Finding the Genes and Mutations Causing Various Coat Colors
a brief explanation of how candidate genes are tested in relation to dog coat colors
This webpage is part of a series on Dog Coat Color Genetics was mounted in 2006 and was last updated on Decmeber 26, 2013 by Sheila Schmutz
Mapping Markers or Genes - Pre the Dog Sequence Assembly
Gene mapping was done by many labs throughout the world, including our own. Some labs concentrated on finding and/or mapping markers that are very polymorphic (have many alleles). Such markers are often "microsatellites". Microsatellites are long runs of 2-4 base paris repeated many times. Several hundred of these markers have been found and mapped in the dog.
Other labs concentrated on mapping genes. Here, I am defining a gene as a section of DNA that codes for a protein or hormone or regulatory peptide. In layman's terms these would be "real genes", not markers. There are two main approaches to mapping these real genes. One is to use a radiation hybrid panel. This means the researchers must find a unique piece of the gene that is different than that of the rodent they used to make their panel. The details for this mapping approach will not be explained further here. This approach places the gene on a particular chromosome and perhaps even a small region of the chromosome, depending how the panel was constructed.
The second approach for mapping real genes is called Linkage Mapping. One needs to find a polymorphsim, or DNA base pair change, often called a SNP( single nucleotide polymorphism) within the gene. The SNP could be in an exon of the gene but is more often in an intron. The dog families are needed to continue. Ideally the litter is large and the sire or dam or both may have had more than one litter so that many pups are available. For the family to be "informative" or useful for gene mapping, either the sire or dam or both must be heterozygous for the SNP in the gene under study. Then one must use the work of other researchers who developed and mapped markers and test if a marker that is polymorphic "co-segregates" with the SNP in the gene.
In the family above, the dam is heterozygous for both the SNP (C or T nucleotide) and the marker (104 bp or 108 bp piece). Therefore she is the "informative" parent and I have underlined the allele she gave to each pup. In 4 of the 5 pups she gave her T and 104 or her C and 108. However Hairy didn't get either of the typical combinations but instead got a C and 104 from her. A recombinant event occurred during the meiosis of the ovum that lead to Hairy. We need to study many such families and then we compare the number of pups that had recombinant events to those that were nonrecombinant, which in this case is 1/5 or 20%. If that same proportion had occurred in the group of families studied, we would conclude this marker and the gene are 20 cM apart. That's actually quite far in genetic terms. At 50 cM, we give up and say we can't detect that the marker and gene are on the same chromosome or that they are not linked.
Mapping traits also requires "informative" families. For example, the gene causing spotting in dogs has not been identified yet. Little called this the S locus. His work suggests that solid color is dominant to spotting. The family below shows that the sire should be heterozygous for spotting since he had one spotted and one solid color parent. Then when mated to a spotted female, they had both spotted and solid color pups. One sees that the sire gave his G nucleotide to every spotted pup and his A nucleotide to every solid color pup. Again if this same consistency occurs in many litters than we conclude the gene with this SNP is a likely candidate gene for spotting. We would also say that the trait spotting is mapped very close to this gene or 0 cM away because there were no recombinant pups. Newfoundlands would be a great breed to study for spotting by comparing the Landseer pups to the solid color pups from a Landseer mated to a solid colored Newfoundland.
Choosing Candidate Genes
Researchers usually choose genes to be "candidate genes" or likely genes for a trait because a very similar trait was shown to be caused by that gene in another species, such as mouse or humans. We might say that a good gene for albinism in dogs is tyrosinase (TYR) because that is one gene that causes albinism in humans, mice and cattle. Or a candidate gene can be chosen because it has been shown to be part of a similar pathway in another species.
Another reason a gene may be chosen as a candidate gene for further study is that the trait was mapped to a marker on a particular chromosome and that a gene also mapped to that chromosomal region just sounds likely. This is often called a "positional candidate gene". For example if spotting had been mapped to chromosome 14 in dogs and a gene known to function in the pigmentation pathway was on that chromosome, one might study it further as a candidate gene. This mapping would have been done today by GWAS (genome wide association study), then by the linkage mapping described above.
Finding the Mutation
Once the trait has been mapped to a particular gene, then it is time to begin to look at the sequence of the gene to try to find the causative mutation. For this, random dogs are used. Dogs with and without the trait are needed. Some of the dogs that do not display a recessive trait, like spotting might still be heterozygous for spotting and therefore have one copy of the mutation but they should never have 2 copies.
Because most genes have several exons and introns, and are very many base pairs in length, it can require a piecemeal attack to actually sequence the entire gene from DNA. However RNA contains only the exons and therefore the sequence is considerably shorter. RNA must be prepared from a rapidly frozen piece of tissue where that gene is being expressed. In the case of coat color, skin biopsy samples or dew claws or docked tails are wrapped in tin foil and immersed in liquid nitrogren immediately. They must be kept frozen in transit to the research lab. RNA is an advantage for research but not handy for collection. Also RNA does not contain the promoter region of the gene and some mutations occur there instead of in the expressed portion of the gene. These are especially difficult to find.
DNA can be obtained from many tissues. Hair roots have DNA so these can be used. White blood cells have DNA. One of the most convenient ways for dog owners to collect DNA is using a cheek brush. These should be used at least 30 minutes after the dog has eaten though so you are collecting its DNA and not the DNA from the soup bone it just chewed.
The table below shows the genes that have been found to cause some or all of the phenotypes Little (1957) assigned to particular loci. Additional genes in the pigmentation pathway have also been mapped but thus far have not been shown to explain any variable phenotype in dogs. The GenBank number relates to the mRNA sequence if one has been submitted or predicted.
|Little's Locus Symbol||Gene||Action||GenBank||Dog||Human|
|A for agouti||ASIP||hair changes color along its length or over parts of the body||NM_001007263||24||20q11|
|B for brown||TYRP1||brown or black eumelanin in dogs||AY052751||11||9p23|
|C for color||SLC45A2||some forms of albinism||NM_001037947||4||5|
|D for dilute||MLPH||"leaden" in mice||AJ920333||25||2|
|E for extension||MC1R||eumelanin or phaeomelanin in dogs||NM_001014282||5||16q24|
|H for Harlequin||PSMB7||Harlequin in Great Danes||GU305913.1||9||-|
|K for blacK||DEFB103||dominant black and brindle in dogs||NM_001129980.1||16||-|
|M for merle||PMEL||merle||NM_001103216||10||12q13-q14|
|S for spotting||MITF||some/all? forms of spotting||AY240952||20||3p|
|R or T?||KITLG/MGF||roan in cattle||AY094361||15||12q22|
|?||KIT||white spotting in pigs and cattle||AY692084||13||4q12|
|?||EDNRB||overo in horses||XM_545664||22||13q22-q31|
|?||TYR||Siamese in cats||AY336053||21||11|
|?||DCT/TYRP2||greying in mice||NM_001048130||22||13q32|
|?||RAB27||ashen in mice||NM_001048130||30||15|
|?||MYO5||dilute in mice||NM_001048130||30||15|
|?||PAX3||spotting in mouse||NM_001014282||37||2|