph at the top for Labrador Retrievers, we can see that most of the values of mean kinship are lower than 0.06. So we can say for this population that on average, the Labradors are less related than first cousins. 

You can do the same thing for the graphs for other breeds, using this chart for the kinship coefficients for each level of relatedness.
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So, looking at the chart for Irish Wolfhounds (bottom right), you can see that most of the dogs have a mean kinship > 0.25; that is, on average most of these dogs are more closely related to each other than full siblings. Imagine a family reunion with a few aunts, uncles, and cousins, and 134 of your siblings. This is a breed with a very high level of average relatedness to each other. Likewise for Cavalier King Charles Spaniels. In the small sample of Havanese, there two clusters of dogs, one with a relatively high level of relatedness to each other, and another with rather low relatedness. 

There are several different ways to understand the genetic diversity in your breed. Here, we show you how you can use the kinship coefficient to provide information about the average levels of relatedness in a population of dogs. This is another tool you can add to your genetic toolbox!

About 80% of the genetics diseases we know about in dogs are caused by a single recessive mutation. Using modern DNA technology that enables us to scan thousands or millions of markers in a dog's genome, we can usually find these broken alleles and develop a test that breeders can use to screen their dogs.

Many disorders, however, are not the result of a single mutation. For these, there might be dozens or even hundreds of genes involved. Creation of a test that will identify an affected dog with no symptoms, one likely to produce affected offspring, is not likely to happen for most of these. This is a huge problem for breeders, because many of these diseases, like cancer, epilepsy, autoimmune dysfunction are very serious and becoming more and more common. Yet they do seem to run in related dogs, so we know genetics is important. How can we manage these diseases if we don't have a DNA test? We can use kinship coefficients. Watch this.

Kinship coefficients provide information about the relatedness of dogs by assessing pairwise genetic similarity. A statistical technique called cluster analysis can use the kinship data to construct a dendrogram - a genomic pedigree - that groups dogs together that are closely related and also indicates the relationships among groups. If a trait or disease has a genetic component, we should expect that clusters of closely related dogs are more likely to share traits than unrelated dogs because they share many of the same genes. ​

​This is a dendrogram constructed using kinship coefficients computed from DNA data for 211 Irish Wolfhounds. The upper figure is the right to the right blown up so you can see a bit easier, and the lower chart is the entire dendrogram. 

Let's take a little tour of the lower chart. You can see that the population of dogs has been separated into two smaller groups at the arrow labeled "A". If we follow the branch that comes off point A to the right, we come to a second branch at the arrow B that creates two subgroups. If we follow that branch at B to the left, we come to branch C that again splits the dogs into two groups. You can see now that is called a dendrogram because it is a picture of a structure that branches like a tree.

Dendrograms are a bit tricky to read properly and getting good at it takes some practice. You need to remember that branches can rotate around any branching point. So for instance, the very top branch that divides at A can be rotated around the junction at A so that the green group on the far left is moved to the far right to sit nest to the turquoise group. Clearly, that green group is not most closely related to the red group that was right next to it at when it was at the left end of the chart. For that matter, we can rotate the two groups on the far right (at arrow D) so  the green group is now right next to the red group. So there are actually many different ways to arrange the clusters that don't change their relationships to each other. This is because relatedness is indicated by the length of the lines that connect groups or dogs. Dogs connected by very short lines are closely related, and dogs that are more distantly related are connected by longer lines. When we swivel groups around branching points, the length of the connecting lines is not changed so the information about relatedness is not changed. (Check out How to read a dendrogram)

Now we have a picture of the genetic relationships among all of the 211 dogs in this sample. We also have information about which of these dogs has been diagnosed with epilepsy. This is indicated by the three asterisks ("***") at the tip of the branch for each dog. For dogs without asterisks, this could be because they have never reported epilepsy or we have no information. So this chart can't be taken as a representation of which dogs have epilepsy and which do not. It only tells us which dogs have reported a diagnosis of epilepsy.

So, what does this graph tell us? We can see that affected dogs tend to cluster together by family groups, which likely reflects a genetic component to disease risk. Unfortunately, there are no large groups where epilepsy is completely absent. This tells you that the genetic predisposition to epilepsy is widely, but possibly not uniformly,  distributed throughout this population of dogs. With some good record-keeping and the addition of data to the database, we might be able to come up with a clearer picture of the patterns of risk that can be used to make breeding choices that will reduce the number of affected dogs.

There are many breeds battling to control genetic disorders for which there are no DNA tests, or the test is unreliable for identifying dogs that will develop the disease (e.g., degenerative myelopathy). For these, kinship analysis could provide very useful information to breeders about risk of disease. Of course, the same technique described here could also be used for diseases caused by single mutations. If incidence of all genetic disorders was mapped on a dendrogram and kept up to date, breeders could notice a problem developing long before it becomes widespread in the breed and difficult to manage because of the high frequency of carriers. 

One caveat about dendrograms from kinship coefficients. The cluster analysis uses the information for all dogs in a sample population to construct a tree that is most likely to reflect their true relationship. If more dogs are added to the population, the groups might be sorted differently because the relationships among them are changed. So to be useful, data for kinship should be updated regularly and needs to accurately represent the genetic breadth of the population of interest. This would be true 

We can produce kinship coefficient from both pedigree and DNA databases. There is some advantage to the DNA data because it will distinguish differences in relatedness among siblings whereas pedigree data does not. On the other hand, the pedigree information is invaluable because it doesn't require collecting a DNA sample and the cost of analysis. If you aren't trying to compare siblings, you need to run only one or maybe two dogs in a litter, which can reduce the expense considerably.
​

Think about the valuable information analysis of kinship coefficients could provide to inform your breeding decisions and managing the genetic diversity of the breed. If you have a pedigree database or genotype analysis from high density SNPs, it would be well worth doing.
​

By Carol Beuchat PhD

This is part 2 of "Cool tricks with kinship coefficients". Check out "Part 1: Is this dog really an outcross?"

One of the major ways genetic diversity is lost from a breed is through selection. In wild animal populations, most animals produce offspring and pass their genes to a new generation. The progeny that inherit the best combinations of genes survive and reproduce themselves, and those that were not as lucky are culled by natural selection. 

In dogs, we often breed only one or two puppies from a litter, selecting those that we feel best suit the goals of the breeder and are good representatives of the breed. But if you started with a sire and dam that you felt were quality dogs, all of those puppies should have "quality" genes but in different combinations, some of which make for a "better" puppy than others. When the pups judged to be less suitable are not bred, we risk losing from the gene pool some of the genes that produced the good quality in the parents. We limit our future breeding options when we lose those genes, and we also remove the opportunity of tossing them together with a mix of genes from another parent dog to perhaps produce something wonderful. You can't breed every dog, and not every dog is worth breeding.

If you breed for both quality (on whatever scale) and health (and of course you do, right?), you should be concerned about the inadvertent loss of genetic diversity that results from selective breeding because eroding the genetic base of the breed will have consequences down the line. But how do you determine which dogs are important for preserving genetic diversity? You can use the kinship coefficient.

The kinship coefficient (K) is an estimate of the genetic similarity of two animals that are related by ancestry. As I explained in Part 1 of this series (Cool tricks with kinship coefficients, part 1: Is this dog really an outcross?), the kinship coefficient of the sire and dam determines the inbreeding coefficient of their litter. Put another way, the inbreeding coefficient of a dog is the kinship coefficient of its parents. (And remember from part 1 that K must always be a comparison of two individuals.) We are going to see how you can use the kinship coefficient to assess the genetic value of a dog to the breed.

Below is the same kinship matrix we talked about in my previous post about kinship coefficients. Again, the white squares on the diagonal are each dog compared to itself (so K = 1), and the other squares are color-
​coded to make it easy to spot combinations that are less (or more) related to each other.

We can use the color coding indicated in the three squares in the upper left corner on the matrix to easily spot dogs that are closely related to other dogs (i.e., green, yellow, and red). If you follow across the rows of dogs 6 and 7 from left to right (or follow down the columns for these two dogs if you prefer), you will see that there are lots of red, orange, and yellow squares, indicating dogs as closely related to 6 and 7 as half-siblings (K = 0.125) to full siblings (K = 0.25). This means that the genes in dogs 6 and 7 are also found in other dogs in the population because they share ancestry.

Now do the same thing for dogs 3 and 4. You will find that most of the squares are green, indicating that they are not closely related to most of the dogs in this group. Interestingly, 3 is as related as a full sibling to 9 and 10 (K = 0.25), which are likewise closely related to each other; similarly, 4 is closely related to 11 and 12, which are likewise  as related as full siblings. It would be interesting to look at the genealogical relationships among these four dogs from pedigrees. 

If dogs 6 and 7 have many relatives in the population and dogs 3 and 4 have very few, then the latter pair are genetically more valuable because they carry genes that are not common among these dogs. It is fairly easy to identify the genetically important dogs in this small example population, but how can we do it with a large population of dogs with complicated patterns of relationship?

The easiest way is by computing the mean (average) of all of the kinship coefficients for each animal. In the figure below, have added a column on the right labeled mK, for "mean kinship". It is easily computed by adding up all the values in a row and dividing by the number of dogs. (The kinship coefficient of each dog with itself is included in the calculation.) Now we have a column of numbers in which it is easy to see which dogs, by the bright yellow squares, are most genetically different from the rest. Although on average, they are as related as half-siblings to the rest of the population, we know that it includes dogs with little or no relatedness, and you should keep this in mind when you are comparing the values for different dogs. It is always worth examining the kinship matrix to see whether a dog with K = 0.125 is closely related to everybody, or instead is unrelated to most of the dogs but closely related to a few that drive up the average (as here).
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Now we can use the data for mean kinship to quickly identify the dogs that are genetically most valuable in a large population.  The simple bar graph organizes the dogs by rank, with the lowest values of K to the left - the dogs with the highest genetic value - and high values to the right (the dogs with lowest genetic value). In this population, there are a few dogs with high genetic value, but most of the rest of the population is closely related.

We should be using kinship coefficients much more than we are, and that is to the detriment of maintaining genetic diversity in our breeds. Breeders should know which dogs are genetically valuable, even if they aren't the top winners in competition, because the genetic variation they carry is the raw material for breed improvement in the future. Lines lost are lost forever. Perhaps breed clubs should have a genetic diversity committee that is responsible for monitoring the population to prevent loss of variation, or this could fall to the health committee because diversity and health go hand in hand. Note that if everybody rushes to breed to the least related dogs, their genes will become common and intermixed in a way that prevents them from being used strategically to manage genetic diversity. Diversity should be exploited strategically. (For the same reason, there should be a "popular sire" monitor, whose job it is to keep an eye out for sires producing more than their fair share of puppies.)

Learn how to use the kinship coefficient to take some of the guesswork out of decisions about genetic relatedness or uniqueness when you are considering your future breeding plans. This simple tool can provide you with a wealth of valuable information!

Learn how you can use kinship coefficients in
​this terrific new ICB course!
​

To learn more about the genetics of dogs, check out
ICB's online courses

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By Carol Beuchat PhD
​

Every few generations, it's a good idea to cross out to a relatively unrelated dog to bring in some new genetic diversity and freshen up your lines. But finding the right dog to do this with can be tricky. A dog that doesn't share any ancestors with the dog you want to breed in the last 5 or 6 generations might have enough shared ancestors farther back in the pedigree to provide a lot of relatedness. Or even worse, maybe you don't have a decent pedigree database so you really have no idea how closely two dogs might be related. This is an especially irksome problem if you're contemplating bringing in an expensive import. You really don't want to get it wrong. 

So what do you do? How can you identify a dog that will be a good outcross for your breeding program?
​

The kinship coefficient is related to the coefficient of inbreeding: the coefficient of inbreeding of a dog is the kinship coefficient of its parents. So, the kinship coefficient between a potential sire and dam is also the predicted inbreeding coefficient of their litter. The kinship coefficient in population genetics uses the Greek symbol phi, as in this figure.
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The kinship coefficient can be calculated from a pedigree database, just as inbreeding coefficients are. But it can also be estimated directly from DNA data - in effect, the "realized" kinship coefficient that reflects the actual level of shared ancestry between two dogs based on their DNA.
​​

Let's look at this very simple pedigree containing 14 dogs. Perhaps you were contemplating doing a breeding between dogs 14 and 7, but you are worried that it might be too close. You want to know how closely related - genetically similar - those two dog are. The kinship coefficient will tell you this. Here's how.
​

The kinship coefficient is always a comparison of two dogs. You can compute the K for each pair of the dogs in the pedigree and display them in the form of a matrix. Each dog is listed down the side and also across the top. The number in the box where the column and row of a pair of dogs intersects is the kinship coefficient of that pair. The white boxes on the diagonal are each dog compared with itself.
 

For example, we can determine that the kinship coefficient between dog 14 and dog 7 is 0.19, or 19%. This means that if you were to choose one allele at random from the pair of alleles at a particular locus on the DNA of dog 14, and likewise choose an allele at random from that locus for dog 7, the probability of choosing two alleles that are identical because they were inherited from a common ancestor is 0.19, or 19%. To put it another way, the chance that the offspring of this pair of dogs will be homozygous at that allele (have two identical copies of an allele inherited from a common ancestor) is 19%. Furthermore, if these two dogs were to produce a litter, the predicted coefficient of the puppies would average 19%.

Computed from a pedigree, the kinship coefficient is an estimate of the average expected kinship coefficient; that is, the shared genetic relatedness because of shared ancestry. But kinship can also be determined directly from DNA genotyping data, such as is provided by Embark. (As far as I know, this is the only company that will allow dog owners to download the raw data for their dogs.) When based on DNA, these are "realized" rather than predicted estimates of genetic relatedness, and they will be different for every dog in a litter.
​

Now, here's the beauty of this. Let's say you're looking for a dog that will be an outcross for a bitch you want to breed, and you're willing to import a dog if you have to. You have computed the kinship coefficients from the pedigree database available for your breed, and there are two dogs overseas - litter mates, in fact - that look promising. Would these dogs be a good outcross for your bitch? Would one of these be a better genetic fit to the bitch than the other?

To answer these questions, you can do direct comparisons of the genomes of these two dogs with the bitch you want to breed by calculating the kinship coefficient for both of the sire-dam pairs. Modern DNA analysis using "single nucleotide polymorphisms" (SNPs) can now provide excellent data with very high resolution that you can use to do this. ("Single nucleotide polymorphism" is a fancy term for loci that are highly variable ("polymorphic"); the abbreviation is pronounced "snips".) We can easily and inexpensively compare hundreds of thousands - even millions - of loci on the chromosomes of a pair of dogs, assess whether the alleles at each of those loci are the same or different. Further, using data for allele frequencies in the larger population we can determine if the shared SNPs are copies of the same allele in a common ancestor; that is, they are "identical by descent" (IBD). This is the technique used for establishing the relationships among many breeds of dogs (Dreger et al 2016), and it is also used to identify genetic mutations associated with diseases in genome-wide-association studies (GWAS).

Instead of estimating kinship from a pedigree database, which will only give you one number that applies to the entire litter, you can estimate the actual degree of genetic similarity between two dogs. In fact, using SNPs you can even see which areas on each chromosome are shared. You can use this information to see if blocks of homozygosity overlap in two dogs, in which case all of the offspring would also be homozygous in the same place. (More on this in an upcoming blog post!) You can specifically look at characteristics of the genome of the two litter mates and determine if there are any ways one might be a better match to your bitch than another. And the kinship coefficient will also tell you the estimated inbreeding coefficient of the litter produced by each potential sire. 

For the preservation breeder, the genomic kinship coefficient from SNP genotype data provides you with the best, most advanced tool available today for genetic management to improve the quality of your puppies while also protecting genetic diversity for the health of your breed in the future. 

We will be learning about kinship coefficients and how to use the kinship matrix in ICB's new course, "Strategies for Preservation Breeding", which starts 10 September 2018. Join other preservation breeders who will be using this powerful tool to improve their breeding program. If you're interested in using genomic kinship coefficients to explore options for your next breeding, contact ICB and we will work with you to set that up. 

Watch this space for more Cool Things you can do with kinship coefficients!

REFERENCES

Dreger, DL, M. Rimbault, BW Davis, A Bhatnagar, HG Parker, & EA Ostrander. 2016. Whole-genome sequence, SNP chips and pedigree structure: building demographic profiles in domestic dog breeds to optimize genetic trait mapping. Disease Models and Mechanisms 9: 1445-1460. (pdf)

Hayward, JL, MG Castelhano, KC Olivera, and others. 2016. Complex disease and phenotype mapping in the domestic dog. Nature Communications 7:10460. DOI: 10.1038/ncomms10460. (pdf)

Li, M-H, I Stranden, T Tiirikka, M-J SevonAimonen, & J Kantanen. 2011. A comparison of approaches to estimate the inbreeding coefficient and pairwise relatedness using genomic and pedigree data in a sheep population. PLoS ONE: Nov 2011; Vol 6:11. e26256. (pdf)

Learn how you can use kinship coefficients in
​this terrific new ICB course!

To learn more about the genetics of dogs, check out
ICB's online courses

***************************************

Visit our Facebook Groups

ICB Institute of Canine Biology
...the latest canine news and research

ICB Breeding for the Future
...the science of animal breeding

Most of the genetic disorders in dogs are caused by recessive mutations, a single broken allele. There are now tests for dozens of disorders that can detect whether a dog has inherited one or two copies of the broken version of an allele. With the DNA testing information, we can reduce or eliminate the risk of producing puppies that will be born with a disease caused by one of these recessive mutation.

Draw a square and divide it in half both ways to produce four compartments. Each compartment will represent a puppy (or a pea seedling, or whatever). Above each of the columns, note the genotype of the father for the gene in question, indicating the dominant allele with an upper case letter and the recessive version with lower case. On the left side, label the rows with the alleles present in the mother. In this example, the father is heterozygous for the B gene (Bb) and the mother is homozygous recessive (bb).
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​Now, to see what we do next, click on this little alien that can't wait to show you the basics -
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The Punnett square works just as well for predictions of two traits at a time. Here's an example of a "dihybrid" cross involving two genes in guinea pigs, one for coat color and the other for coat length. The Punnett square makes it easy to map out all 16 possible combinations of inheritance in just a minute or two.
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Have a go at checking the red, blue, and greed Ridgebacks and see if you get the same results for each pair of parents on the left. And below are some other examples you can play with. By the time you've worked through these, you'll be a pro!