Ink Spots, Paw Prints, Homozygous—What in the world do all these terms mean?

Much has been talked about and published concerning the recent discovery of identifying tobiano paints able to produce paint foals every time. This will be an attempt to simplify the terms to real world examples and explain "Informative" and "Uninformative" and other terms associated with these highly desirable, 100% paint producing horse.

Homozygous: When two genes are identical, for this discussion the individual has two tobiano genes. Example- Homozygous Tobianos will always pass a tobiano gene to an offspring (genes TT).

Heterozygous: When genes at the same location are not the same- and the parent can pass either gene (genes Tt). An Example- a tobiano foal with one tobiano parent and one quarter horse parent can only have one tobiano gene, regardless of any external markings!

Ink Spots, Paw Prints: These areas are usually in the ‘white’ on a tobiano and are small dabs of spots or color (1-3 inches) with prominent "blue zones" (white hairs on black pigmented skin) surrounding the ‘spot’. Usually there will be groups of them, looking like dog or cat tracks. These tend to show up on homozygous individuals, but also happen on heterozygous (one tobiano gene) individuals.

Informative: The blood markers of an individual can be used as indicators to test their offspring for homozygous tobiano status.

Uninformative: The blood markers of this individual do not confirm the tobiano status of their foals. (See Stallion C.)

Directly from UC Davis, the requirements for a homozygous tobiano are as follows:

• Pedigree: both parents tobiano.
• Phenotype: tobiano with secondary body spotting ("ink spots," "paw prints").
• Test breeding: no solid color offspring from solid color mates.
• Studbook data: no solid color offspring.
• Genetic marker analysis: research at UC Davis has shown it may be possible to follow the inheritance of the tobiano chromosome in pedigrees through analysis of records of parents and offspring for linked genes (ALB and GC).

There is a requirement for a homozygous tobiano to have either tobiano or tobiano-overos (toveros) parents. Some overo paints have been miss-identified as tobiano or toveros . Before paying a large sum for a homozygous individual, be sure to check the offspring production of the parents if a picture is not available. They should produce tobianos on solid crosses. (I know of one ‘tovero’ that is most definitely an overo, and has never produced a tobiano on solid mares.) If the pedigree of an individual is one tobiano and one quarter, solid or overo parent—then the individual can have only one tobiano gene. (The exception, of course, is when the pedigree has an error.)

Horses were originally parentage verified through blood typing. At one point a study was done blood typing a group of tobiano paints, breeding them and then blood typing the resulting foals. Even though the actual tobiano gene has not been identified, this research was able to trace the blood markers passed from parent to offspring. These studies led to the discovery of blood markers that are ‘linked’ to the tobiano gene. The theory is the genes producing blood type are very close in location to the tobiano gene. When the gene for a blood type is passed, the tobiano gene located next to it will be passed to the foal. This is called ‘linkage’ and makes the identification of tobiano status possible in foals from informative parents. It is possible for these genes to be ‘unhooked’ and not passed together and so test breeding on solid mares is used to confirm the status of individuals thought to be homozygous.

Typical blood markers are as follows:

All combinations are found, A-F, A-S, B-F and B-S, but some blood markers are more prevalent in a population than others. These markers can be found on a blood type card. (But not the tobiano gene—it is yet to be found directly.)

The tobiano gene will be represented as:

Usually an individual will have the A - F markers linked together or B – S , But there are A – S and B – F markers as well. 90% of the Tobiano horses have the tobiano gene linked with the marker combination of Albumin = B and GC = S (a B-S marker). Next most ‘common’ would be the A-F linked with the tobiano gene. A-S linked with a tobiano gene is very rare (less than 1%), B-F linked with tobiano is only rare. The practical application of this knowledge is to assume the markers A-S and B-F will not have a tobiano gene associated with them. There will be exceptions, but the vast majority (95%) of tobiano paints will have either a B-S or A-F marker associated with their tobiano gene.

Just because a tobiano paint has the blood markers B-S, B-S will not make them homozygous. If you were to blood type a set of quarter horses, chances are you will find the B-S marker represented in many of them - without the tobiano gene, of couse. B-S is a common blood marker and many breeds have it in profusion. Even with two tobiano parents and the B-S, B-S markers, you still are not guaranteed a homozygous individual. Lets try to illustrate this through real examples. All stallions are Tobiano paints.

All of these stallions are black or grullo tobiano individuals.

Which stallions are Informative? Stallions A, B, and D are informative, and stallion C is not—unless he turns out to be homozygous. Why? Let’s follow the genetics:

Stallion D: Has two tobiano parents. However he has sired solid foals. He therefore has only one tobiano gene. I know of three sons from quarter horse mares that have been blood typed, and all have the B-S marker from the sire, Stallion D. Two have A – F and one has B – F as the other marker. It is a safe bet that all tobiano foals produced from solid mares will have the B-S blood marker from this sire. The down side is if the solid mare has one or two B – S blood marker(s) and the tobiano foal receives this marker, then the tobiano foal will be BB-SS/T? and uninformative.

Stallion A: Has one tobiano parent (stallion D) and one quarter parent. He is still informative. The two blood markers he can give to his offspring are B-F and B-S, making him F/S informative. (This is an alternative to A/B informative, both are legitimate.)

Stallion B: Has two tobiano parents, but blood typing indicates he is probably NOT homozygous. He is a grandson to stallion D and still needs to be test bred.

Stallion C: Has two tobiano parents, blood markers are inconclusive to show tobiano status. He is uninformative. His bloodlines go to Dixie’s War Drum, and the tobiano gene from this line is associated with the A – F blood marker. If he is bred to 15 solid mares and produces 15 tobiano foals then he would be considered homozygous and informative. All tobiano foals sired by this stallion would have at least one A-F /T combination.

Let’s look at several mares:

This is an Informative cross that produces unique blood marker combinations that allow us to identify each type of foal: Solid, Heterozygous Tobiano and Homozygous Tobiano. This Stallion is F/S informative.

This is also an Informative cross producing unique blood marker combinations that allow us to identify each type of foal: Solid, Heterozygous Tobiano and Homozygous Tobiano.

This is an informative cross producing F/S informative tobiano foals and homozygous tobiano foals. As you can see, any foal with the blood marker B-F will have a 't' or non tobiano gene and be heterozygous.

This cross also produces homozygous and informative foals. The informative foals are of the well-known A/B informative type.

This is one of the more interesting crosses. The uninformative stallion produces A/B blood marker foals, but you can NOT tell them apart—they could be homozygous or heterozygous. The heterozygous foal can be informative, but only through test breeding to the correct informative individuals. Still confused? Think of it this way- both homozygous and heterozygous foals have the same blood markers, A-F and B-S (aka. AB-FS). But the tobiano gene is expressed with both foal types—so how do you tell which foal you have ? The problem lies with Stallion C-- you don’t know which A-F combination was given to the foal—the one with the tobiano gene or the one without.

 The answer to this question lies with which blood markers your stallion has. For the A-F/t : B-S/T (aka…AB-FS/Tt) individual like stallions A and D with one tobiano gene, solid mares having AA or FF blood markers will produce informative foals. These mares will also be able to produce foals that will tell you which marker the tobiano gene is linked with if the stallion has these blood markers but the linkage with the tobiano gene is uncertain. Informative AB-FS/Tt tobiano mares or Homozygous tobiano mares will produce homozygous foals that are identifiable through blood markers.

For Stallion C: breeding him to AA-FF solid mares would be counter productive. The tobiano foals will be uniformative (see foal in red above) BB-SS solid mares would produce foals of AB-FS blood type, but the informative ones would be obvious—they would be tobianos and you would know that gene was linked with the A-F blood marker. With Stallion C you will not be able to tell the homozygous foal from the heterozygous tobiano foal when breeding to informative tobiano mares, only guess because of 'Ink Spots'. If your stallion is already homozygous with the BB-SS markers, you want AB-FS informative tobiano mares or homozygous mares to produce homozygous individuals. Good Luck and Happy Breeding!!!

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