Written originally in Finnish for Shibasanomat 2/2020. The English version kindly translated by Topi Kuusinen.

   This part of the gene test series discusses coat colours. The public database of dogs by MyDogDNA is a good place to study the colour genetics of Shibas, because the data usually includes, in addition to the test results, a picture of the dog as well as the official name. Currently the database includes 51 Shibas with public data.

The colour tests included in the MyDogDNA test panel. The loci variation within the breed, so they explain the different basic colours of Shibas.

   Some of the terms commonly used with Shibas are different from those in the panel, e.g. genetic sable is the same as a red Shiba. All Shibas have the same gene forms i.e. alleles for a number of colour loci, whereby these alleles are said to have fixed in the breed. Currently, there are three loci describing the changes of coat colour in Shibas (loci is plural for locus, and locus means the address of a gene or other sequence of DNA in the chromosome) that have been verified: A locus, E locus and the S locus for white spotting.
   Note that gene tests do not "read" the whole gene. They are designed to detect certain mutations only. Other mutations may be present in the gene yet gene tests do not know to look for them. Is is often unclear whether the detected mutation causes the associated trait, or if it is just a coincidental marker mutation inherited together with the actual causal mutation.

A-locus and the base colour of coat   

   Colour locus A, i.e. agouti, gives a Shiba either red, black & tan  or sesame base colour. A cream Shiba is also one of these three as far as A locus is concerned, even though with cream the A locus colour cannot be expressed.
   It is currently believed that at least three alleles are possible for the A locus in Shibas, the official names of the alleles differing a bit from those commonly used. Allele Ay is connected to sable colouring, but in Shibas this means red colour. Allele at, i.e. tan points, is known as black and tan in Shibas. The coat colour allele aw, found in wolves and called both agouti and wolf-grey, is connected to the agouti sesame colour in Shibas. A fourth allele could be the hypothetical Ays, which may explain the red sesame colour.
   The result of the gene test shows the gene type inherited from both the dam and the sire. In a red Shiba, at least one of these is always Ay. For aw sesames the possibilities are aw/aw or aw/at. A black and tan Shiba has inherited the at allele from both parents, so its genotype is at/at. In practice, the test result for A locus is interesting when one wants to know whether a red Shiba carries B&T or sesame colour, because these both are recessive under red coat colouring. Visual recognizing of especially a carrier for sesame (Ay/aw) can be impossible. Carrying a colour has an effect on the possible colours of puppies produced by the dog.
   Fun fact: In most test laboratories the allele aw of the A locus is defined by elimination. If the three other alleles (Ay, at and a) do not match, the result is interpreted as allele aw. In principle this method can produce a strange test result, if the genotype of the dog is very unusual.

E-locus and cream

   The E locus at the top of the list is related to whether the base colour of A locus can be expressed. This is called epistasia, the effect of a gene to another gene. In a Shiba the options are “capital E”, i.e. coloured, or “small e”, which is expressed as cream. If a Shiba has inherited the “capital E” from at least one of its parents, it will express the colour of A locus. A Shiba with the genotype E/e has inherited colour expression from one of its parents and the cream allele from the other parent. In other words such a Shiba carries cream. Carriers of cream can often be easily discerned by their larger and brighter urajiro.  
   A cream Shiba can be born from a combination of two carriers for cream if the puppy happens to inherit the “small e” from both parents. In this case the colour of A locus can’t be expressed. A Shiba with genotype e/e is genetically a “recessive red” and “diluted red”, because in reality the cream (white) of a cream Shiba is a red pigment with a very light tone.
   The terms recessive and diluted red are in this context only related to the red pigment controlled by the E locus. As far as A locus is concerned, a cream Shiba can be any of the three: red, B&T or sesame. The occurrence of the cream allele in Shibas tested by MyDogDNA (including both carriers and cream-coloured dogs) is nearly 25%. 
   It seems most likely that the Shiba gene pool lacks the E locus alleles causing the black mask (Em), sighthound domino colouring (Eg), and ancient red colouring (eA). However, new and hidden E locus alleles - currently undetected by any gene test - may still appear.

S-locus and the pinto Shiba

   In a developing embryo the pigment cells migrate from the top of the head and back towards the extremities. If the migration of these cells is intercepted for any reason, the skin of the dog will have areas with no pigment. The color of non-pigmented area is bright white with crisp borders in contrast to the urajiro, where the hue of the red pigment is almost off-white. Regardless of genes, small white markings can form on the chest, toes or the end of the tail, and this is called residual white. 
   In Shibas, large white markings outside the urajiro area are usually connected to genetic white spotting, i.e. the S locus. The "normal" allele for the S locus is S, but Shibas can also have the allele sp, called piebald. This gene form interferes with the spreading of pigment cells.
   A Shiba of genotype sp/sp can have elbow-length white socks, a spot in the neck or the body or even patterning reminiscent of that of an American Akita. In a Shiba, excessive white markings are called pinto colouring, and according to the breed standard it is a disqualifying fault.
   A carrier of pinto colouring (S/sp) does not necessarily have any white markings. White ankle-length socks, a stripe on the muzzle or a spot hidden in the urajiro on the chest can, however, indicate a pinto carrier. This means that the white pinto colouring (=missing pigment) related to the S locus is different from the cream Shiba related to the E locus (=very light hue of the pigment). A cream Shiba can also have pinto colour or carry the pinto gene, even though the pinto-white areas can’t be seen in the light coat colour of a cream Shiba.

    Like all other recessive traits, a pinto puppy is possible only if both parents carry the pinto colouring. In addition to the aesthetic problem, the white pinto colouring can cause health problems, because on the head it can lead to innate deafness of the dog. The occurrence of the pinto-causing allele sp is currently about 7% in the Shibas tested by MyDogDNA.
   The white socks and the white spot on the back of Shiba shown in the photo are strong indicators of white spotting or carrying it. Despite this the gene test did not detect any known alleles for white spotting in the S locus (test result is the S/S of full colouring). However, something has affected the pigmentation in embryonic stage.
   It is likely that the S locus and white spotting do not always “function” in Shibas and other indigenous Japanese breeds as expected. In some cases, a Shiba with obvious white, long socks is according to the gene test a non-white spotted S/S, and a completely coloured Shikoku or Akita can have the genetic form of sp/sp.

Some more information about different sesame Shibas

   There are at least two different genetic bases for the sesame colour of Shibas. The well-known type of sesame colour is the aw sesame detected by the gene test. In this form the colouring is caused by the aw allele of A locus. In this kind of sesame, the black tipping of the coat in expressed on the same areas as in a B&T Shiba, extending to the top of the muzzle and near the ankles on the front part of forelegs. The hue of an aw sesame can vary from a reddish and brownish to a very dark hue, which are usually called black sesame.

   A less well-known sesame type is likely based on the Ays gene form (Ay sesame), which causes the red to be shaded by black. Current gene tests cannot detect Ays - the result is always red Ay. In this case the black shading totally covers the back and head of the dog, but does not extend far down to the legs or the top of the muzzle. On the forehead there usually is a sharp “widow’s peak” pointing towards the muzzle. The hue can be reddish brown, reddish grey or even slightly yellowish. Such a sesame has been called a red sesame (even though an aw sesame can also look reddish), a widow's peak sesame (even though this pattern is not necessarily visible) and an Ay/at sesame (even though the colouring also seems to be possible for a Shiba having the genotype Ay/aw). Therefore the best name for this colour could perhaps be sable sesame or Ays sesame.

   In other dog breeds this colour is usually called shaded sable. The genetic cause is still unknown, however, separate modifier genes or epigenetic modifications (= same DNA sequence but different activity of gene expression) have been suggested. New research has found specific gene regulation modules on A locus, which seem to differ between sable and shaded sable dogs. It is not yet clear if this new A locus system actually detects the hypothetical Ays allele.

   Typically the black shading of a sable sesame appears on an originally red-looking puppy only after the first puppy coat has been shed. In gene test, the sable sesames seem to have the genotype E/E Ay/at or E/E/ Ay/aw, which means that they don’t usually carry cream. It is supposed that the sable sesame phenotype is inherited with the Ays gene form, which is recessive to red (Ay) but dominant to aw-sesame (aw) and black-and-tan (at). It follows that a breeding of Ays sesame and a black-and-tan should result in puppies, which are either Ays sesame or black-and-tan. It is not yet fully clear how cream carriers or "double-Ays" Shibas look like. Breeders of Ays sesame Shibas have reported that the hypothetical Ays gene form is actually inherited in a predictable fashion.

   There are regularly new, interesting observations of the characteristics of sesame Shibas on the public sesame Shiba group of Facebook.

So which one is "the real" sesame?

   The answer is very simple: both sesame types are just as real sesames as long as the Shiba looks like a correct sesame Shiba. The breed standard for the Shiba, or the small Japanese dog, was written in Japan almost 100 years ago. The sesame colouring was never defined by gene tests or genotypes, but rather how the coat colours appeared to the eye. Balance - not too much and not too little - was likely the key again, and the ideal sesame colouring should not be too dark nor too light.
   If we are precise, a sesame Shiba can never be "a genetic sesame" as such a term does not exist in dog coat color genetics. Ays type sesame likely corresponds to a genetic shaded sable, while aw sesame refers to a genetic agouti.

Sable sesame or sashige?

   In addition to an Ays sesame, a Shiba with the gene test result E/E Ay/at can be bright red or shaded with black only on the neck and back; in Japanese “sashige”, dirty red. Even a red "carrying red" (genotype Ay/Ay) sometimes has some black on the back or tail. There’s a rule of thumb for discerning between a sashige and a sable sesame. If you have to think about it, the colouring is sashige, not sable sesame. The difference is most clear when viewed from the front. The odds are on the side of a sashige as well, as sable sesames are very rare.

 A fatal hereditary disease for Shibas 

   In the year 2000 a Shiba was diagnosed for the first time in Japan with a rare hereditary disease that was known in different breeds but until then had not occurred in Shibas. This concerns a so-called storage disorder (metabolic disease) by the name of GM1 gangliosidosis which is not curable and is fatal within one and a half years.

   The disease was detected by a group of veterinary physicians working with Dr. Osamu Yamato at the Hokkaido University in Sapporo. Within a short time these scientists had discovered the cause of this disease in Shibas (a gene mutation) and since then have developed a new diagnostic method. They assume that "there may be a high prevalence of carriers all over Japan" and therefore "preventive measures should be taken against GM1 gangliosidosis in Shiba dogs to eradicate this disease or at least to reduce the prevalence of carriers". The researchers are also concerned that this new disease might already have reached America, Europe or Australia.

  What is gangliosidosis? 

   Gangliosidosis belongs to a group of hereditary diseases known as "lysosomal storage diseases". Lysosomes are specific structures inside the cell in which, as in a "sewage plant", many substances are degraded or altered. Molecules carrying out this degrading or altering are called enzymes. With a lysosomal storage disorder, substances that due to an enzyme deficiency cannot be processed are stored in the lysosomes. In the case of gangliosidosis, gangliosides (fat-sugar compounds) build up in the brain cells. Thereby vital cell functions in the brain are mutilated resulting in severe disorder symptoms.

   Gangliosidosis occurs in two different forms. The GM1 gangliosidosis is caused by an inherited deficiency of the beta-galactosidase enzyme. In this form the neurological symptoms begin slightly later (approx. at 4 months) and proceed more slowly. With GM2 gangliosidosis the beta-hexosaminidase enzyme is missing, the disease pattern normally appears earlier and aggravates quicker. Even though both forms of gangliosidosis have similar symptoms, they are evoked by completely different defects of two specific lysosomal enzymes. These genetic defects known as mutations are due to a modification in the genetic code.

   What makes both of these diseases pernicious is that they are inherited covertly (autosomal recessive). With this transmission, the illness only breaks out when both formations of a critical gene are defective (as opposed to autosomal dominant inheritance where the illness breaks out when only one critical gene is defective.) The heredity course of gangliosidosis follows the classical laws of genetics. An individual always inherits a gene copy from the dam and one from the sire. A recessive heredity course means that dogs carrying just one copy of the defective gene (i.e. they are heterozygote) are clinically healthy but are carriers. Carriers themselves will never get this disease but there is a 50% chance that they will pass the defective gene on to their offspring. Only if two copies of the defective gene (from dam and sire) exist, i.e. the puppy is homzygous, will the disease break out. If carriers are mated, 25% of their offspring will statistically get gangliosidosis, 50% will be carriers and 25% will be free of the defective gene.

   Unfortunately, there is no effective treatment for this disease. The disease manifests itself in very tiny puppies, and no care brings results - the disease only progresses, eventually the puppy ceases to recognize the owner. To see how a beloved dog dies before our eyes is a huge psychological shock for the owner. All these feelings result in strong anger, the owner involves the registering organization in the situation, and the breeder or pet store faces serious problems.

Preventive measures.

   More than 10 years ago it became possible to do genetic analysis. The advantage of genetic analysis is not only that it is possible to diagnose dogs with a manifested disease, but also that it is also possible to identify carriers of this disease. For analysis, a small amount of genetic material is sufficient. And it can be obtained not only from the blood - a very small amount, but can also be extracted from the mucous membrane of the oral cavity or from saliva.

   Dogs that were identified as carriers were excluded from the ranks suitable for breeding, due to which the possibility of spreading this disease was completely excluded. However, if the individual is a carrier, but her exterior and disposition are impeccable, in order to preserve these advantages, the carrier is crossed with a healthy dog, and all born puppies are examined. Thus, it is possible to select individuals who have inherited the best qualities and with a good genetic code - they will be able to participate in further breeding. Also, in the case of a large number of carriers, not all carriers should be excluded from the number of dogs for breeding. Otherwise, the coefficient of close kinship will increase and there will be a risk of manifestation of some hidden unknown genetic disease. Therefore, it is necessary to avoid a sharp increase in the coefficient of closely related relationships, in other words, crossing close relatives. After all, a sharp sudden exclusion of a known hereditary disease can cause the opposite effect - the spread of another hereditary disease.

Carrier and ethics of animal treatment.

   A dog that has been identified as a carrier by genetic examination has an absolutely healthy body and as a pet is no different from normal dogs. It is better not to use carriers for breeding, however (as noted above, there are many cases when, under certain conditions, such a dog should be used for crossing with a normal dog), you can get such a dog as a pet. If you accidentally find out that your pet is a carrier, there is nothing wrong with that. But if you bought a carrier for further breeding or if the disease is transmitted further, a very serious situation will arise: such a breeder will lose the trust of the entire association. My guess is that some owners disagree about buying a carrier as a pet, and I want to emphasize that these are incompetent dog owners whose dogs are probably overlooked. In fact, I believe that there are no animals or humans with a complete absence of any genetic diseases, all have a proportion of mutated genes. If man takes artificial measures against the spread of carriers, then there will be a very serious problem from an ethical point of view, and there is a possibility that in the future such a nursery or organization will not even survive. Within the international community, it is required that breeders and organizations be attentive to the ethical treatment of animals.