The Canine Genome Project Sheds Light On Mysteries

Reprinted by permission from the February 2004 issue of the AKC Gazette

The U.S. National Human Genome Research Institute, a division of the National Institute of Health (NIH), is funding a $50-million project to sequence the canine genome. By June, the complete genetic code of Tasha, a female Boxer, will be sequenced at the Whitehead Institute of the Massachusetts Institute of Technology.

DNA is made up of four chemical letters, or nucleotides, represented by the letters A, T, C, and G. It is the ordering of 2.4 billion of these chemical letters that comprise the canine genome. Of interest to researchers are variations of single letters within genes, called SNPs, or single nucleotide polymorphisms. These SNPs can help differentiate individuals, breeds, and copies of normal or defective genes.

All genes come in pairs-one from the father and one from the mother. The researchers wanted to select a dog with the most homozygosity, or least variation between the maternal and paternal genes in all of its gene pairs. The breed of dog was not crucial, as all dogs are more than 99 percent identical at the DNA level. Tasha was chosen from more than 120 dogs and 60 breeds. She had one SNP per 20,000 nucleotides, while the average dog has 1 SNP per 1,000 nucleotides.

DNA 101
The genes that make a dog reproduce as a dog are the same across all breeds. The genes that create the differences between the breeds, but allow the breed to reproduce itself are homozygous within the breed. Then there are genes that create the differences among individuals within a breed. These variable gene pairs are what are estimated when computing a pedigree inbreeding coefficient.

Based on the pedigree relationship of her sire and dam, Tasha was not the most inbred dog of all of those examined. A sampling of Boxers whose pedigrees were more inbred than Tasha found that they had less SNP homozygosity. A pedigree inbreeding coefficient is only an estimation of average homozygosity. When the sperm and egg from Tasha's parents combined, they just happened to carry a large proportion of the same genes to combine into homozygous gene pairs. Contrary to what is reported in the press, the Boxer was not the most inbred breed based on the genetic sequence analysis of the breeds studied. Many breeds showed more average homozygosity. But no other individual dog showed the homozygosity of Tasha.

The researchers are using a process called the whole genome shotgun approach to sequence the canine genome. Chromosomes of DNA are broken into smaller, more manageable pieces. These pieces have their nucleotides sequenced. The small pieces are then compared and pieced back together to create the order of the nucleotides on the chromosomes.

The canine genome project is creating what is called a 6.5x sequence of the dog genome. This means that so many small pieces of Tasha's DNA are being examined that piecing them together should cover all of her chromosomes over six times. The location of each DNA letter will be determined an average of 6.5 times. This should provide a high degree of accuracy, and the most detailed genetic map of a dog to date.

Drs. Ewen Kirkness and Craig Venter, at the Institute of Genomic Research, have already created a 1.5x genome map of Venter's standard Poodle, Shadow. This rough framework study sequenced approximately 2 million fragments of Shadow's DNA to cover 80 percent of the entire genome at least one time. From their study, they estimated that dogs and humans have 18,473 genes in common, which is more than the 18,311 genes found to be in common between humans and mice.

After the researchers at the Whitehead Institute finish sequencing Tasha's genome, they will also sequence 100,000 DNA fragments from each of 10 additional dogs, encompassing nine other breeds, and a wolf. These breeds were selected based on their being unrelated to Boxers, and each other, to attempt to sequence and identify genes that create the differences between breeds. Included are the Beagle, due to the great amount of biomedical research done on the breed, and the wolf, used in this project for conservation as well as evolutionary studies. The researchers expect to find an average of 25,000 novel SNPs for each breed. The number of SNPs found for each breed will vary based on the average level of homozygosity within each breed.

The canine genome map can be compared with those of man, mouse, and other species to identify common genes that code for different traits, or disorders. By knowing the gene that causes a disorder in other species, researchers can study this "candidate gene" in the dog to see if it is the gene causing the disorder in specific breeds.

Up to now, the most success in identifying defective genes in the dog is for simple, one-gene Mendelian traits. However, large proportions of disorders have complex inheritance, or inheritance involving more than one gene pair. These include many cancers, heart disorders, and skeletal disorders. Thanks to the canine genome project, the cause of these disorders will be easier to find.

Jerold Bell is director of the clinical veterinary genetics course at the Tufts University School of Veterinary Medicine.

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