UC Davis School of Veterinary Medicine Veterinary Genetics Laboratory

Genetic Diversity Testing for Black Russian Terriers

(Phase 2 - Preliminary results/Research)

Overview

The Veterinary Genetics Laboratory (VGL), in collaboration with Dr. Niels C. Pedersen and staff, has developed a panel of short tandem repeat (STR) markers that will determine genetic diversity across the genome and in the Dog Leukocyte Antigen (DLA) class I and II regions. This test panel will be useful to Black Russian Terrier breeders who wish to track and increase genetic diversity of their breed as a long term goal.

Genetic testing of Black Russian Terriers is now in the development phase -please see Enrolling a Breed. During this phase, we continue to test Black Russian Terriers to build genetic data necessary to provide breeders with an accurate assessment of genetic diversity in their breed. We encourage breeders to submit samples from active dogs to further build the database. The goal is to test enough dogs so that no new alleles or DLA haplotypes are recognized.

Price: $50.

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Results reported as:
Short tandem repeat (STR) loci: A total of 33 STR loci from across the genome were used to gauge genetic diversity within an individual and across the breed. The alleles inherited from each parent are displayed graphically to highlight heterozygosity, and breed-wide allele frequency is provided.

DLA haplotypes: STR loci linked to the DLA class I and II genes were used to identify genetic differences in regions regulating immune responses and self/non-self recognition. Problems with self/non-self recognition, along with non-genetic factors in the environment, are responsible for autoimmune disease.

Internal Relatedness: The IR value is a measure of genetic diversity within an individual that takes into consideration both heterozygosity of alleles at each STR loci and their relative frequency in the population. Therefore, IR values heterozygosity over homozygosity and uncommon alleles over common alleles. IR values are unique to each dog and cannot be compared between dogs. Two dogs may have identical IR values but with very different genetic makeups.

Introduction

The genetic information used to formulate the enclosed tables and graphs came from DNA samples of 93 BRT. We believe that the dogs currently tested represent most of the genetic diversity that still exists in the breed. These pages will be updated as more dogs are tested, so allele and DLA haplotype frequencies may change over time.

The Canine Genetic Diversity Test

STR markers are highly polymorphic and have great power to determine genetic differences among individuals and breeds. The test panel contains STRs that are recommended for universal parentage determination for domestic dogs by the International Society of Animal Genetics (ISAG) with additional markers developed by the VGL. The diversity of alleles at each of the genomic STR loci and their frequency in the population were used to calculate the internal relatedness of each dog and for the BRT population as a whole (Fig. 1). Internal relatedness calculated for over 93 BRT ranged from <-0.3 (most outbred) to >0.3 (most inbred).

Figure 1. Distribution of IR estimates in 93 Black Russian Terriers based on intra-breed diversity (red line), compared with IR adjusted for diversity lost during breed development (blue line).  Diversity lost as a result of breed development was determined by comparing allele frequencies at the same loci between in Black Russian Terriers and randomly breeding village dogs from the Middle East, SE Asia, and the Pacific Islands.

The DLA Haplotype

In addition to the markers used to estimate relatedness, which reflect genome-wide diversity, a set of STRs associated with specific genes in the DLA region (the canine Major Histocompatibility Complex) was identified and used to measure genetic diversity associated with immune function (see sidebar). We have identified 9 different STR-associated DLA Class I and 7 DLA Class II haplotypes in BRT (Tables 1 and 2). Various Class I and II haplotoypes are also linked to each other forming 9 extended haplotypes. DLA-Class I and Class II STR-based haplotype frequencies in BRT are also provided in the Tables 1 and 2.

Dog DLA and STR haplotype diversity.

The DLA consists of four gene rich regions making up a small part of canine chromosome 12. Two of these regions contain genes that help regulate normal cell- (Class I) and antibody-mediated (Class II) immunity. Polymorphisms in these regions have also been associated with abnormal immune responses responsible for autoimmune diseases. The Class I region contains several genes, but only one, DLA-88, is highly polymorphic (with many allelic forms) and is therefore most important for immune regulation. Specific alleles at the four STR loci associated with the DLA88 are linked together in various combinations, forming specific haplotypes (Table 1). Groups of genes and their alleles that are inherited as a block, rather than singly, are called haplotypes. The class II region also contains several genes, three of which are highly polymorphic, DLA-DRB1, DLA-DQB1 and DLA-DQA1. Specific alleles at STR loci associated with each of the three Class II genes are strongly linked and also inherited as a single block or haplotype (Table 2). One haplotype comes from each of the parents. The linkages between alleles within Class I or II regions are very strong; while linkages between regions of the DLA that are more distant from each other, such as Class I and II, are weaker. There are almost two million base pairs separating the class I and II regions, thus allowing for some genetic recombination to occur. This recombination is most apparent between the common DLA class I and II haplotypes, forming unique "extended DLA class I-II haplotypes. Extended class I-II haplotypes are inherited as a single block of genes.


Tables 1 & 2: DLA Class I & II Haplotype Frequencies in Black Russian Terriers

DLA Class I Haplotype Frequencies (Updated Nov 20, 2017)
Black Russian Terrier (n=108)
1006387 375 293 1800.032
1014375 373 287 1780.042
1016382 371 277 1780.009
1091381 371 277 1810.505
1092376 379 277 1810.255
1093386 379 277 1800.093
1094395 375 277 1760.019
1095382 371 277 1840.014
1096395 375 277 1820.005
1160386 369 289 1760.028
DLA Class II Haplotype Frequencies (Updated Nov 20, 2017)
Black Russian Terrier (n=108)
2005339 322 2800.019
2007351 327 2800.032
2023341 323 2820.005
2031339 322 2820.023
2032339 323 2800.093
2033339 323 2820.509
2037341 327 2800.292
2060343 323 2840.028

 

The STR-based haplotype nomenclature used in this breed diversity analysis is based on numerical ranking with the first haplotypes being identified (in Standard Poodles) being named 1001, 1002, ... for class I haplotypes and 2001, 2002, ... for class II haplotypes. It is not unusual for various dog breeds to share common and even rare haplotypes, depending on common ancestry. Therefore, identical haplotypes in other breeds are assigned the same number. The numerical nomenclature used by VGL for DLA class I and II haplotypes does not correlate with numerical rankings used by others.

After a sample is submitted for genetic testing, the identity of the dog and owner will be replaced by a laboratory barcode identifier. This identifier will be used for all subsequent activities.Example certificate for an outbred dog After testing, each owner will be provided with a certificate that reports the internal relatedness, genomic STR genotypes and DLA class I and II haplotypes for the dog(s) tested. The internal relatedness value for the dog being tested is related to the population as a whole.

An example certificate for an inbred dog

 

 

 

 

The goal for BRT breeders should be to produce a greater and greater proportion of puppies with IR scores less than 0, and with time even lower scores. This will be difficult to achieve given limited genetic diversity genome-wide and in the DLA. The small founder population that went into creating the breed is the most likely explanation for this limited genetic diversity.  Even though genetic diversity is limited, it is nonetheless important to properly manage the diversity that exists. Maintaining existing diversity will require using different combinations of breeding stock, including even those from inbred lines with high IR values. IR values, because they reflect the unique genetics of each individual, cannot be used as the criteria for selecting ideal mates. Mates with identical IR values may produce puppies significantly more or less diverse than their parents. Conversely, a mating between dogs with high IR values, providing they are genetically different, may produce puppies having much lower IR scores than either parent. A mating between a dog with a high IR value and a low IR value, providing the latter has few alleles and DLA haplotypes in common, will produce puppies much more diverse than the highly inbred parent. Breeders should also realize that a litter of puppies may have a wide range of IR values, depending on the comparative contributions of each of the parents. The more genetically diverse and different the parents, the greater the range of IR values in their offspring.

Potential sires and dams should be first screened for genetic differences in the genome and in the DLA regions by first comparing allele differences at each STR locus, and then at the DLA class I and II haplotypes. Some thought should be given to rare vs common alleles. This information is included on all certificates and on the website. This preliminary comparison will identify promising pairings and if desired, genetic information on the potential sires and dams can then be used to calculate actual IR expectations for their puppies. Puppies, once born, should be tested for their actual IR values, which will reflect the actual genetic impact of each parent on internal diversity. Considerations of mate choices for genetic diversity should be balanced with other breeding goals, but improving genetic diversity in puppies should be paramount.

An additional goal of this study is to contribute this genetic information to a web repository, hopefully under the control of the registry. This information could be incorporated into a mate selection service that will allow a breeder to identify, among all of the dogs tested, potential mates that would be most ideal for increasing genetic diversity in their litters.

What we have learned for our studies based on 92 Black Russian Terriers

The genetics of BRT based on autosomal markers are typical for many of the pure breeds (Table 3). The average number of alleles (Aa) per each of the 33 genome-wide STR markers is 6.27, of which 3.48 alleles effectively define the genetic differences between individuals (Ae).  The observed heterozygosity (Ho) and expected heterozygosity (He) are nearly identical, indicating that the population is in near Hardy-Weinberg equilibrium (random breeding).  This is reflected by the slightly negative value for F (an inbreeding coefficient).  The fact that F is slightly negative but near to zero means that there is a small subpopulation of dogs that are more outbred than dogs in the population as a whole.  This genetic information indicates that the breed as a whole has a small genetic base but that breeders are doing a good job of selecting mates that are as unrelated as possible.

Table 3. Genetic assessment based on 33 STR markers on 25 autosomes.
N Aa Ae Ho He F
BRT 93 Mean 6.273 3.483 0.689 0.682 -0.012
SE 0.335 0.186 0.020 0.019 0.011

A principal coordinate analysis (PCoA) plot shows the distribution of dogs as they relate to each other (Fig. 2).  The actual distribution is three dimensional (i.e., the relationship of individual dogs to each other forms a ball), but for graphic purposes the relationship of individuals is plotted in the two dimensions that most accurately displays their three dimensional relationships (coordinates 2 on y-axis and coordinate 1 on x-axis).  The bulk of the population of BRT is found around the center of the plot with a smaller number of more genetically differentiated dogs forming more distant genetic outliers.  The fact that all 93 individuals were found in the same plot indicates that they are all members of the same breed.  The presence of genetic outliers indicates that some individuals in the breed are somewhat less related to the bulk of the population than others, which was suggested by the slightly negative F value.

Figure 2.  A principal coordinate analysis (PCoA) plot showing how individuals within the population of 93 BRT have genetically differentiated from each other.  

The values of Aa, Ae, Ho, He, and F are helpful in defining the genetics of a population as it currently exists.  However, considerable genetic diversity is lost during the evolution of any breed.   An estimate of how much genetic diversity has been lost can be obtained by comparing the alleles found and their frequency with that of indigenous village dogs that currently exist in the Middle East, SE Asia and Island Pacific nations.  This population of dogs is genetically diverse, randomly breeding, and share common ancestry to virtually all existing breeds.  Based on studies we have conducted, village dogs from these regions still contain almost all of the genetic diversity that existed in dogs before the last several hundred years, during which time strong positive selection by humans has slowly, and sometimes rapidly, created our modern breeds.  

Figure 1 (above) plots the internal relatedness (IR) of the 93 BRT before (red) and after (blue) adjustment for genetic diversity lost during evolution of the breed to its present state.   IR is based on data from the 33 genomic STRs and is a more complex measure of heterozygosity that gives more value to less common alleles.  It is an indicator of the genetic similarity of an individual dog’s parents.   A value of -1 would indicate that the parents were totally different, while a value of +1 would indicate that an individual’s parents were genetically identical. The unadjusted IR values peak at a little less than 0.0, indicating that the population is a little more outbred than inbred.   An IR value of 0.25 would be expected if the parents of an individual BRT were full siblings.  This means that hardly any of the BRT in the population is inbred to this level, as based on existing genetic diversity currently in the breed. However, when the population is adjusted to measure diversity lost in breed evolution using village dogs as a standard, the peak shifts to the right and is near to 0.25, with a number of dogs with values up to 0.4.  This means that most BRT are related to the same level as offspring of village dogs that were full siblings,  and assuming that the full-sibling pair were offspring of relatively unrelated dogs.  Adjusted values over 0.25 would mean that the parents of the full-siblings were more closely related than random village dogs.

Black Russian Terriers are known to suffer from several health problems common to larger dogs, including hip dysplasia, elbow dysplasia, and gastric torsion (bloat), which can be considered non-immunologic heritable disorders of ancestral origin and of complex genetic nature.  Hypertrophic osteodystrophy, skin and intestinal allergies, and autoimmune diseases such as Addison’s disease are immunologic heritable disorders that are also of a complex genetic nature.  Simple recessive genetic traits for which tests are available include hyperuricosuria (uric acid stones), juvenile laryngeal paralysis and polyneuropathy, and progressive retinal atrophy.   The presence of both ancestral and more recent complex and simple genetic disorders in BRT is not unique and identical, similar or different disorders occur in many other pure breeds.  They are indicators of lost genetic diversity and inadvertent positive selection pressures for desirable traits.

Veterinary Genetics Laboratory, Tel 530-752-2211, Email VGL