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 and was last updated on Decmeber 26, 2006 by Sheila Schmutz
Mapping Markers or Genes
Gene mapping is being done by many labs throughout the world, including our own. Some labs concentrate 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 concentrate 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 the 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. 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.
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, are 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.
How Can Dog Owners Participate in Research?
Dog owners can be a vital factor in the progress of research. If they keep careful records of traits in the parents and pups, these can be used to determine how traits are inherited. Traits that have a complex inheritance like hip dysplasia are much more difficult to study than the simple spotting example described above. However traits like perk or flop ears, spotted or not, dew claws or not, could potentially all be single gene traits and mapped using the approach described.
If you have a litter of pups from parents of two opposite phenotypes that includes pups of both phenotypes, and access to DNA from both parents, please write a detailed desription about each member of the family including the identity, such as: Ranger, sire, 4 yrs old, black & white spotted; Sally, dam, 3 yrs old, solid black; pup 1, male, solid black, pup 2, female, black & white spotted, etc. We need to be able to track the alleles back to the parent with the dominant phenotype that carried the recessive phenotype, so that parent is essential. The litter must still be with you, the breeder.
Some researchers obtain most or all of their families this way. Others prefer to use litters produced in kennels they know or in a research facilty so that they can record all of the data about each pup. I will be happy to try to put you in touch with a researcher who is willing to work with breeders on a particular coat color if you send a detailed description of the litter you have immediately available. The litter must have at least 3 pups of each coat color to be useful. This often means that the pups are less than 4 weeks old so that DNA brushes could be mailed to you, potentially from another country, before your pups leave for their new homes. Photos of each pup must also be taken and of each parent. If the grandparents are available, that is even better. Litters with too many related traits happening at once are not usually useful at this stage of our research (i.e. spotted and not, brown, black and red coat colors). Try not to get too specific........i.e. differentiating spotting patterns into 8 different categories. This research is in the early stages and we need to broadly define traits before we get down to dissecting spotting into piebald, Irish, Dalmatian type, etc.
We are especially interested in litters of red and cream pups right now, from breeds such as Golden Retrievers and/or Labrador Retrievers that specialize in yellow. Please email email@example.com if you are willing to help, and DNA brushes will be mailed to you if you currently have such a litter and are willling to send DNA and photos from each.
Note that although we appreciate your offer of an individual dog or dogs you own, we only want litters for our studies. If you are not the owner/breeder, please ask the owner to write instead. Finally, please do not post this call for samples on any chat list or message board. Breeders sincerely interested in coat color will find us. We do not have time to respond to flurries of chat list offers or explain why we need litters, including specific colors at a particular time. When and if we need individual samples, a call will be made by a breed club reprsentative on our behalf explaining in detail the type of sample needed at that particular time in a study.
Breed Clubs who want to better understand the genetics of coat color in their particular breed are encouraged to write for an information sheet explaining the samples required and the funding needed to undertake such a study. A breed club official should write, ideally suggesting a club coordinator for such a study.
If you are a researcher and want to be listed here, please send the coat color or pattern you are studying and I will post that and your email here also.
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||?||some forms of albinism||-||-||-|
|D for dilute||MLPH||"leaden" in mice||AJ920333||25||2|
|E for extension||MC1R||eumelanin or phaeomelanin in dogs||NM_001014282||5||16q24|
|K for blacK||unpublished||dominant black and brindle in dogs||-||-||-|
|M for merle||PMEL17||merle||-||10||12q13-q14|
|S for spotting||MITF||some/all? forms of spotting||AY240952||20||3p|
|H?||PAX3||spotting in mouse||NM_001014282||37||2|
|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|
|?||SLC45A2||Palamino in horses||NM_001037947||4||5|
|?||RAB27||ashen in mice||NM_001048130||30||15|
|?||MYO5||dilute in mice||NM_001048130||30||15|