Genetics And Breeding Strategies:
Essays For The Dog Breeder
By Dr. Susan Thorpe-Vargas
Reprinted with permission.
Mapping out the dog's genetic future
Author's Note: This chapter is highly technical and will be of most interest to students of genetics.
In 1990, the greatest intellectual task ever attempted by humans began. Even more of a challenge than walking on the moon, the Human Genome Map Project staggers the imagination in terms of concept and complexity.
The ultimate intent of the project, completed in 2001, was to ascertain the definitive sequence of the more than 3 billion base-pairs comprising the human genome. A genome is all the genetic material in the chromosomes of a particular organism; its size generally is given as its total number of base pairs. This enormous effort has "spilled over" to other species, and dogs will reap the benefits. Building a road map of the dog's makeup through the Canine Genome Project eventually will lead to genetic tests that in turn may eradicate many genetic diseases. These results, which haven't the social, ethical and legal implications that muddy the waters of the human genome work, may be used to enhance the quality of our dogs' lives and help us back out of the genetic cul-de-sac in which we now find ourselves.
Pet owners spend billions of dollars every year diagnosing and treating genetic diseases afflicting their pets. We now have in our hands the elementary tools to prevent or ameliorate our dogs' physical suffering. Recently, in a truly international effort, the dog community took another small step forward: the publishing, far earlier than ever expected, of the first canine linkage map. Not only will this endeavor help to discover the basis for many genetic diseases in dogs, but the effort will spillover into human disease also.
The first of many hurdles
One of the biggest hurdles to overcome when mapping a genome, human or canine, is to assign a gene or genetic marker to a particular chromosome. Unfortunately assigning a genetic marker has been much more difficult because most of the canine chromosomes are the same shape and many are quite similar in size. Remember, besides the coding regions (i.e. genes), chromosomes include noncoding regions within the gene that act like spacers between the coding sequences. In addition, between the genes are long stretches of noncoding areas. It is in these sections that Mother Nature has given us a gift to help map the canine genome.
Interspersed along the entire length of the genome are regions called microsatellites. These areas of DNA consist of tandem repeats (identical or nearly so) of a short basic repeating unit, such as TGTGTGTGTGTGTG...ATTATTATTATTATT... etc. They can be mono-, di-, tri- or tetranucleotide blocks, and are referred to as short tandem repeat polymorphic (STRP) markers. Considered in evolutionary terms, these regions tend to show a higher percentage of variations, therefore even closely related individuals will exhibit differences. These variations can be as simple as a change of one base-pair, called a point mutation, or as different as the deletion or addition of base-pairs. For example, these repeats usually appear in blocks that vary from 10 to 30 units long. A puppy could inherit a (TG)10 from its dam and a (TG)14 from its sire. If the pup carries enough of these parental type alleles, it is possible to ascertain parentage. However, further variations in additional markers would be necessary to differentiate between siblings.
Identifying genetic markers
It has been suggested that it will require several thousand microsatellites to saturate the canine genome. This means that there will be a marker about every 3 megabases (a megabase is 1 million base-pairs). This will ensure that once these markers have been identified, at least one of them will be associated with, and inherited along with, a specific gene. Once a marker has become linked to a particular gene that has been characterized for a specific trait or disease, it then can be used as a diagnostic tool to screen for a desired characteristic. It would also be useful in identifying a carrier (or an affected individual) of a genetically transmitted disease. This would be extremely valuable information, as many inherited diseases are of the late onset type, meaning the disease does not become evident until the dog is well past the age where it might have been used for breeding.
Another handy tool for the geneticist was discovered some 30 years ago. Scientists were able to isolate several proteins from various strains of bacteria, named restriction enzymes because they cut DNA at specific sites. The normal function of these enzymes was to protect the bacteria from attack by phage (viruses that infect bacteria) or other foreign DNA. Each restriction enzyme recognizes a particular double-stranded DNA sequence. This specificity has been extremely useful for mapping the genome. Hundreds of restriction enzymes have been isolated. Depending upon the source, these enzymes "see" restriction sites that vary from four to eight base-pair recognition sites.
Some rare-cutter enzymes cut DNA very infrequently, which results in a small number of very big pieces. The use of simple sequence repeats in identifying canine polymorphic markers has been a fairly recent innovation. Prior to this, a technique called restriction fragment length polymorphism (RFLP) markers were used to construct gene maps. Using restriction enzymes that recognize base-pair sequences, it is possible to cut DNA into various lengths. These segments can be separated by gel electrophoresis. DNA carries an overall negative electric molecular charge. Under the influence of an electric field, the different fragments migrate toward a positive charge at a speed that corresponds to their molecular weight. Since the shorter fragments travel faster than the longer pieces, it is possible by using this technique to differentiate between segments that differ by as little as one nucleotide.
RFLP thus provides the basis for a technique called DNA fingerprinting that can establish a parent-progeny relationship. The chief disadvantage of this procedure is that it is extremely labor intensive (read: expensive) and requires a great deal of genetic material. Tandem repeat markers have an advantage over RFLP because they can be assayed by polymerase chain reaction (PCR) and have a higher polymorphic information content (PIC). For this reason they have become the basis of the DNA parentage verification tests in use today.
PCR is a technique that increases a specific section of DNA about 1 million times. Since it is an automated procedure, the reaction can be repeated as many times as needed to obtain ample DNA for that area being investigated. The DNA is then separated using gel electrophoresis, and because the variations in length correspond to those of the repeat sequence, it is possible to recognize individual differences. The main drawback of this procedure is that the primers used in PCR amplification for a dog are not always the same for other mammals, so unique markers must be developed for every species.
The term PIC is a little more complex. If a marker is to be useful, it must be unique. As the number of variations within each marker increases, it becomes more and more individualized and therefore has a higher polymorphic information content. This is a little like saying my house is on First Street, then adding that it is on the corner of First Street and B Avenue. If next I say it is on the northwest corner, it is easier to locate. Then if I add that it is a white house with green shutters, etc., you can see that each little bit of information increases the ability to find my house. It is these characteristics that make markers useful for parentage verification and for the purposes of positive identification of the animal. Remember it is possible to see chromosomes only in certain phases of the cell cycle. The best time to see them is during metaphase. Normally, chromosomes exist in a dispersed state that cannot be seen with an ordinary light microscope. Just before the cell divides, it tightly gathers up its chromosomes. While in this state of metaphase, it is possible to take a picture of all the chromosomes in the cell. As you recall this picture is referred to as the karyotype, and in this picture, we can see the number of chromosomes, their size and their physical appearance.
Standardization of the canine karyotype was necessary before researchers could relate genes or genetic markers to their chromosomal origins. Development of chromosome-specific markers will ensure that all of the canine chromosomes will be represented within the map and that the linkage groups are correctly orientated. This difficult barrier has been overcome by some brilliant work in England. Knowing the dog has 39 haploid chromosomes (half of 78 is 39), researchers used a male dog in their experiment because it is easy to see the Y chromosome. That left 38 chromosomes to identify.
The researchers first separated the chromosomes by their DNA content and the use of two fluorescent dyes that distinguish the base-pair ratio by preferentially staining either A-G or C-T rich regions. Using a technique called dual-laser flow cytometry, they were able to resolve their sample into 32 different components. Twenty-two of the portions contained single chromosomes, and the remaining eight had two each. Thus, all of the chromosomes were accounted for.
To identify the chromosome type, they then used these fractions to "paint" a normal metaphase chromosome spread (a cell that has been "fixed" chemically so it no longer cycles) and highlight the chromosomes using another technique called FISH (fluorescent in-situ hybridization).
Hybridization is a very important concept to understand because it is the basis for many of the methods used to study DNA. Recalling that the two possible DNA base-pairs are C-G and A-T, if you have a DNA strand that reads AATGGCTAT, its complimentary strand would have a base-pair sequence of TTACCGATA. In FISH, complementary strands of DNA or RNA preferentially bind to each other. If one of the strands is tagged with a fluorescent dye, it can be used to locate its equivalent complement on another DNA strand. Other types of probes use radiographic or immunological labels. Hybrid probes will be addressed in detail when we discuss mapping strategies.
Mapping our way
Just like maps we use to find our way around town, genetic maps establish spatial organization and symbolize a wide variety of information. They also are similar in that there are different types of genetic maps, each with a corresponding range and level of precision.
The karyotype (also known as a cytogenic map) is the lowest resolution of what is known as the physical map. The highest resolution would be to know any posttranscriptional modifications (changes in the RNA after DNA transcription) once we know the entire base-pair sequence. Another type of genome map is a linkage map. The final genetic map will be a synthesis of physical and linkage maps. This new map will let us know which chromosome a gene is on, how many base-pairs separate each genetic marker, their positions relative to each other and ultimately the complete basepair sequence. Once the entire sequence has been resolved, we will need to find all the genes and use this information to determine their function. The medical applications of this map alone are overwhelming. These data, used as diagnostic tools to identify deleterious mutations, combined with future gene therapy technologies, could lead to the eventual eradication of genetic disease. We also could learn how certain behavioral traits are transmitted, which is of especial interest to dog breeders.
Making the linkage
In 1865, the father of modern genetics, a young monk named Gregor Mendel, published a paper in which he described the inheritance of certain traits he had observed while growing peas. In choosing which attributes to follow, Mendel was very lucky that he chose the characteristics he did, as they all turned out to be on different linkage groups. As a rule, we equate linkage groups with individual chromosomes, and the number of linkage groups corresponds to the haploid number of chromosomes. Thus, the dog has 39 linkage groups.
Mendel's observations led him to postulate two "laws". The first law says "particular factors" (genes) come in different forms (alleles). When gametes are formed, these alternative alleles are inherited independently from each other. Mendel's second law predicts that different genes (i.e., different traits that are not on the same chromosome) will assort themselves into two different types of progeny in statistically equal amounts. These two types are the parental type and the recombinant type. However, when the genes that code for those traits are on the same chromosome, the percentage of recombinant types would be less than the anticipated 50 percent. American geneticist Thomas Hunt Morgan suggested this lowered recombination rate simply was a function of how far apart the genes were from each other. The closer together they were, the more likely they would stay 'linked.' We can use this information to predict the relative distance between the loci of two genes. Today we measure the distance that separates genetic markers in centimorgans (cM). Two loci are said to be 1 cM apart if they are separated by a recombination event one percent of the time. This roughly corresponds to a physical distance of one million base-pairs.
The next step in the mapping process is to determine the linear order of the genetic markers. For instance, let's say Gene A is 5 cM from Gene B and Gene B is 7 Cm from Gene C. If we then find out Gene A is 12 cM from Gene C, we can assume their relative positions are Gene A.....Gene B.......Gene C.
It would be nice if it were this easy. Unfortunately it is not. Coding regions, also know as exons, are just too far apart to be linked conveniently, and so we need to use other types of genetic markers. Another problem is that, compared with bacteria or fruit flies, the dog has too few progeny to generate the statistical recombination data needed. Humans have even fewer offspring.
The discovery and use of microsatellites, the genetic markers in the noncoding introns of the gene, has overcome this barrier. So far, about 1800 canine microsatellites have been characterized. At this time, markers have been found for 98% of the canine genome.15 In order to be useful, these markers must be similar within species, breed and family groups, yet be different enough (polymorphic) to detect the differences among individuals.
To repeat, microsatellites exist as di-, tri- and tetra repeat patterns, but because of founder effect and the tight "linebreeding" inherent in the purebred dog, the most useful microsatellites for elucidating the canine linkage map have been tetra repeats. Although these areas are not genes, differences in the number of copies of the basic repeat unit also are called alleles (length polymorphism). Because of technological advances, it is fairly easy to ascertain the difference between two genotypes, and these procedures are the basis for the most commonly used parentage tests now available. The more alleles a microsatellite has, the more likely it is to be useful.
Mutations that occur within these regions do not cause changes in the dog's appearance, behavior or health; however, linking these genetic markers to disease alleles or genes that characterize a specific trait will lead to diagnostic tests to identify carriers or affected individuals. Several of these tests already are available. These microsatellites are especially useful for identifying the carrier status of genetic disorders that arise from mutations at different sites within the same gene. This is why breed-specific tests often are required for the same disease.
Recombinant DNA technology promises to make higher resolution linkage maps possible. A lab in France has used radiation to fragment human chromosomes and has fused these fragments with cells from other species. These hybrid cells can be manipulated so that only specific human chromosomal components are retained. Determining the frequency of genetic markers that stay together after being irradiated places their order and the distance between them at a finer resolution. These techniques also have overlapped into the canine mapping effort. Work is progressing rapidly on a radiation hybrid panel specific to the dog.
The physical mapping realm
In addition to the linkage maps, there are several types of physical maps:
Keep in mind that the lowest-resolution physical map is called a cytogenetic map (karyotype). During the metaphase and the interphase stage of the cell cycle, it is possible to stain the chromosomes with various dyes that result in distinctive banding patterns. Using radioactive or fluorescent labels it is possible to assign genes or other identifiable DNA fragments to their respective chromosomes and to estimate the distance between them, measured in base-pairs. Improved FISH methodology now allows identification of genetic markers from as close as 2Mb to 5 Mb apart (one Megabase, or Mb, equals approximately 1 cM). With FISH, we can observe chromosomal mutations and abnormalities associated with disease states. German researchers have discovered a translocation on the first canine chromosome (a type of mutation - see Ch. 2) that is linked to mammary tumors in dogs. Cytogenetic analysis may prove useful for comprehending the underlying genetic mechanisms for other types of cancers for dogs and humans.
Complementary DNA (cDNA) maps
Although two genetic markers may have a recombination rate higher than 50 percent, this does not preclude them from being on the same chromosome. This further complicates the mapping issue. The trick is finding out which of the 39 unique dog chromosomes to assign a particular gene to. One of the methods used depends on knowing the protein the gene is responsible for making, then working backward to figure out the approximate DNA sequence. Using a tagged complementary hybrid probe made from a synthetic DNA sequence, it is possible to see where the gene is located on the chromosome.
Another way to map a gene to a chromosome is to know the base-pair sequence of the gene that codes for the same trait in a related species. For example, all mammals have some genes in common. We even share conserved sequences - base sequences in a DNA molecule that have remained essentially unchanged through evolution - with the lower orders of animals. Although entire chromosomes are not conserved among species, parts of chromosomes, called syntenic groups, are.
Homologous genes and genetic markers from the human mapping project have been beneficial to the canine map effort. In turn, the canine map has been expected to be useful to the Human Genome Project. Knowing the function or position of a certain gene in one species makes it a possible candidate gene for the same ailment or trait in another species. One such ailment, Severe Combined Immunodeficiency (SCID), is caused in humans and canines by a mutation in one of the proteins that form the receptor site for interleukin-2. Interleukin-2 is a chemical messenger that improves the bodies response to disease. This specific defect causes a profound inability to mount both a cell-mediated and humoral (antibody) immune response. It frequently is called the "Boy in the Bubble" disease because of the movie about a boy with this affliction who lived in an isolation bubble.
Several different laboratory techniques are used to localize differences to a smaller region of the genome. Once such an area has been identified, it is possible to use automated sequencing methods to distinguish any base-pair mutations. If these mutations result in an amino acid substitution within the coded protein product, it becomes a likely suspect. The candidate gene approach can save a lot of time, not only by providing a model for the progression and course of a disease, but by suggesting treatment strategies.
A better technique for obtaining finer mapping details is the contig map, produced by cutting a chromosome into very small pieces, cloning these pieces and constructing an overlapping clone "library."
This kind of cloning is a recombinant technique that involves inserting a DNA segment into another host cell, called a cloning vector, and using that cell's own replication apparatus to generate multiple copies of foreign DNA. This provides large amounts of experimental material.
Cloning vectors often are bacteria such as E.coli, but recent technological advances have made it possible to clone larger segments of DNA by using an artificial cloning vector packed into a lamda phage. This virus normally infects bacteria and inserts its own DNA into that cell's genome, where it is replicated along with the normal cellular DNA. Using nature's own tricks has often worked well for us in this endeavor. Once contig mapping of a particular section of the genome is accomplished in one laboratory, the resulting genetic library can be published so other researchers can use the same information. A common reference system called sequence-tagged sites makes this sharing of information possible. STS are short DNA fragments (200 to 500 base-pairs long) whose unique base sequence and location make them useful landmarks. A variation of this method is to sequence cDNA partially instead of random sections of DNA. Complimentary DNA is a special type DNA that is synthesized from a messenger RNA template. This is reverse of the process as it normally happens. Since they are "tagging" a transcription product of an expressed gene, they are called expressed sequence tags, or EST. These are especially useful for finding candidate genes.
We have only just begun
Current mapping strategies, as brilliant and innovative as these techniques are, still have left gaping holes that need to be filled. Genetic mapping is technology-driven, but technology costs money and time. Faster, more precise mapping methods are needed. Funding for the canine project generally takes a back seat to funding for the human mapping effort and for species that are more agriculturally and economically important than the dog. But it must be done as this information will provide us with the basis of all genetic testing and strategies for coping with genetic disease.
Where is the money to come from? It is most likely that if genetic testing is embraced by the dog fancy, market pressures will result in development of new tests, some of the profits from which will be used to fund further research. If breeders do not test for genetic disease, who will?
For breeds at great risk, certain breeding strategies such as introgression, a very complex method that involves going back to the original stock and selecting for or against a particular gene trait, should be considered. We need to contemplate opening up the studbooks. At present, studbooks are closed. The only way to get back some of our lost genetic diversity is to breed those dogs that made up the breed originally. The AKC could have a set procedure for going back to the original foundation stock, even when there is no actual breed registry in the original country. As it now stands, each breed club is being asked to "reinvent the wheel" in this endeavor. The Samoyed, Saluki and the Basenji are perfect examples of this predicament because seldom have tribal peoples from whence these dogs came, maintained written records. Some hard questions also need to be asked about the validity of our pedigrees and what must be done to protect those records. The fancy must address these problems if our beloved animals are to have a viable future. We are the custodians of our various breeds, thus the responsibility for finding answers to these genetic problems is ours.