By Dr. Margrit Miekeley
Scientists are zooming into the Saluki DNA, a lecture given during the Seminar of the Saluki World Congress 2008 in Mustiala, Finland. Conclusion of some scientifi c consideration with a transfer into breeding practice.
Fig. 1: Comparative analysis between the human and canine genome.
Genes are not abstract but are a biological reality. As they are effective over boundaries and make manipulations possible, it is impossible to lock them up in laboratories. Genes lie on certain spots (loci) next to one another on the chromosomes in the nucleus of the cell and are visible with an electron microscope when stained. Genes at the same locus but with different expression are called alleles.
Egyptian mural wall paintings from about 3,000 BC show that hounds had been bred for many years. These hounds called Tesem were used for chasing. They had erect ears and curled tails. Modern genetic research of today is mainly based on mitochondrial studies and on comparison of the gene diversity. Dogs were derived from Asian gray wolf about 16,300 years ago (Pang, et al. 2009). The oldest ancient breeds are from Asia and present the greatest variability and similarity to the wolf. As hereditary traits are in permanent connection to environmental alteration, there is a continuous interplay between genes and external infl uences. In the sense of evolution gene mutations provide a means for rapid adaptation to altered circumstances. Consequently, gene reactions are without limit and borders (see Miekeley 2007).
When breeding dogs many questions will come up, that require an understanding of the genetic process.
Of course, every breeder will know that breeding purebred dogs is a selection of desired characteristics found within their breed standard. Therefore the breeder should have a grasp of “practical genetics” before his fi rst litter at least.
Fig. 2: Dogs were derived from wolves, as newest data show their origin in South East Asia (Pang, et al. 2009)
Today geneticists describe a breed standard as “homozygous and heterozygous”. What does it mean? Both terms “homozygous and heterozygous” describe the condition of a gene-locus. If two alleles, one on each of the chromosome pair, are identical, the haplotype for that locus is homozygous. But if they are different, they are heterozygous. A doubling of recessive genes carried by each of the parents for the same defect gene becomes homozygous and may then cause hereditary diseases in dogs.
By breeding constantly on the same traits, the genes will not only become homozygous, but also eradicate certain alleles, which the standard of a breed does not stipulate. This loss of certain genes can cause a lot of apparent diseases in a line. If some of the lost genes were associated with polygenetic conditions it could result in a change in their appearance and affect the individual. Thus, over generations of breeding the genetic diversity of purebred dogs has become more and more reduced by the selection on certain traits and is even becoming worse. This is particularly true when inbreeding occurs without natural selection in a small isolated population (Trumler, 2000, 8. ed. p. 40 and Gallant, 2008, p. 112).
The Oriental sighthound breed is very old and based on the newest data it is suggested it originated in eastern Asia. It appears to demonstrate a high percentage of homozygosity. For this reason it would seem that this breed needs no further gene doubling by further maintenance of inbreeding methods. Today this method becomes more and more disputed, though practised for a long time to achieve a desired kennel-type. If there is a strict selection for vitality in line-breeding, it is really possible to postpone the period of time for a “blood-refreshment”. The behavioural scientist Eberhard Trumler (2000) gained all his knowledge about dogs by his own experiments, and he noted that effective selection measures should be taken in a litter, e.g. the offspring should have good pigmentation (a lot of black colour) and has to prove vitality. But the pet breeder is not willing to follow these demands, when a pup is born weak, it will be reared by bottle. Johan and Edith Gallant consider this action a capital sin of modern dogdom, because natural selection has been switched off. They feel breeders should not interfere unnecessarily and “in nature, only mothers with a solid nervous constitution and normal reproductive behaviour qualify for breeding.” (see Gallant, 2008, p. 132).
Anyhow, “inbreeding (incest) is not a method to produce hereditary defects, but will turn up existing damages” (Trumler, E.; 2000, p. 253). But inbreeding can never be a method to “clear” a line of the recessive defect genes by eradication. This must be declined because of animal welfare and ethical reasons. So it must not only be necessary to have a wide knowledge on lines and genealogical tables when breeding hounds, but it is also necessary to have a basic knowledge of genetic process. Above all, breeding animals demands a great responsibility for the creature.
With our love of our animals in mind, what can be done to avoid or to restore a genome that has suffered from inbreeding for generations? Particularly when the health of an individual may be badly compromised by gene loss. Vanished characteristics could result in faulty metabolism of the individual? Metabolism regulates the biochemical processes necessary for life of an individual. If functional mistakes occur a homozygous organism is more likely to have diffi culty in infl uencing and balancing regulations. The reason for this disadvantageous phenomenon lies in the composition of the dog’s genome, that is aligned for heterozygosity. Malcolm B. Willis, 1992, stated that all mammals need a certain level of heterozygosity. When heterozygosity is lost in an individual, particularly the genes associated with vitality, fertility and physical fi tness, it will be exposed to every environmental infl uence. It is known, that a homozygous organism has only 20-30 buffer
substances, while a heterozygous one may have as many as 120 and 180. When this involves the immune system, (leukocytes vs antigens) gene loss results in interference with white blood cell multiplication resulting in an infection. This can have dramatic consequences. Therefore, a dog bred on heterozygosity will have a sounder immune system than one bred on homozygosity, and thus be able to better adapt to environmental infl uences. It will have a stronger power of resistance.
Geneticists do not only demand giving up methods of inbreeding to preserve the genetic diversity, but also to avoidance of champion or Super dog breeding (Krautwurst 2002). That means, that champions should not be bred any more exclusively with champions. This reduces not only gene-diversity, but also the dog’s physical health.
Fig. 3: Mapping disease genes in dog
Although, it is possible to calculate the inbreeding coeffi cient and the loss of the ancestors before mating, these calculations must be seen as a taxation, as they generally do not go far back enough in the generations. Modern molecular genetics involves research to recognize markers for certain hereditary disease in the canine genome (see Miekeley, M.: DNA-Tests in Molecular Genetics and Holistic Health Care in Hound-Breeding, in “Unsere Windhunde” 2/2004, p. 16). Today it is important to develop special molecular genetic tests, to qualify the inbreeding-status and the degree of relationship in future. A preventive method to reduce the danger of illness in purebred dogs is to analyse the micro-satellites of chromosomes also. These are sure indicators to fi nd out the degree of relationship and the level of homozygosity of certain genes (Streitberger, et al; 2007).
Breeders should consider doing an outcross from a different strain as a means to carry out “blood refreshment”. Saluki breeders have the rare opportunity of securing this gene pool enrichment from imports from the countries of origin. Some Western breeders consider COO imports for mating as a high risk, because their ancestors are not known. However, many direct imports probably show a high degree of heterozygosity of genes and thus they will pass on more diversity and possibly also a higher variety of characteristics. Though the offspring may not correspond to all points of the breed standard ideally, this can be offset by the increased fact of vitality and fi tness.
Fig. 4: A desert bred smooth saluki at the 1st Saluki Beauty Contest 2006 in Abu Dhabi with henna on his legs.
Practical experiences in dog-breeding have shown, that the number of puppies increases after an outcross. That suggests that fertility has increased. In addition, in maturity, the dogs show more substance and height after such an outcross. A low number of pups and frequent barreness of a bitch can be an indication of inbreeding depression (Trumler 2000). Knowing that purebred hounds possess a high level of homozygous genes from the beginning, breeders should not fear the risk of taking a direct import into the line and nor that they will lose their favoured type. Also, the opponents of COO import matings are afraid of unknown genes coming from those unknown ancestors. Like all matings, recessive genes for certain unwanted traits or diseases can be passed on for generations, but both parents must be the carrier for such genes to be expressed.
So one should bear in mind that the direct import can never be the only trigger for an illness that has a recessive autosomal inheritance. The best indicator for vitality is the dog´s good stamina and performance that is proved during chasing. A high level of heterozygosity in genes means more buffer-substances to create a functional immune-system. This is becoming more and more important with the increasing amount of viral infections and high environmental pollution. So a powerful defence-mechanism is of superior importance for a body, but more details about this topic later on.
Fig. 5: Bahraini salukis having fun in the desert of Dubai
During recent years, I have visited the United Arab Emirates often and could admire many different Saluki-types with a suspected diverse genome, They should be seen as a “treasure-house” for genetic resource. Genetically, it is of high concern to perpetuate real desert-bred Salukis in the countries of origin because it is there that these hounds will likely be tested for their natural abilities of the breed’s “Ancient Heritage”, I note fortunately that I have not seen in the UAE Salukis with exaggerated breeding forms, e.g. with over-angulated rears, with a falling back or a swan-neck. Obviously some of the Salukis are still be used for chasing as demonstrated by their cropped ears and henna on their legs. If these imports may have for our taste - an “unnoble head”, a lack of elegance in conformation, or a few markings which may not correspond to the European standard, compare them to the western hounds with those breeding exaggerations in the phenotype which do not correspond to the standard that much or that little either. Breeding on excessive traits, which are of no importance for the function, affects the health of the animal. The phenotype and the genes always correspond to each other (Dan Belkin 1993). In the age of globalisation we should appreciate to opportunities to have an exchange of experiences and also of genes between Occident and Orient.
Resulting from a long lasting gene impoverishment and increasing homozygosity in hounds, the white blood cells (leukocytes) are no longer able to proliferate. This allows infectious intruders to go undetected and unattacked to kill them. This all opens the door to many different diseases. The question is: are there any biological explanations for this phenomenon?
Fig. 6: MHC gene cluster, the "Molecule of the Month"
What is the essential role of the genes in the so called MHC complex which has been found in all vertebrates? These are polypeptides (proteins) anchored in the cell membrane. Infectious agents bind to these proteins of the MHC-molecules and may alter them. The T-cells, a special kind of leukocyte, are able to recognize the cells with MHC protein/foreign agent as non-self. Because the receptor on the cell surface no longer fi ts, the pathogens and infected malfunctioning cells with the strange protein will be killed by cytolysis. The MHC region plays an important role in the immune system of all mammalian genomes. If the system is properly functioning, it can control autoimmunity and reproductive success. Certain MHC-genes in the genome of all mammals have attracted the attention of evolutionary biologists. These genes present at three loci demonstrate a high level of allelic diversity which are associated with proper functioning. The most diverse locus in the human genome is the MHC with 500 known different alleles. The MHC-genes are the most polymorphic members of the mammalian genome needing this high level of diversity for proper function. Why does this complex need diversity? A powerful immune system that is based on diversity is fl exible for balancing reactions. But a malfunctioning immune system can produce autoimmune disorders, which attack the individual itself. These include autoimmune haemolytic anaemia (AIHA), diabetes, and many others.
Some of the newest research during the last decade indicates that a natural balancing system exists between the MHC complex and reproductive behaviour. The diversity of the MHC-genes plays a major role is selecting a reproductive partner in humans. Research suggests that humans, fi sh and mice are able to recognize by smell the structure of the MHC-genes of a potential partner. This undoubtedly has played an important role of survival of the species.
Fig. 7: MHC genes and autoimmune diseases - Inbreeding and "popular sire effect" destroy the dog's MHC diversity predisposing it to autoimmune diseases.
A study of female students near their time of ovulation showed a preference for the body-odor of men whose MHC complex genes were very different from their own (Wedekind, 1997). A female makes her choice of a male, when she decodes the gene register of a potential partner with her nose. That all means, the more diverse the genepool of the parents, the fi tter and healthier their offspring will be – and it is the best way to expand the gene spectrum for a new profi le with powerful resistance to pathogens. In general, a woman’s own smell has always been rejected (except during pregnancy or being on the pill). Thus, great variability of the MHC super types is not only necessary for a powerful capacity of immunity, but also for optimal sexual success. This fact may not only infl uence the choice of the partner, but suggest it may also determine whether a pregnancy will be successful or not. Dogs as mammals, probably demonstrate the same phenomenon with regards to breeding: If there is a similarity in the structure of the MHC- genes, the bitch might refuse a male for mating and she will be barren. Likewise the bitch might reject a pregnancy as an “irritating transplant” (Wedekind 2000).
At the Seminar of the Saluki World Congress, summer 2008 in Finland Prof. Hannes Lohi, the University of Helsinki, presented the lecture “Zooming into the Saluki DNA: the genetic diversity and the health of the breed”. He introduced LUPA, an organization with 22 members from 12 European countries which supports research projects on dog diseases. (LUPA is very much like the AKC’s Canine Health Foundation). Many of these studies fi nd markers for cancer, cardio-vascular disorders, infl ammatory disorders, neurological disorders and monogenic diseases. Many of diseases are the consequence of a malfunctioning of the immune system showing a reduced diversity of the MHC genes with homozygosity. He emphasized the importance of a diverse MHC. In the study, he compared DNA analyses, from COO imports salukis with those from western bred salukis. He found that COO import salukis had a higher level of heterozygosity in expression of the MHC-genes than those seen from long consolidated Western strains. Prof. Lohi noted, that he considered a DNA analysis as an open book, because it is able to detect hidden defects and reveals convincing stories about this breed.
Fig. 8: LUPA is doing dog disease research in 12 different European countries
LUPA reflects the companionship between dogs/wolves and humans, as Romulus and Remus - the founders of Roma who survived by being fed by a female wolf. Why are scientists simultaneously studying diseases in dogs? Humans and dogs share a very similar genome and develop the same diseases. Doing research on dog diseases gives information about human diseases and vice versa. Further, dogs have a unique population history (pedigrees) and breed structure as well as a short life span. Dogs can simplify the genetic studies on humans. Dogs are ideal to make studies on phenotypic variability in size, colour, behaviour and character. Hounds became superior companion for human geneticists, so they are certainly more than “man’s best friend”.
Fig. 9: LUPA - an organization for dog diseases research - a female wolf is feeding Remus and Romulus - a symbol for LUPA and for holistic consideration of nature.
Variety and diversity in morphology are principally demanded in nature (Willis 1992). But in the breeding of purebred dogs we fi nd reduced variety and diversity in breeds where a group of genetically similar animals have descended from a few ancestors. This natural law has been ignored unfortunately and consequently the 400 breeds that have been created during the last 400 years have produced almost 400 diseases during this period (Lohi 2008). Some of these are e.g. cataract, epilepsy, atopic dermatitis, mammary tumours, which are the same diseases in humans. But the most common ones in dogs are cancer, epilepsy, allergies, heart disease, vision disorder, hip dysplasia and thyroid disorder. This fact places dogs as a species with the second largest number of known genetic diseases.
So the structure and condition of the MHC-genes can give the geneticists important information about the dog’s genotype and health. Prof. Lohi confi rmed that inbreeding can result in reduced diversity of the MHC method resulting in a decrease in the dog’s health as a result of a weakened the immune system. The Finnish Saluki Club like the AKC is supporting studies on genetics and the search to fi nd markers for certain severe diseases that threaten this wonderful saluki breed. At the Saluki World Congress 150 saluki blood samples were collected for genetic testing. In 2004, the Club made an anonymous survey of 507 salukis to fi nd out which diseases occur mostly in the breed. The results indicated autoimmune diseases, Carcinoma and Heart Disorders.
The Finnish University has established a large dog DNA bank with more than 20,000 blood samples from 200 breeds. This will be a great resource for different genetic studies. The Finnish Saluki Club has contributed samples from almost 300 Salukis to this DNA bank. This will enable the scientists to initiate research including the genetics of autoimmune disorders and genetic diversity of the breed. They have also studied the possible association of the immune system. These studies include the characterization of the genes in the MHC. The structure of the MHC genes will be compared between healthy and ill dogs. As the MHC genes form a unique gene cluster in the genome, so each gene can have several variants and combination of these variants, which secures powerful immunity.
Prof. Lohi warns breeders about the consequences of a long lasting inbreeding method. Of concern is the use of popular sires for mating. This practice can destroy or diminish MHC diversity of future generations of a breed. Many might display a predisposition for autoimmune diseases (Wachtel 1998; Krautwurst 2002; Gallant 2008). Once more it must be said, the more homozygosity in the MHC the higher is the chance for illness. Over 30 different autoimmune diseases occur in humans, AND these same diseases are becoming more common in dogs. At the end of the lecture, it was noted that genetic diversity of the immune genes can be analysed by special DNA tests. When a marker for an autoimmune disease is found, it should be possible to create a test to identify carrier dogs in a breed. These tests can be used when planning breedings. Using saluki samples will allow for progress and advantage for the breed with a small population. This research will refl ect on breed health and reveal important stories of the breed. How happy we saluki-fanciers can be!
During the time since Prof. Lohi’s lecture, I have continued communications with him and his research crew via the internet. Some more information and papers were easily ordered. When questions arose regarding problems in a friend’s salukis, blood samples were collected from three female salukis by a vet and were sent to the laboratory of the Helsinki University for testing.
After some weeks the response from the scientists arrived with the results. All three females have, the same structure of the MHChaplotypes present in duplication on loci DQA and DQB but differ on one locus (DRB). That means homozygosity on two loci. In his Helsinki presentation Prof. Lohi reported that of the 94 Salukis participating in the MHC study – 72.3 % showed the same haplotypes on loci DRB, DQA and DQB. He suggested that perhaps this may be a sign of a long lasting line-breeding. A practical question would be: What could be done to restore the wanted diversity of the MHC genes in future? Dr. Lohi’s suggestion is “To avoid homozygosity of the MHC-genes you should fi nd a stud-dog with different haplotypes on the three mentioned loci”. Given the high incidence of these same haplotypes (72.3 %) at these three loci in western salukis,this will probably not be easy. Of course, a potential sire should also be tested fi rst to know whether the bitch and dog would be genetically fit parents together.
Fig. 10: A vision is to be seen - humans are healed of disease by dogs.
Research into canine disease is ongoing in many places. Even with tests to identify many diseases and to fi nd DNA markers for them, breeders must have a more open attitude to illness and share information about severe diseases. Sincerity among the breeders is necessary. Here, I quote Dr. Friedmar Krautwurst’s statement: “The fi rst main principle is the demand for honesty in a breed. Those breeders (better to say producers), who deny defects in their litters or breeds are a shame for the whole breeding organisation and should not participate further more. In ANY breed it is possible for defects can occur, this is relating to genetic laws. But if this is spread widely and misused by ignorant laymen to the character assassination under breeders, then it is a violation of any breeding cooperation, so the clubs should punish this accordingly” (Krautwurst, 2002, p.183).
So Scandinavian saluki breeders are obviously avantgardists, fortunately, because they are able now to present the quality of their Salukis by advertising a litter as follows: “The planned litter has a high DLA/MHC diversity.” DLA means dog leukocyte antigen, that is necessary to have a powerful immune system with diverse MHC genes. Hopefully, this method will become more common as owners test to fi nd the best fi tting partners to avoid diseases in Salukis or in other dog breeds. And this will also help humans.
If there is any interest to participate with samples in the projects of Prof. Lohi at the University of Helsinki, it is kindly asked to determine one partner of a country who will contact the scientist. Otherwise the activities in research could be disturbed.
As the scientists need urgently further DNA samples that can be obtained from saliva, cheek swabs or blood, you can fi nd more information by contacting the following links:
Dr. Margrit Miekeley has held a particular attraction to sighthounds since her childhood and has kept them since 1971. After teaching biology for 20 years, Margrit returned to university where she undertook a research study in biology to graduate with her doctorate. Her love applies to all animals and plants, but especially to her dogs. She has experience in breeding and rearing animals and genetics. After her retirement from teaching the mother of two sons published several articles in dog magazines. Her book “A Life with Sighthounds and other animals. Stories and experiences” was published in 2005 by Kynos and has already sold out.
Fig. 11: The authoress and her young saluki - looking into the horizon of research.
The images 1, 2, 3, 7, 8, 9 and 10 were presented during Prof. H. Lohi’s lecture given on the Saluki World Congress 2008 in Finland. All pictures © Prof. H. Lohi with his kind permission to publish.
The images 4, 5 and 11 © Dr. Margrit Miekeley
Image 6: Molecule of the month by http://www.pdb.org.png
Text © Dr. Margrit Miekeley
All texts and images, except otherwise noted, copyright © Dr. Margrit Miekeley