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 Italian Greyhound (IG) breeders who wish to track and increase genetic diversity of the Italian Greyhound breed as a long term goal.
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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.
The genetic information used to formulate the enclosed tables and graphs came from DNA samples of 329 dogs from North America, the UK, and Continental Europe including outlying countries such as Romania, Russia, and the Ukraine. We believe that the dogs tested represent almost all of the genetic diversity that still exists in the breed. This data will be updated as more dogs are tested, so allele and DLA haplotype frequencies may change to a limited extent over time. The breed appears to have reasonable breed-wide diversity, but this diversity is very unbalanced. As a result of genetic bottlenecks and potential popular sire effects, a majority of IGs are relatively inbred and contain a minority of the existing genetic diversity. This has resulted in an increased incidence of known simple recessive disease traits (e.g., PRA, glaucoma, enamel hypoplasia) and a wide variety of autoimmune diseases. Additional disorders of a heritable nature exist in the breed but the exact form of inheritance and causative mutations are yet to be determined. These disorders include spontaneous limb bone fractures in young dogs, Legg-Perthe’s disease (aseptic necrosis of the femoral head), patellar luxation, hip dysplasia, congenital megesophagus, progressive periodontal disease, porto-systemic shunts, masticatory myopathy, vitreous degeneration, cataracts, lens luxation, color dilution alopecia, epilepsy, and allergies. These various disorders appear to have resulted from both ancient and relatively new mutations that have been concentrated in certain lines as a result of inbreeding. The hope is that breeders will use genetic diversity testing, along with pedigrees, to re-establish genetic diversity across the breed by careful mate selection, while continuing to investigate diseases that appear to have a genetic basis.
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 for the IG population in the USA and Europe (Figs. 1a & 1b). Internal relatedness for IGs from North America, UK and Continental Europe ranged from <-0.2 (most outbred) to >0.4 (most inbred).
Figure 1a. Distribution of IR estimates in 212 Italian Greyhound from USA based on intra-breed diversity (solid line or Red line), compared with IR adjusted for diversity lost during breed development (dashed line or blue line). Diversity lost as a result of breed development was determined by comparing allele frequencies at the same loci between Italian Greyhound and randomly breeding village dogs from the Middle East, SE Asia, and the Pacific Islands. These village dogs are the most genetically diverse population that has been studied and are the ancestors of Italian Greyhound.
Figure 1b. Distribution of IR estimates in 174 Italian Greyhound from Europe based on intra-breed diversity (solid line or Red line), compared with IR adjusted for diversity lost during breed development (dashed line or blue line). Diversity lost as a result of breed development was determined by comparing allele frequencies at the same loci between Italian Greyhound and randomly breeding village dogs from the Middle East, SE Asia, and the Pacific Islands. These village dogs are the most genetically diverse population that has been studied and are the ancestors of Italian Greyhound.
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 18 different STR-associated DLA Class I and 17 DLA Class II haplotypes in Italian Greyhounds (Tables 1 and 2). Various Class I and II haplotoypes are also linked to each other forming 28 extended haplotypes. DLA-Class I and Class II STR-based haplotype frequencies in Italian Greyhound are also provided in the Tables 1 and 2.
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.
|DLA Class I Haplotype Frequencies (Updated Mar 17, 2017)|
|Italian Greyhound (n=550)|
|1008||386 373 289 182||0.1345|
|1012||388 369 289 188||0.0100|
|1016||382 371 277 178||0.0691|
|1030||380 373 293 178||0.0218|
|1036||389 365 289 180||0.0009|
|1040||380 371 277 186||0.1091|
|1044||375 373 291 178||0.2273|
|1048||380 370 289 184||0.0127|
|1049||380 370 289 186||0.0009|
|1050||380 371 289 182||0.0009|
|1051||380 371 289 184||0.0036|
|1052||380 372 289 184||0.1945|
|1053||382 377 277 186||0.0900|
|1054||382 379 277 184||0.0136|
|1055||386 373 289 180||0.0009|
|1056||386 373 289 190||0.0064|
|1058||387 378 287 186||0.0082|
|1059||390 371 291 182||0.0936|
|1065||380 371 277 181||0.0009|
|1104||386 383 289 186||0.0009|
|DLA Class II Haplotype Frequencies (Updated Mar 17, 2017)|
|Italian Greyhound (n=550)|
|2003||343 324 282||0.0082|
|2015||339 327 280||0.0064|
|2017||343 322 280||0.2273|
|2023||341 323 282||0.0218|
|2029||337 324 268||0.0936|
|2030||339 322 268||0.0009|
|2031||339 322 282||0.0682|
|2032||339 323 280||0.0500|
|2033||339 323 282||0.0073|
|2034||341 322 280||0.2273|
|2035||341 323 280||0.0864|
|2036||341 327 276||0.0655|
|2037||341 327 280||0.0109|
|2038||345 324 280||0.0100|
|2039||345 327 276||0.1127|
|2040||345 327 280||0.0018|
|2041||349 321 280||0.0009|
|2044||343 324 268||0.0009|
|2031||01301/00101/00201 or dqb002v|
Table 2 demonstrates the strong relationship between the STR-associated DLA class II haplotypes and the official international DLA class II designations for alleles within the DRB1, DQA1, and DQB1 genes. One (or more) STR haplotypes is associated with each of the official DRB1/DQA1/DQB1 haplotypes identified in Italian Greyhound. 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. 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.
The goal for IG breeders should be to produce a greater and greater proportion of puppies with IR scores less than 0, and with time even lower scores. There appears to be ample genetic diversity in the breed to achieve this goal over a number of generations. This 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 provide a web repository of genetic information on hundreds or thousands of dogs. 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. This service could be on a subscription basis and hopefully key information on potential mates would also be included. This might include pictures of the animal, age, phenotypic traits such as height, coat color or pattern, behaviors, show and field characteristics, health status, physical location, breeding fees, etc. The service would only identify the most appropriate mates and owner contact information. It would be solely up to breeders to identify the best possible genetic matches, to make a secure contact, and enter into actual breeding arrangements. This service, if initiated, would be independent of any official registry.
What We Have Learned From Our Studies
We were pleased with the participation of IG breeders in the USA and Europe in our genetic diversity study. European samples came from countries as distant as Russia, Ukraine, Romania, Germany, France, Italy and the UK. Although we expected that European and US IGs would be somewhat genetically distinct, we found that the two populations were more diverse from each other than expected. A Principal Component Analysis plot showed that although there was some overlap, with more European dogs in the US than US dogs in Europe, the two populations have been bred in isolation from each other for some time.
Figure 2. Principal component analysis (PCA) plot of all dogs tested from Europe and the US. Each red diamond or black triangle represents a dog in the study. PCA takes into consideration the relatedness of individuals in a population based on allele frequencies, in this case for the 33 STR loci. The closer individual (or groups) dogs graph to each other, the closer their relationship.
We also looked at the genetic heterozygosity present in both populations using F-Statistics. IGs from the USA utilized somewhat more alleles at each of the 32 STR loci than European dogs (6.2 vs 5.6). However, the European dogs were somewhat more heterozygous than US dogs (Ho=0.68, SE 0.025 vs Ho=0.598, SE 0.032). The F value (a coefficient of inbreeding) was higher (more positive) in the US dogs than in the European dogs (F=0.039, SE 0.008 vs F=0.002, SE 0.013), indicating that the European dogs were less inbred than the US dogs. Taken as a whole, it appears that the European dogs have a slightly smaller gene pool, but that mate selection has been more random.
We are still analyzing possible genetic associations with inbreeding and disease traits that are potentially heritable. The IG has the greatest variety and incidences of potentially heritable diseases than any breed that we have studied (see introduction). This observation is admittedly based more on dogs from the US than from Europe, but it has been hard to get accurate disease information from Europe. It appears, however, that European dogs suffer from many of these same disorders, but we cannot say whether it is more or less. Although some of these diseases have resulted from spontaneous mutations within the breed (e.g., enamel hypoplasia, glaucoma, PRA), followed by inadvertent positive selection for certain desirable phenotypic traits, others may have been introduced by outcrossing in the breed’s history (e.g., color dilution alopecia, congenital megesophagus, Legg-Perthe’s disease, aseptic necrosis of the femoral head, patellar luxation, hip dysplasia, epilepsy). Some of these heritable disease traits are the result of simple recessive mutations, while others appear to involve many genes and gene pathways. The most worrisome "polygenic" or complex genetic traits fall under the category of autoimmune disease (and probably the related problem of allergies). IGs have one of the highest incidences of autoimmune disease and it manifests in virtually every clinical form known to humans or dogs. Clinical forms of autoimmunity in the breed include Addison’s disease (hypoadrenocorticoism), immune mediated thyroiditis (hypothyroidism), immune mediated polyarthritis and meningitis, systemic lupus erythematous, systemic vasculitis, panniculitis, masticatory myositis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, lupoid onychodystrophy, pemphigus, rheumatoid arthritis, and immune mediated orchitis (male sterility).
Will better selection of mates increase genetic diversity and lower IR scores and significantly reduce the incidence of these major health problems? We believe that it will, but the proof will come when and if it is widely applied. The number and complexity of heritable disorders in the breed will not make it easy to find causes, develop tests, and eliminate bad genes. Therefore, the best immediate solution is to dilute out these undesirable traits making it less likely to mate dogs that carry them. There is still considerable genetic diversity in both US and European IGs to allow for a significant reapportionment of existing diversity. The genetic difference between European and US dogs also offers the additional option of "outcrossing." However, the reluctance to mix these two lineages of dogs has to be overcome.
It should be re-emphasized that the parentage markers recommended for dogs by the International Society for Animal Genetics (ISAG) are incorporated into the present 33 STR loci panel used for genetic diversity testing. The allele sizes have been adjusted, when necessary, to conform to the allele sizes standardized by ISAG. Therefore, this genetic diversity test can be used for parentage confirmation or in lieu of an internationally recognized parentage tests using the ISAG canine markers.