The purpose of this article is to provide a mini-course in genetics that will serve the reader well during upcoming articles on open registries and the Canine Genome Project, and why they are so important to the future of the breeds we love. Both the American Kennel Club and United Kennel Club are moving in the direction of genetic identification and registry.
Pull a few hairs out of your dog’s tail. At the bottom of a few of those hairs you will find a tiny root. That root contains about 40,000 cells. Within each of those cells is about 6 feet of genetic material called DNA–your dog’s entire genome. The information encoded inside the nucleus of that cell is a unique “blueprint” of what makes up your dog. This blueprint is absolutely specific to your particular animal, and thus identifies your dog unconditionally among all other living things, animal and vegetable.
Have you ever had a litter sired from more than one male? Have you ever been disappointed in the results of a mating between your bitch and the “top” stud dog? Well, the technology is available now top positively and inexpensively identify the sire of all your puppies, but parentage identification is just a small part of what is possible.
In the not-so-distant future we will be able to compare the genetic similarity between two prospective breeding pairs. Imagine being able to see how closely related two dogs are before you breed them. It is possible that what you thought was a tight line-breeding, when looking at just the respective pedigrees, would actually be a greater outcross for a particular trait.
What if you could screen your dog for all sorts of genetic diseases, or double up on the probabilities of a trait’s expression such as herding instinct or scenting? Even though the technology has not yet reached this stage, it is coming. With the human, canine, porcine, mouse and other genome projects under way, the breeding game has now progressed to the next level.
Are you mystified by the genetic code? Do you blanch at words such as allele, dominant, intron & exon? Do you think of microsatellites as small orbs circling the Earth? The time is coming when such words will be part of the everyday vernacular. The study of genetics has previously been the domain of specialists, but it is rapidly becoming part of the responsible dog breeder’s repertoire.
The study of genetics is much like learning a foreign language. it really isn’t all that difficult to become conversational once you master the rules and become comfortable with a new vocabulary.
Each cell within the body is composed of cytoplasm, a jellylike layer of material that surrounds the nucleus. Within the nucleus are a number of threadlike chromosomes that are almost entirely made up of two kinds of chemical substances, proteins and nucleic acids.
Nucleic acids have at least two functions: to pass on hereditary characteristics and to trigger the manufacturing of specific proteins. the two classes of nucleic acids are the deoxyribonucleic acids (DNA) and the ribonucleic acids (RNA).
DNA, the genetic building block, is made up of substances called nucleotides, each of which consists of a phosphate, a sugar known as deoxyribose and any one of four nitrogen-containing bases. These four nitrogenous bases are adenine (A), thymine (T), cytosine (C) and guanine (G). Canine DNA is about 6 billion nucleotide pairs long.
In mammals, the DNA molecule appears as two complementary strands that are wrapped around each other like the railings of a spiral ladder, known more formally as the double helix of Crick and Watson. The strands (sides of the ladder) are composed of alternating phosphate and sugar molecules. The nitrogen bases, joining in pairs, serve as the rungs.
Each base is attached to a sugar molecule that is linked by a hydrogen bond to a complementary base on the opposite strand. These bases are complementary because only adenine pairs up with thymine, and only cytosine pairs up with guanine; thus the pairs are AT and CG. For example, if one were looking along a strand of DNA, that is reading DNA linearly, looking down the two strands, the first segment pair might look like this:
When DNA is duplicated during cell division, the chain of nucleotides is synthesized from the 5′ (5 prime) end to the 3′ (3 prime) end. This terminology should be considered as a way to spatially orient oneself along the DNA strand. The 5′ end is referred to as upstream and the 3′ inch end is referred to as downstream. The two strands are held together by weak electrical bonds between the bases on each strand, thus forming basepairs (bp). Each strand has its own polarity opposite of the other. Thus if you turned the strands upside down, the picture would not change. An easy way to visualize the opposite polarity aspect of the two chains is to think of two identical snakes intertwined around each other but facing opposite directions (head to tail and tail to head). Thus each half of the double helix can serve as a genetic template of its complementary half.
Before a cell can express a particular gene, it must first transcribe that specific part of the DNA into messenger ribonucleic acid (mRNA). This is similar to the formation of a complementary strand of DNA during the division of the double helix, except that RNA contains uracil (U) instead of thymine as one of its four nucleotide bases. In the process of transcribing DNA into mRNA, all the T bases are converted to U bases. These bases–C,G, A, and U– are the alphabet of the genetic code. A sequence of AGATC in the coding strand of the DNA produces a sequence UCUAG in the mRNA.
When a cell is expressing a particular gene, that means it is producing either a specific protein or polypeptide (a short sequence of amino acids). It is also able to do this by translating a codon composed of three bases. For example, CUU stands for the amino acid leucine. CUA, CUG, and CUC also “code” for leucine, so there is some redundancy in the system. Notice in this example that only the base is different (A vs. G vs. C). The term degeneracy is used when a change in a base does not affect the amino acid being added to the peptide.
Breaking Down the Gene
So much for DNA and RNA. What is a gene? a gene is the basic unit of inheritance. Each one carries a set of directions for producing either a protein or a polypeptide. If all goes well, a complete set of genes–one half from each parent–is inherited. If the two copies of each gene are exactly alike, the progeny are homozygous at that locus. If the gene inherited from one parent is different from the gene inherited from the other, the progeny are heterozygous. Different forms of the same gene are called alleles.
In the dog, the various genes are located among 78 different chromosomes. What we don’t know is how many genes exist, although a rough estimate has been made that there may be about 100,000. We also don’t know where on the various chromosomes specific genes are located. In fact, we have just recently karyotyped the canine. This means that we are able to differentiate between specific chromosomes. This will be valuable information when we finally are able to map the canine chromosome. Such a genetic map will now only allow us to determine the position of genes relative to each other, but also will tell us their approximate distance apart on the helix.
At the molecular level, a gene is that portion of DNA that codes for a specific polypeptide. It also includes regions preceding and following (known as the leader and trailer) as well as noncoded regions within the gene called introns that act like spacers between the coding sequences known as exons. Between the genes are long stretches of noncoding areas, and 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
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, so even closely related
individuals will exhibit differences. These variations can be as
simple as a change of one basepair, called a point mutation, or as
different as the deletion or addition of basepairs. 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 markers would be necessary to differentiate
between siblings. (Even though these regions are not considered
genes in that they do not code for polypeptides, different forms
of these areas also are called alleles.)
Identifying Genetic Markers
It has been suggested that it will require about 1,000 microsatellites to saturate the canine genome, so that there will be a marker about every 3 megabases (a megabase is 1 million basepairs). This will ensure that once these markers have been identifies, 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 could be used as a diagnostic tool to screen for a desired characteristic or to identify 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. This usually means the disease does not become evident until the dog is well past the age where it might have been used for breeding.
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 special enzymes that recognize basepair sequences, it is possible to cut DNA into various lengths. These segments can be separated by gel electrophoresis because DNA carries an overall negative electric molecular charge. Under the influence of an electric field, the different fragments migrate toward the 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 also 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).
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 A 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.
The Genetic Crystal Ball
Given the advances in identification, the United Kennel Club, wanting to avoid the obvious pitfalls in the American Kennel Club’s pedigree honor system, has contracted with Zoogen of Davis, Calif., to use its genetic identification services for registration purposes. The authors have reported previously in DOG WORLD that the Canadian government will not allow importation of dogs under the age of 10 months for resale if the claim that the dogs are purebred is supported only by AKC registration. There are estimated to have been several thousand bogus AKC registrations. No one really knows the extent of the fraudulent registrations.
The AKC, in its February meeting, agreed to cooperate with the Institute for Genetic Disease Control in Animals in assembling a health and information database. The AKC-GDC plan proposes that sometime in the next three to five years, the LGDC would be placed under the aegis of the AKC, and its work would parallel the AKC’s registration program. In an effort to strengthen the registry, the AKC hopes to announce a pilot program sometime this spring to incorporate DNA testing in an effort to support and expand its registration facility and discourage fraud.
VetGen, a company associated with the University of Michigan and Michigan State University, currently offers identification and pedigree validation services, and also is able to test for several genetically transmitted diseases. An example of such a program already in place is the progressive retinal atrophy (PRA) screening under the auspices of the Irish Setter Genetic Registry through Purdue and Cornell universities. This is just the beginning. In the future we can anticipate that even polygenic diseases such as hip dysplasia ultimately will be avoided by standard DNA testing. Prevention of genetic disease, drug design, therapy protocols, identification and parentage verification are just a few of the many beneficial options that soon will be available to breeders and pet owners.
It is fortunate for us that the dog is the ideal animal for this type of genetic analysis. No other species has such variations in body type, traits and behaviors. Compare the appearance of a Newfoundland to a Tibetan Terrier, or the scenting abilities of a Bloodhound to a Samoyed. Contrast the phlegmatic behavior of the St. Bernard to the scrappiness of the whole Terrier Group. For this reason, test matings between different breeds are proving very useful for mapping studies. Information gleaned from this canine genome research can be applied to the humane genome and other mammalian genome projects because many genes have been highly conserved throughout evolution.
Exciting advances in the field of genetics are opening up opportunities for the breeder that until now have only been dreamed of in our attempts to produce the perfect dog. For those interested in pursuing this subject of genome projects, whether animal or vegetable, much information is available through various sites on the Internet. The keyword “genome” will provide plenty of sites to visit. Additionally, the Department of Energy puts out “Human Genome Program: Primer on Molecular Genetics.” There also is the very readable “Exons, Introns, and Talking Genes” by Christopher Wills, a professor at the University of California at San Diego.
The authors would like to thank the following for their time and valuable assistance: Wazyl Malyj, University of California at Davis; Dr. Jasper Rine, University of California at Berkeley; Dr. Elaine Ostrander, Fred Hutchinson Cancer Institute, Seattle; and from the University of Oregon at Eugene, friend and teacher Dr. Jim Long, and teachers Dr. George Sprague and Dr. Tom Stevens.
Statistician and retired Marine officer John Cargill wears many hats, among them that of an Akita Breeder. He is presently involved with the Osler Institute in continuing medical education in Terre Haute, Ind.
Susan Thorpe-Vargas has been accepted in the doctoral program for immunology at an Oregon University. She has an extensive chemistry and lab background and has been involved in numerous Environmental Protection Agency clean-up sites. She also raises and shows Samoyeds.