UC Davis School of Veterinary Medicine Veterinary Genetics Laboratory

Genetic Diversity Testing for Miniature Poodles

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 within the genome and in the Dog Leukocyte Antigen (DLA) class I and II regions. This test panel will be useful to Miniature Poodle breeders who wish to determine the amount of genetic diversity that exists in the breed and to compare that diversity with that of Standard Poodles.

Breeders and owners have submitted enough DNA samples from 110 Miniature Poodles to create a preliminary baseline for genetic diversity in the breed. However, given the high amount of genetic diversity that has been determined so far, more dogs need to be sampled determine the full range of that diversity. Therefore breeders and owners should continue to submit samples until the goal of at least 200 dogs is reached.

Price: $80

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Results reported as:
Short tandem repeat (STR) loci: A total of 33 STR loci from across 25 of 39 (panel 1) or 58 of 39 (panel 2) chromosomes 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: Seven additional 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. IR values can be adjusted using data from highly random bred village dogs from across the Middle East, SE Asia and Island Pacific nations. Adjusted IR values, known as IRVD, are estimates of the amount of genetic diversity that has actually been lost over the entire period of breed evolution and not just from the time founding dogs were selected and registries were closed.

Introduction

Previous genetic testing established Miniature Poodles to be a distinct breed compared to Standard Poodles, although the genetic imprint of Standard Poodles can be seen by shared genomic alleles and DLA class I and II haplotypes. This research can be openly accessed at: http://cgejournal.biomedcentral.com/articles/10.1186/s40575-015-0026-5, and http://www.vetmed.ucdavis.edu/ccah/local-assets/pdfs/Miniature_Poodle_genetic_comparison_May-23-2012.pdf.

The Miniature Poodle has a lower incidence of autoimmune disease than Standard Poodles and appears from these previous studies to be more genetically diverse. The Poodle Club of America standards determine “varieties” of Poodles by size alone and all sizes are AKC registered as Poodles. They are then assigned a variety according to their size as Toy, Miniature or Standard Poodles.  Although breeders are free to cross Miniature and Standard Poodles, there has been reluctance for breeders to do so.  However, there are some breeders who believe that genetic diversity, and therefore health problems, could be improved by increased cross-breeding of Standard and Miniature Poodles.  The purpose, therefore, of this study is actually twofold: 1) to determine the genetic diversity in Miniature Poodles from disparate regions of the world to help breeders better manage existing diversity, and 2) to compare and contrast existing genetic diversity both in the genome and in the DLA class I and II regions between Miniature and Standard Poodles.  For this reason, the various genetic determinations made for Miniature Poodles in this study will be presented separately and with comparisons to results obtained for Standard Poodles.

The Canine Genetic Diversity Test and What It Tells Us

A. Thirty three vs 58 STR loci genome panels

STR markers are highly polymorphic and have great power to determine genetic differences among individuals and breeds. The VGL has been experimenting with two different STR marker panels. Panel 1 has markers on 25/39 canine chromosomes and panel 2 has markers on 39/39 chromosomes. Twenty five of the 33 STR markers on panel 1 are also found on panel 2. Twenty markers on each panel are recommended for universal parentage determination for domestic dogs by the International Society of Animal Genetics (ISAG).  The panel of 58 genomic markers covers all 39 canine chromosomes instead of the 25 chromosomes of the 33 marker panel.  The test panel used for baseline genetic testing of Miniature Poodles used two different panels, panel 1 (110 dogs) with 33 STR markers and panel 2 with 58 STR markers (99/110 dogs). Testing has shown that the 33 marker panel gives results that are very close to the results from the 58 marker panel in the various genetic assessments (Fig 1). Therefore, most of the genetic assessments will be from the original 33 genomic STR loci (Table 1).

B. The use of genomic allele frequencies to assess genetic diversity in a population

The diversity of alleles at each STR and their frequency in a population can be used to make several genetic assessments such as  principal coordinate analysis (PCoA),  genetic assessment indices (average alleles/locus, average effective alleles/locus, observed heterozygosity, expected heterozygosity, index of inbreeding [F]), internal relatedness (IR), and adjusted IR (IRVD).  The first 33 STR markers listed were used in the original panel. Genomic STR loci and their frequencies for Standard and Miniature Poodles can be found at: genomic STR loci (Table 1).

Table 1: STR alleles from 33 genomic loci and their frequencies in different populations

(link to table 1)

C. Differences in population structure as determined by principal coordinate analysis (PCoA)

Figure 1 shows PCoA results for the same population of Miniature Poodles using allele frequency data obtained from either the 33 or 58 STR marker panels.  Both panels provide comparable results.

Figure 1.  A comparison of PCoA of Miniature Poodles using a 33 vs 58 STR loci panel; the 33 STR loci are found on 25/39 canine chromosomes (top plot), whereas the 58 STR loci panel has markers on 39/39 chromosomes (bottom plot). The results are comparable.

Principal coordinate analysis can also be used to determine how two populations have genetically differentiated from each other. Figure 2 shows a PCoA plot of 110 Miniature, 898 Standard and 57 Standard/Miniature Poodle crosses tested using 33 genome-wide STR markers. The two varieties of Poodles are clearly related, given their close proximity to each other on the plot, but are genetically distinguishable.  Only three dogs registered as a Standard Poodle were found among the Miniature Poodle population. The Standard/Miniature Poodle crosses and a number of registered Standard Poodles bridged the two populations, as expected. Registered Standard Poodles that segregated with the crosses may well have had both varieties at some point in their pedigrees, but such ancestry was not divulged.

Figure  2.  PCoA plot of Miniature Poodles (n=110), Standard Poodles (n=898) and Standard-Miniature cross (n=57) based on 33 STRs.

D. Assessment of population genetics using heterozygosity measurements

The standard genetic assessments such as those first proposed by Wright can be determined from alleles and their frequencies at each STR locus in the genome.  These measurements include average # alleles/locus (Na), average # effective alleles/locus (Ne), observed heterozygosity (Ho), expected heterozygosity (He) and a coefficient of inbreeding (F) (Table 1). The average number of alleles/loci for the Miniature Poodles was 7.58, which is higher than many other pure breeds. The average number of alleles/locus that contributed the most to genetic diversity (effective alleles) was 4.06, again higher than other pure breeds that have been studied to this point. The observed and expected heterozygosity (Ho=0.712 and He=0.720) were also higher than many other breeds. The values for Ho and He were not significantly different from each other, creating a breed-wide coefficient of inbreeding (F) close to zero (0.010).  Therefore, these data indicate that Miniature Poodles have a relatively high genetic diversity and that breeders are doing a good job of maintaining Hardy-Weinberg equilibrium (random breeding).  Standard Poodles had somewhat higher average alleles/locus (8.91) than Miniature Poodles, but the number of effective alleles per locus was 3.49 for Standard Poodles vs. 4.06 for Miniature Poodles, and the Ho (0.654) and He (0.680) was lower for Standards than Miniatures. The coefficient of inbreeding F was 0.038, which was also higher than for Miniature Poodles. Therefore, Miniature Poodles were genetically more heterogeneic than Standard Poodles.  The average effective alleles per locus was higher for the Standard/Miniature Poodles than for Standard Poodles (3.76 vs 3.49) and the F value was -0.058 vs 0.038 demonstrating that the crosses were more outbred than the population as a whole.  

Table 2.  F-Statistics comparison among Miniature, Standard Poodles, and Miniature-Standard cross based on 33 STR markers

Pop

N

Na

Ne

Ho

He

F

ST

Mean

898

8.909

3.489

0.654

0.680

0.038

SE

0.473

0.210

0.019

0.019

0.005

ST-Mini

Mean

57

6.879

3.756

0.752

0.709

-0.058

SE

0.285

0.195

0.021

0.016

0.012

Mini

Mean

110

7.576

4.057

0.712

0.720

0.010

SE

0.477

0.263

0.018

0.018

0.009

E.  The use of genomic STR loci allele frequencies to determine internal relatedness (IR)

The genetic assessments given in Table 1 refer to the population as a whole, and not to individual dogs.  Moreover, assessments based on allele frequency alone do not weight the contributions of common vs rare or uncommon alleles to genetic diversity.  Internal Relatedness (IR) is a calculation that gives more weight to rare and uncommon alleles and has been often used as a measure of the genetic differences of an individual’s parents.  IR values are therefore a measure of heterozygosity contributed by each parent. The lower the IR score, the more outbred the individual, and the higher the score, the more inbred.  IR scores of all individuals in a population can be graphed to form a curve ranging from -1.0 to +1.0.  A dog with a value of -1.0 would come from parents that were totally unrelated at every locus, while dogs with an IR value of +1.0 have parents that were genetically identical at every locus.

The mean of  internal relatedness calculated for 110 Miniature Poodles from North America and other parts of the world  was 0.038,  with individuals ranging from -.176 to +0.389 (Fig. 3). IR values as high as +0.389 were uncommon and most of the breeds had values below +0.20.  A value of +0.25 would apply to offspring of parents that were full siblings, provided that the parents of the full siblings were randomly bred.  IR values >0.25 occur when the parents of the full sibling parents were themselves highly inbred.  Like in any pure breed, there are always a few individuals that are highly inbred.  

F. IRVD values as a measure of genetic diversity lost during the entire period of breed evolution from earliest ancestors to present

The IR values can be adjusted in such a way as to provide an estimate of total genetic diversity lost from the earliest ancestors of the breed to present time. This is done by using allele frequencies obtained from DNA of present day village dogs from the Middle East, SE Asia and Island Pacific nations. These dogs are the most random bred and genetically diverse population that has been studied to date and is the origin of almost all modern breeds. The adjusted IR value is known as IR-village dogs or IRVD.

The IRVD values for Miniature Poodles are shown in Fig. 3 (blue line). The mean IRVD was 0.149 for the population as a whole individuals ranging from -0.085 to 0.525 (Fig. 3). The shift to the right in IRVD values was not nearly as pronounced as it has been for several other breeds that are participating in genetic diversity testing at the VGL and indicates that Miniature Poodles have retained a greater amount of the overall diversity still present in village dogs.

Figure 3.  IR and IRVD values for 110 Miniature Poodles.

G. DLA Class I and II Haplotype frequencies

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, which contains the canine Major Histocompatibility Complex, can be used as proxy to represent gene diversity associated with immune function (see sidebar). Four STR markers are used to identify DLA class I haplotypes and three STRs for DLA class II haplotypes.

We have identified 27 distinct DLA Class I and 20 distinct DLA Class II haplotypes in Miniature Poodles (Table 3). These STR-based haplotypes are strongly associated with known functional haplotypes that have been determined by sequencing of DLA-88, DRB1, DQB1, and DQA1 genes.  It is likely that a small number of additional haplotypes, especially of low frequency, will be recognized as the goal of 200 dogs being tested is reached.

Miniature and Standard Poodles share many class I and II haplotypes, but also possess haplotypes unique to each breed. Miniature Poodles have 10 class I and nine class II haplotypes that were not found in Standard Poodles. Standard Poodles have even more unique class I and II haplotypes, but some of these may be identified among Miniature Poodles when more dogs are tested. Unique haplotypes tend to be uncommon

Standard Poodles have a marked imbalance in DLA class I and II haplotypes, with a high incidence of the 1001, 1002, 1003 class I haplotypes and 2001 class II haplotypes. The 1018 DLA class I and 2003 class II haplotypes are higher than expected by chance in Miniature Poodles. The prominence of a relatively small number of DLA class I and II haplotypes in breeds with a broad genetic base, such as Standard and Miniature Poodles, is usually a result of an artificial genetic bottleneck that occurred in the past associated with either a popular sire or closely related bloodline.

 

Table 3: DLA Class I & II Haplotype Frequencies in Miniature Poodles

DLA Class I Haplotype Frequencies (Updated Nov 20, 2017)
Miniature Poodle (n=234)
1001380 373 281 1820.015
1002380 365 281 1810.002
1003387 375 277 1860.011
1004393 379 277 1830.004
1005389 371 277 1810.053
1006387 375 293 1800.002
1009382 377 277 1840.085
1011376 365 281 1800.002
1012388 369 289 1880.043
1013392 373 289 1860.165
1014375 373 287 1780.004
1016382 371 277 1780.024
1018375 373 287 1860.233
1020388 369 289 1840.002
1025380 365 281 1860.004
1028376 369 291 1860.047
1031382 371 277 1860.051
1032382 377 277 1780.006
1033382 379 277 1810.006
1036389 365 289 1800.045
1040380 371 277 1860.006
1045376 371 277 1860.002
1054382 379 277 1840.002
1068380 373 287 1810.015
1105382 379 277 1780.064
1106395 379 277 1780.024
1107376 375 293 1830.030
1108382 371 277 1800.011
1109381 379 291 1860.015
1110382 371 289 1840.011
1111387 378 287 1820.009
1112393 371 277 1810.004
1168382 379 289 1860.002
DLA Class II Haplotype Frequencies (Updated Nov 20, 2017)
Miniature Poodle (n=234)
2001343 324 2840.017
2002343 327 2800.004
2003343 324 2820.504
2004351 327 2680.002
2008339 327 2760.085
2009351 324 2800.011
2011345 322 2840.002
2012345 322 2800.066
2014339 322 2840.024
2015339 327 2800.049
2016339 323 2840.030
2021339 324 2680.064
2022339 327 2820.002
2024343 323 2800.009
2025351 321 2800.045
2028345 327 2880.006
2032339 323 2800.011
2037341 327 2800.009
2040345 327 2800.002
2053343 324 2800.015
2066339 324 2800.030
2067343 322 2840.011
2100341 324 2820.002

Another way to assess genetic diversity in the DLA class I and II regions is to apply the same types of statistics used to assess diversity across the genome as reported in Table 2.  Table 3 shows this same type of genetic diversity assessment using allele frequencies at each of the 7 STR loci that are associated with a much narrower, but very critical, part of the genome, i.e., DLA class I and II regions on chromosome 12.  Miniature Poodles possess somewhat fewer average alleles per locus than Standard Poodles (7.00 vs. 7.86), but the average number of effective alleles per loci is higher (3.53 vs. 2.76).  Therefore, a greater proportion of alleles are contributing to genetic diversity in the DLA in Miniature Poodles than in Standard Poodles.  The observed and expected heterozygosity (Ho and He) are higher in Miniature Poodles than in Standard Poodles and are virtually identical in value, giving an inbreeding coefficient (F) in these DLA regions that is slightly less than zero.  This indicates that DLA class I and II alleles are in Hardy-Weinberg equilibrium (random breeding), which supports the earlier F values based on genomic markers. In contrast, Ho and He values for Standard Poodles are not in balance, giving an inbreeding coefficient (F) of 0.044.  Therefore, a subpopulation of Standard Poodles is more inbred in the DLA class I and II regions than the population as a whole.  This is logical given the marked imbalance in DLA class I and II haplotype frequencies in Standard Poodles.  The Standard Poodle/Miniature Poodle crosses are intermediate in most values between the two breeds and the inbreeding coefficient F is negative, mirroring what was seen with the genomic markers; crosses are more outbred than either parental population, as would be expected for matings between two genetically disparate breeds.

Table 4.  Assessment of genetic diversity within DLA region using the frequencies of alleles for each of the 4 STR loci associated with DLA class I and the 3 STR loci associated with class II. 

Population

N

Na

Ne

Ho

He

F

ST

Mean

897

7.857

2.760

0.554

0.579

0.044

SE

0.202

0.937

0.449

0.060

0.063

0.005

ST/Mini

Mean

57

6.000

3.241

0.679

0.652

-0.036

SE

0.000

0.926

0.428

0.063

0.053

0.026

Mini

Mean

110

7.000

3.532

0.690

0.691

-0.002

SE

0.000

0.816

0.460

0.027

0.034

0.020

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 3). 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 3). 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.


 

Certificates for sharing of genetic information

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 genotyExample certificate for an outbred dogpes and DLA class I and II haplotypes for the dog(s) tested. The diversity of alleles at each of the genomic STR loci and their frequency in the population were used to determine the genetic relatedness and diversity. The internal relatedness value for the dog being tested is related to the population as a whole.

An example certificate for an inbred dog

 

How are DNA based genetic markers best used by breeders?

The goal for Miniature Poodle breeders should be to maintain the large amount of genetic diversity that exists in the breed and to continue to randomly select mating pairs. IR values, because they reflect the unique genetics of each individual, cannot be used as the criteria for selecting ideal mates. A breeding pair with identical IR values can have genetically distinct parents and 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 is much different in genomic allele and allele frequencies and DLA haplotypes, 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 as a result of genetic recombination. The more genetically diverse and different the parents, the greater the range of IR values in their offspring.

In brief, 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 individual 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.

Genetic information on Miniature Poodles and Standard Poodles will be extremely useful for those breeders interested in outcrossing between the two varieties of Poodle.  Outcross pairs should be chosen to provide maximum genetic differences in puppies over parents (i.e. low IR scores) and genetic diversity testing used to identify which puppies would be most valuable for further outcrossing or backcrossing.  The goal of outcrossing and backcrossing should be to regain the desired phenotype, while maintaining the greatest amount of new diversity.   

Heritable diseases problems of the Miniature Poodle

Miniature Poodles, like many small dogs, are long-lived. The breed suffers from a number of conditions that are common among miniature breeds such as patellar luxation, aseptic necrosis of the femoral head, collapsing trachea, and periodontal disease.  Cushing’s syndrome and heart valve degeneration with increase in frequency with age. Intervertebral disc rupture can be a problem in individuals with shorter legs and longer backs. Cystic calculi are also a problem in the breed. Hip dysplasia occurs, but is uncommon.  Autoimmune disorders are also relatively uncommon compared to breeds such as the Standard Poodle, but can include type 1 diabetes, immune mediated thrombocytopenia or hemolytic anemia, and granulomatous meningoencephalitis.  Otitis externa is a problem as it is in any dogs with this type of coat, drooping ears, hair growth down into the ear canal and skin allergies. Cancer such as lymphoma is one of the leading causes of death, but not different in frequency than dogs in general.  Skin tumors such as basal cell carcinomas are also a problem in the breed.  Epilepsy is increasing incidence in many breeds. Obesity is a problem in Miniature Poodles as it is in many pure and random breeds of dogs.  Progressive retinal atrophy and von Willebrand’s disease type 1 are simple recessive genetic disorders in the breed. Cataracts often occur within the first three years of life, also suggesting a heritable origin. 

Conclusion

Miniature Poodles have managed to maintain a great deal of genetic diversity compared to other breeds.  This can be attributed to a wide genetic base that probably involved a number of breeds,  their popularity and large population size favoring random mate selection,  a relatively loose standard including many coat colors and a range of sizes and body types and careful breeding  by owners.

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