Availability of DNA tests

Nowadays, scientific articles do not often publish mutations that are also present in other breeds. Therefore, mutations that are described and validated in one breed can also be found in other breeds. The occurrence of these mutations in other breeds is determined by laboratories that carry out the tests. It is difficult to estimate how high the reliability is for a certain test for a particular breed.

The above basis applies in general to hereditary diseases. Hereditary diseases are passed on from one generation to another through defective genes. Nevertheless, inheritance of a disease remains a biological process, and therefore exceptions are still possible. The specific information for a test gives more information about possible deviations.

Our tests do not make recommendations on breeding decisions

Our DNA tests offer you insight into the composition of your animal’s DNA. If possible, we will try to inform you of the optimal choice of tests for your personal situation. You should be aware that our laboratories are not responsible for the breeding decisions that you take. This is due to the complexity of the variation in tests and breeds. We recommend that, for advice concerning breeding decisions, you contact your international or national breed association or vet.

Within a combination package, a single result for a DNA test may be missing from the report

It is possible that a low percentage of results may be missing if a combination package is requested this can be caused due to technical reasons. We consider a combination package ‘fully reported’ if only one or two of the markers within a combination package are missing after retesting. No refund will be provided for missing tests within a combination package.

Combination packages are not 100% complete

Our combination packages will never be 100% complete due to the large number of publications in the scientific literature. We update the packages twice a year, whereby we use the following criteria: a) changes to the breeds in which a DNA test has been validated, b) the type of disease and c) technical criteria.

There is never a fool-proof link between test results and disease symptoms

In general, our tests are based on scientific publications. In these articles, a disease or condition is described with detailed information about the symptoms and background of the DNA test. The test does not guarantee that the animal still has the possibility to develop symptoms. The symptoms can be caused due to unknown genetic mutations. However, after testing positive for a disease an animal will not always develop symptoms. We therefore recommend that you contact your local or international breed association, Management Board or vet for breeding or veterinary advice.

History

Since the 19th century experiments have been conducted on the heredity of various organisms. The heredity was determined by observations of organisms – that the next generation gets one copy from each factor from each parent, and subsequently passing the factor on to following generations (Durmaz et al., 2015). The factors include for example colour, height, or shape of the organism. Pioneers Gregor Mendel and Augustinian Friar were scientist studying genetics scientifically. Gregor Mendel performed breeding experiments with hybridizing pea plants, in which different traits were traced. The traits included colour of the plants and round or wrinkled peas. The pioneer, after reporting the first breeding experiments, died in 1884. Little did he know that he would end up in biology textbooks.

Astounding results were observed by Mendel, the scientist saw traits were independently transmitted from each other (Dijk, Weissing, & Ellis, 2018). The independent transmission of traits is based on the position of genes on the corresponding chromosome. The progeny receives half of the chromosomes of both parents. If the gene is positioned on a chromosome – which is not passed down the lineage – the progeny does not express the gene. Therefore, if an experiment is conducted on various traits encoded by the corresponding genes. The progeny expresses different variation of traits in contrast to the parents.

Although, Mendel started the experiments on heredity of organisms. The scientist did not introduce the words “genetics” or “gene”. Later in the 20^th, the scientific community century begun to focus on more breeding related experiments, and thereby referring to the results indicated by Mendel. The heredity of organisms would be called “genetics” and the factor that expresses the trait of a species was described as “gene” (Portin, Wilkins, 2017). It was the start of a new discipline in the scientific community.

Introduction to genetics

The introduction of the study genetics leaded to genetic research on a more molecular level. The molecular level experiments were more focussed on the structure and biosynthetic pathways that are needed to express a certain trait. In the first stages of genetic research on various structures and biosynthetic pathways, scientists suggested corresponding proteins were responsible for the induction of the perceived traits. However, following-up research leaded to the – todays well known double helix structured DNA – to be the encoding factor that expresses the perceiving trait.

Nowadays, DNA structures, which have the typical double helix structure, are seen everywhere. Genetic research elucidated more specification on the structure of the DNA strand and stated DNA was an information molecule (Travers & Muskhelishvili, 2015). The DNA strands are made up of so called “nucleic acids”, which are based on four nucleotides adenine (A), thymine (T), cytosine (C) and guanine (G). Groups of nucleic acids, three nucleotides, encode for the amino acids and amino acids are consecutive the basis of entire chromones. As it has been highlighted in modern society are the Homo Sapiens exist of 46 chromosomes. The chromosomes are the building blocks of the human genome.

Mutations and phenotypes

Progressive research broadened the insights on the DNA structures of various species. The DNA structure consists of information molecules, which encode for structural or active biosynthetic systems were the organisms are made up on. Genetic research has indicated changes on the prescribed encoded DNA strand. The changes are called mutations. Mutations are alterations in the DNA strand. The mutations can change a trait such as eye colour, skin colour or height. These traits are all observative characteristics that can be seen by the eye, also called phenotypes. Therefore, when a gene is mutated, the phenotype also changes. Besides, there are non-observative characteristics, which are alternation of the gene that are not visible by the human eye. Mutation for example organ failures, diabetes, or heart defects.

Mutations are commonly experienced as something that should not occur. However, there are multiple outcomes at alternations of DNA, the mutation did not express in a coding region, and therefore no phenotypical changes are witnessed. The alternation has taken place in an active coding region, and subsequently effecting the phenotype of an organism. These are the most common interpretations of DNA alternations.

Implementations of DNA alternations

Implementations of DNA mutations is commonly used in modern society. DNA mutation can be used as genetic markers for the identification of genetic variation, hereditary carriers and dominant inherent. Genetic variation in animals is experienced in everyday life, since every animal has a unique genotype that encodes for a unique phenotype that can be seen. Heredity carriers are more scientifically substantiated as where in the phenotype is not visible by the human eye. In general, the terms recessive and dominant are mostly used. Recessive means the organism has inherited the recessive allele (certain region of DNA) and dominant indicates the organisms has inherited the dominant allele.

The Hereditary carrier

The hereditary carrier is an organism which has inherited a recessive allele for a specific trait, but generally does not express the trait. Although the trait is not expressed by the organism, the organism is able to pass the allele on to the next generation. This way, a specific mutation can be present in multiple generations without noticing. Another possibility is in which the organisms have a dominant inherited allele. When an organism has a dominant and recessive allele for a specific allele, the dominant allele will be expressed. Nevertheless, if a hereditary carrier inherits a recessive allele for the specific trait it carries. This will result in the expression of the inhibited trait.

Punnet Square

The well-known Punnet Square identifies the percentual change of an organism to be homozygote dominant (AA), homozygote recessive (aa) or heterozygote (Aa) (Edwards, 2012). If both parents are carriers and heterozygote the outcome would be 25% homozygote, 25% homozygote and 50% heterozygote. Resulting an allele mutation on the dominate allele would lead to 75% expression on the next generation. However, if the allele mutation was on the recessive allele only 25% of the next generation would express the recessive allele. In addition, spontaneous alternations can also cause genetic variation on alleles, and therefore lead to unexpected results. As for example the Punnet square is used to determine the percentual chance of the lineages genotype. A spontaneous alternation can change a phenotype, for example the hair colour. The linage can have different phenotypes then the ancestors if the breeding continues with the mutation.

Karyotyping

Alleles are specific regions on the chromosome of an organism. The chromosome can be visualized using the technique karyotyping. During karyotyping all the chromosomes are coloured, and subsequently counted and examined using a microscope. Malfunctions in the chromosome assembly can be identified as irregularity of chromosomes or sometimes the number of chromosomes can be reduced or increased. Karyotyping is one of VHLGenetics genotyping techniques.

Business view

VHLGenetics DNA testing is performed at two laboratories. The head office is in Wageningen, the other laboratory is in Germany. DNA tests are performed under various accreditations, certifications, and memberships of organizations such as ICAR and IS. The main goal of VHLGenetics is to provide optimal DNA services for their customers. The core competence is the standardization of work processes in the laboratories. This while remaining flexibility in adding new tests and technologies to the portfolio. The DNA services have been developed from knowledge and experience gained in the last 30 years. DNA services are offered in a wide variety including plants and animals. The service involves mainly KASP, real-time PCR, capillary electrophoresis, and Thermo Fisher Scientific Targeted Genotyping by Sequencing®.

The DNA-profile of one individual is identical in each part of the body. It does not make a difference in a comparison if a DNA-profile of an individual is based on hairs, blood, swabs, semen or tissue.

 Because large variation is present in the DNA, it is almost not possible that two randomly selected individuals have identical DNA-profiles. Each individual will have his or her own DNA, which will differ on one or more points from other individuals. One exception on this are identical twins or clones, which have completely identical DNA patterns.

As with STRs, the basis is the same. However, more genetic markers are tested because the information content per markers is lower for SNPs.

The genetic variation which is present in an animal, originates from both parents. Half of the variation is originating from the father, whereas the other half comes from the mother.

For parentage verification, typically 200 up to 400 genetic characteristics are visualized. In this process the actual genetic composition (A,C,G, or T) is being measured. The composition/variant  in an offspring must correspond to the composition/variant in the mother and father that were provided for comparison. In two examples, it is shown how the basic rules are applied in parentage verification.

In the above table an example is provided of a correct parentage. In this table, the DNA is shown of three individuals: an offspring (left column), a potential mother (middle column), and a potential father (right columns). In each line one variant is shown. In this case all variants in the offspring are present in the parents: the parentage is correct.

In the second table an example is provided of an incorrect parentage. In this table, the DNA is shown of three individuals: an offspring (left column), a potential mother (middle column), and a potential father (right columns). In each line one variant is shown. In this case several variants are present in the offspring which are not are present in the parents: the parentage is not correct.

When 200 up to 400 different genetic fragments are checked, the chance that an incorrect parentage is not detected becomes very small. The genetic fragments which are used for parentage verification and identification provide no information on properties such as color and quality of an animal, plant or human, since the fragments are non-coding.

In 2008 a colt with a striking white-spotting coat colour was born out of two solid-coloured bay Franches-Montagnes parents. The coat colour looks like a combination of white-spotting and coat colour dilution and it was named ‘‘macchiato’’. A clinical examination revealed that the macchiato stallion was deaf and had a low progressive sperm motility.

The Coat Macchiato (Splashed White)  test (P593) tests for a mutation in the MITF gene. This test detects two variants (alleles). The allele M is dominant. One or two copies of the M allele result in the Macchiato coat colour. The allele N is recessive and does not have an effect on the basic colour.

The Coat Macchiato (Splashed White) test encloses the following results:

White patterning in horses is known as Dominant White or White. Dominant White patterns are variable, ranging from minimal Sabino-like spotting to all-white horses. The eye colour of Dominant White horses is brown. There are about 20 different mutations identified that are associated with white patterns, all mutations are found in the KIT gene. Except for W20, most of the known Dominant White mutations arose recently and are restricted to specific lines within breeds. The Coat Colour Dominant White 3 test (P592) tests for the mutation known as W20 in the KIT gene. This test detects two variants (alleles). The allele W20 is dominant. One or two copies of the W20 allele have a subtle effect on the amount of white expressed. It appears to increase the expression of white in combination with other white pattern genes. The allele N is recessive and does not have an effect on the basic colour.

The Coat Colour Dominant White 3 test encloses the following results, in this scheme the results of the Coat Colour Dominant White 3 test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

White patterning in horses is known as Dominant White or White. Dominant White patterns are variable, ranging from minimal Sabino-like spotting to all-white horses. The eye colour of Dominant White horses is brown. There are about 20 different mutations identified that are associated with white patterns, all mutations are found in the KIT gene. Except for W20, most of the known Dominant White mutations arose recently and are restricted to specific lines within breeds. The Coat Colour Dominant White 1 test (P591) tests for the mutation known as W18 in the KIT gene. This test detects two variants (alleles). The allele W18 is dominant. One or two copies of the W18 allele result in horses that display some degree of white spotting but the specific pattern cannot be predicted. The allele N is recessive and does not have an effect on the basic colour.

The Coat Colour Dominant White 1 test encloses the following results, in this scheme the results of the Coat Colour Dominant White 1 test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

The Silver dilution gene dilutes the black pigment but has no effect on the red pigment. The effect of the Silver dilution gene can vary greatly. The mane and tail are lightened to flaxen or silver gray, and may darken on some horses as they age. A black horse will be diluted to chocolate with a lightened mane and tail. A Bay horse with Silver dilution will usually have a lightened mane and tail, as well as lightened lower legs (places with black pigment). A horse can also carry mutations for other modifying genes which can further affect its coat colour.

The Coat Colour Silver dilution test (P784) tests for the genetic status of the PMEL17 gene. This gene has two variants (alleles). The dominant allele Z results in the dilution and the recessive allele N does not have an effect on the basic colour.

The same mutation responsible for the coat color Silver is also associated with Multiple Congenital Ocular Anomalies (MCOA) Syndrome, a wide range of ocular defects that occur in the anterior and posterior parts of the eye. The severity of the syndrome is dose related, so horses with 1 copy of allele Z have fewer severe signs than those with 2 copies of allele Z.

The Coat Colour Silver dilution test encloses the following results, in this scheme the results of the Coat Colour Silver dilution test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

The Pearl dilution gene lightens the coat colour of the horse by diluting the red pigment. A chestnut basic colour is diluted to a pale, uniform apricot colour of body, mane and tail. Skin coloration is also pale. Pearl dilution is also referred to as the ‘Barlink Factor.’ The Coat Colour Pearl dilution test (P783) tests for the genetic status of the SLC45A2 gene. This gene has two variants (alleles). The allele Prl, causing the Pearl dilution is recessive. This means that only horses with two copies of the Prl allele have a lightened coat, mane and tail, in addition to bright eye colors. The dominant allele N does not have an effect on the basic coat colour.

Pearl dilution interacts with Cream dilution to produce pseudo-double dilute phenotypes including pale skin and blue/green eyes. Therefore if a horse has one copy of the Prl allele and Cream dilution (Cr allele) is also present, this results in a pseudo-double dilute, also called pseudo-cremellos or pseudo-smoky cream

A horse can also carry mutations for other modifying genes which can further affect its coat colour.

The Coat Colour Pearl dilution test encloses the following results, in this scheme the results of the Coat Colour Pearl dilution test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

The Dun dilution gene lightens the coat colour of the horse by lightening the body colour, leaving the head, lower legs, mane and tail undiluted. Dun is also typically characterized by “primitive markings”, allmost all dun horses possess at least the dorsal stripe, but the presence of the other primitive markings varies. Other common markings may include horizontal striping on the legs, transverse striping across the shoulders, and lighter guard hairs along the edges of a dark mane and tail. Dun diluted coat colour with primitive markings is considered the “wild-type” colour and is found in wild equids such as przewalski horses. Dun dilutes both red and black pigment, and the resulting colors range from apricot, golden, dark gray, olive and many more subtle variations. A horse can also carry mutations for other modifying genes which can further affect its coat colour. The Coat Colour Dun dilution test (P660) tests for the genetic status of the TBX3 gene. This gene has three variants (alleles); allele D is dominant over the alleles nd1 and nd2; allele nd1 is dominant over nd2. The dominant allele D results in Dun dilution with primitive markings. Allele nd1 does not dilute the coat colour of the horse, primitive markings are present but the expression is variable. Allele nd2 does not have an effect on the basic colour.

The Coat Colour Dun dilution test encloses the following results, in this scheme the results of the Coat Colour Dun dilution test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

The Champagne dilution gene lightens the coat colour of the horse by diluting the pigment. The skin of Champagne-diluted horses is pinkish/lavender toned and becomes speckled with age; the speckling is particularly noticeable around the eye, muzzle, under the tail, udder and sheath. The eye colour is blue-green at birth and darkens to amber as the horse ages. Champagne has the following effects on the basic coat colours of horses:

Chestnut/Sorrel -> Gold champagne:   a gold body color and often a flaxen mane and tail. Gold champagne horses are visually similar to palomino horses.

Bay/Brown -> Amber champagne:       a tan body color with brown points (sometimes referred to as amber Buckskin).

Black -> Classic champagne:               a darker tan body with brown points.

A horse can also carry mutations for other modifying genes which can further affect its coat colour. The Coat Colour Champagne dilution test (P853) tests for the genetic status of the SLC36A1 gene. This gene has two variants (alleles). The dominant allele Ch results in the dilution and the recessive allele N does not have an effect on the basic colour.

The Coat Colour Champagne dilution test encloses the following results, in this scheme the results of the Coat Colour Champagne dilution test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

The cream dilution gene has an effect on both red and black pigment and dilutes the basic coat colour to lighter coat shades. In several breeds this is considered a desirable trait. The Cream dilution gene is responsible for the palomino, buckskin, smoky black, cremello, perlino and smoky cream coat colours. A horse can also carry mutations for other modifying genes which can further affect its coat colour. The Coat Colour Cream dilution test (P713) tests for the genetic status of the MATP gene. The MATP gene has two variants (alleles). The allele Cr is semi-dominant. One copy of the Cr allele dilutes the coat colour with a single dose, resulting in palomino, buckskin or smoky black. Two copies of the Cr allele dilute the coat colour with a double dose into cremello, perlino or smoky cream. The effect on black pigment might be very subtle. Horses with two copies of the Cr allele are also called “double-dilutes” or “blue-eyed cream” and they share a number of characteristics. The eyes are pale blue, paler than the unpigmented blue eyes associated with white color or white markings, and the skin is rosy-pink. The allele N is recessive and does not have an effect on the basic colour.

The Coat Colour Cream dilution test encloses the following results, in this scheme the results of the Coat Colour Cream dilution test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

Each horse has a basic colour, which can be black, bay/brown or chestnut. These basic coat colours are controlled by the Extension and Agouti genes. The Agouti gene (A-locus) controls the distribution of black pigment. The pigment can be uniformly distributed or distributed to the “points” of the body (mane, tail, lower legs and inside the ears). The Agouti gene has no effect on horses that are homozygous ee for the Extension gene as black pigment has to be present for agouti to have an effect. The Coat Colour Agouti test (P907) tests for the genetic status of the Agouti gene. The Agouti gene has two variants (alleles). The dominant allele A restricts black pigment to the points of the horse (for example in bays and buckskins) and the recessive allele a uniformly distributes black pigment over the entire body. Only when the horse has two copies of the recessive allele a (homozygous aa), the black pigment is evenly distributed. The black pigment is distributed into the points if at least one copy of the allele A is present. All horses, regardless of their coat colour do have the genetics for the distribution of black pigment, but it’s not always physically visible.

The Coat Colour Agouti test encloses the following results, in this scheme the results of the Coat Colour Agouti test are shown in combination with the possible results for the Coat Colour Chestnut test:

Splashed white is a variable white spotting pattern characterized by a large blaze, extended white markings on legs, variable white spotting on belly, pink skin and often blue eyes. In other cases, the unpigmented areas are quite small and cannot be distinguished from horses with other more subtle depigmentation phenotypes. Splashed white horses are sometimes deaf, however most splashed white horses are not deaf. Hearing loss is due to the death of the necessary hair cells, caused by the absence of melanocytes in the inner ear. Although the majority of splash horses have pigment around the outside of the ear, the pigment must occur in the inner ear to prevent hearing loss. There are several different mutations identified that are associated with splashed white patterns. The Coat White Spotting 3 test (P514) tests for the mutation known as SW3 in the MITF gene. This test detects two variants (alleles). The allele SW3 is dominant. One or two copies of the SW3 allele result in splashed white. It is speculated that two copies of the SW3 allele are lethal (the foal dies). The allele N is recessive and does not have an effect on the basic colour.

The Coat Colour White Spotting 3 test encloses the following results, in this scheme the results of the Coat Colour White Spotting 3 test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

Splashed white is a variable white spotting pattern characterized by a large blaze, extended white markings on legs, variable white spotting on belly, pink skin and often blue eyes. In other cases, the unpigmented areas are quite small and cannot be distinguished from horses with other more subtle depigmentation phenotypes. Splashed white horses are sometimes deaf, however most splashed white horses are not deaf. Hearing loss is due to the death of the necessary hair cells, caused by the absence of melanocytes in the inner ear. Although the majority of splash horses have pigment around the outside of the ear, the pigment must occur in the inner ear to prevent hearing loss. There are several different mutations identified that are associated with splashed white patterns. The Coat White Spotting 1 test (P512) tests for the mutation known as SW1 in the MITF gene. This test detects two variants (alleles). The allele SW1 is dominant. One or two copies of the SW1 allele result in splashed white. The allele N is recessive and does not have an effect on the basic colour.

The Coat Colour White Spotting 1 test encloses the following results, in this scheme the results of the Coat Colour White Spotting 1 test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

Roan is a white patterning coat colour trait of intermixed white and coloured hairs in the body while the head, lower legs, mane and tail remain colored. Roan horses are born with the pattern, though it may not be obvious until the foal coat is shed. The white and coloured hairs are evenly mixed in horses that inherit the classic Roan gene, which can differentiate this from several mimic patterns called roaning. Roaning patterns tend to be uneven in the distribution of white hairs and the inheritance of roaning has not been defined. The mutation causing the Roan coat colour has not yet been identified. The Coat Roan test (P659) tests for DNA markers that are associated with Roan coat colour in several breeds, the DNA markers can be used to determine if a horse has the Roan mutation and how many copies. This test detects three variants (alleles), Rn, Rn* and N. The allele Rn is dominant. One or two copies of the Rn allele result in a Roan coat colour. The allele Rn* is very uncommon and not always associated with the Roan coat colour, this allele has only been observed in Tennessee Walking horses and Rocky Mountain horses. The allele N is recessive and does not have an effect on the basic colour.

The Coat Colour Roan test encloses the following results, in this scheme the results of the Coat Colour Roan test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

The genetic variation which is present in an animal, originates from both parents. Half of the variation is originating from the father, whereas the other half comes from the mother.

For parentage verification, typically 20 up to 40 genetic characteristics are visualized. In this process the length of genetic fragments is being measured. The measured length of a genetic characteristic in an offspring must correspond to the length in the mother and father that were provided for comparison. In two examples, it is shown how the basic rules are applied in parentage verification.

In the figure an example is provided of a correct parentage. In this figure, the DNA is shown of three individuals: an offspring (upper line), a potential mother (middle line), and a potential father (bottom line). In each line one genetic marker is shown. Two DNA fragments are visible as peaks. The first fragment of the offspring is originating from the father (length of the fragment is 150), whereas the second fragment comes from the mother (fragment length 152). In this case both fragments of the offspring are present in the parents: the parentage is correct.

In the second example a situation is shown where parentage does not qualify. The three lines are shown in the order of offspring, potential mother and potential father. Again in each line one DNA marker is shown, where two DNA fragments are visible as peaks. The second fragment of the offspring is present in the mother (fragment length 152), whereas the first fragment in the offspring (fragment length 150) is NOT present at the assigned father. In this case, one fragment is present at the offspring, which is not present in either of the parents: the parentage does not qualify.

When 20 up to 40 different genetic fragments are checked, the chance that an incorrect parentage is not detected becomes very small. The genetic fragments which are used for parentage verification and identification provide no information on properties such as color and quality of an animal, plant or human, since the fragments are non-coding.

When the length of a number of DNA fragments is measured for a sample, a DNA-profile is established. This pattern is unique for a specific individual person, animal or plant, so that in cases of doubt DNA-profiles can be compared to confirm if two samples originate from the same individual.

Complexity of genetic material

The body of an organism consists of a large number of cells, which contain a full and complete set of genetic material. Genetic information is present in the nucleus of a cell. The genetic information is stored in chromosomes, which are translated by the body in useful data (proteins). This happens constantly in all cells. The general code is called DNA.

Chromosomes exist of long DNA-strains which are wound around each other very tightly. When a chromosome is studied in detail, it is possible to look at the composition of DNA in the form of A, T, G, or C. These A, T, G and C are the building blocks from which the DNA is constructed. Sometimes stretches of repeats are present (e.g. CACACA) – such stretches are indicated as microsatellites (also known as STRs). Other variation such as G/A or C/G is indicated as Single Nucleotide Polymorphism (SNP). The order and composition of DNA are the basis for all kinds of applications.

For the typing of the composition of genetic characteristics it is possible to use hairs, feathers – to be drawn with roots –, blood, milk, tissue etcetera. The usability of sample material depends on the test which is carried out. The use of ‘fresh’ material provides the best result.

Techniques

Genetic variation can be visualized with a number of different techniques. Frequently a technique is used, where DNA is multiplied (PCR). DNA can be made visible through three steps:

• DNA-extraction, where the cells are broken into small pieces. The DNA is present in an aqueous solution, which is necessary to enable a successful PCR-reaction,
• Selective multiplying of DNA, where PCR is used to multiply small specific pieces,
• Analysis of DNA on a machine, with which DNA is visualized. For that purpose, fluorescence is incorporated during PCR.

The final result of these steps results generally in the detection of the variation in STRs or SNPs. By examining a number of STRs or SNPs, a genetic bar code is generated. This bar code can be used for a number of different tests, among which are lineage, identity of samples, etcetera. In a number of examples these applications are described below.

The Appaloosa spotting pattern, also known as Leopard Complex spotting (LP) includes a highly variable group of white spotting- or depigmentation patterns in horses. Appaloosa horses have three additional identifiable characteristics: mottled skin around the muzzle, anus and genitalia, striped hooves and white sclera round the eyes. The Appaloosa pattern is the result of an incompletely dominant mutation in the TRPM1 gene, also known as the LP gene. The LP gene allows for the expression of the various leopard complex spotting patterns while other genes determine the extent (or amount) of white. The CSNB / Leopard Spotting test (P311) tests for the status of the LP (TRPM1) gene. This gene has two variants (alleles). The allele LP is incomplete-dominant and expression of the Appaloosa pattern is variable, ranging from absent to extremely white patterning. At least one copy of the LP allele allows the expression of the Appaloosa pattern. The amount of white present is not dosage related, horses with two copies of the LP allele can have minimal expression of white patterning. The recessive allele N does not have an effect on the basic colour. The variability in the amount of white on Appaloosa-coloured horses is controlled by other genes, one of which is PATN1. Horses that have one copy of the LP allele, in combination with at least one copy of the PATN1 allele most often have a Leopard or a near Leopard pattern. Horses that have two copies of the LP allele in combination with at least one copy of the PATN1 allele most often have a Few-spot or near Few spot pattern. Horses that have two copies of the LP allele suffer from Congenital Stationary Night Blindness (CSNB), which is the inability to see in low to no-light conditions.

The CSNB / Leopard Spotting test encloses the following results, in this scheme the results of the CSNB / Leopard Spotting test are shown in combination with the possible results for the Coat Colour Appaloosa Pattern-1 (PATN1) test.

The Appaloosa spotting pattern, also known as Leopard Complex spotting (LP) includes a highly variable group of white spotting- or depigmentation patterns in horses. Appaloosa horses have three additional identifiable characteristics: mottled skin around the muzzle, anus and genitalia, striped hooves and white sclera round the eyes. LP is the result of an incompletely dominant mutation in the TRPM1 gene, also known as the LP gene. The LP gene allows for the expression of the various leopard complex spotting patterns while other genes determine the extent (or amount) of white. One of the genes that is associated with increased amount of white in in LP horses has been identified (RFWD3) and has been termed Pattern-1 (PATN1) for first pattern gene. The Coat Colour Appaloosa Pattern-1 (PATN1) test (P305) tests for the status of the PATN1 gene. This gene has two variants (alleles). The dominant allele PATN1 results in an increased amount of white in horses that carry at least one copy of the LP allele on the LP gene. The recessive allele N does not have an effect on the basic colour. Horses that have one copy of the LP allele, in combination with at least one copy of the PATN1 allele most often have a Leopard or a near Leopard pattern. Horses that have two copies of the LP allele in combination with at least one copy of the PATN1 allele most often have a Few-spot or near Few spot pattern. Horses that have at least one copy of the  PATN1 allele but do not have a copy of the LP allele will not have a Appaloosa spotting pattern but can pass on the PATN1 allele to their offspring.

The Coat Colour Appaloosa Pattern-1 (PATN1) test encloses the following results, in this scheme the results of the Coat Colour Appaloosa Pattern-1 (PATN1) test are shown in combination with the possible results for the LP Gene.

The Tobiano coat pattern usually involves white on all four legs below the hocks and knees and rounded white spots on the body with sharp, clean edges. The head is dark, with white markings like those of a solid colored horse. The white on the body will generally cross the top-line of the horse. The skin underlying the white spots is pink and under the colored areas it is black. The eyes are usually brown, but one or both may be blue or partially blue. The tail can be two colors, a characteristic seldom seen in horses that are not tobiano. A horse can also carry mutations for other modifying genes which can further affect its coat colour.

The Coat Colour Tobiano test (P903) tests for a genetic factor that affects the function of the KIT gene. This gene has two variants (alleles). The dominant allele TO results in the Tobiano pattern and the recessive allele N does not have an effect on the basic colour.

The Coat Colour Tobiano test encloses the following results, in this scheme the results of the Coat Colour Tobiano test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

The Overo coat pattern is a white spotting pattern with white patches on the side with a “frame” of colour surrounding the white. A horse can also carry mutations for other modifying genes which can further affect its coat colour. While Overo coloured horses are desirable, the mutation that causes the overo colour is linked to a fatal condition known as Overo Lethal White Syndrome or OLWS. A foal with OLWS is born all- white and dies of complications from intestinal tract abnormalities. The Coat Colour Overo test (P902) tests for a genetic factor that affects the function of the EDNRB gene. This gene has two variants (alleles). The allele O is semi-dominant. One copy of the O allele results in horses with overo coat pattern. Two copies of the O allele result in a lethal white foal (OLWS). The allele N is recessive and does not have an effect on the basic colour.

The Coat Colour Overo-factor test encloses the following results, in this scheme the results of the Coat Colour Overo-factor test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

A horse that inherits a Grey coat colour can be born in any colour. The grey gene causes progressive depigmentation (fading) of the hair and is considered to be the “strongest” of all coat colour modifying genes. The depigmentation process may last for years, but once the hair is depigmented, the original colour will never return. Some grey horses become completely white whereas others will keep tiny non-faded spots (also called fleabites). A horse can also carry mutations for other modifying genes which can further affect its coat colour.

The Coat Colour Grey test (P807) tests for the genetic status of the STX17 gene. This gene has two variants (alleles). The dominant allele G results in the Grey coat colour and the recessive allele N does not have an effect on the basic colour. The dominant allele G has a duplication of a part of the DNA. The test does not discriminate between horses carrying 1 or 2 copies of  the duplication (N/G or G/G). All horses carrying the duplication will turn grey.

The Coat Colour Grey test encloses the following results, in this scheme the results of the Coat Colour Grey test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

Sabino is a general description for a group of similar white spotting patterns. The sabino pattern is described as irregular spotting usually on the legs, belly and face, often with roaning around the edges of the white markings. A mutation has been discovered that produces one type of sabino pattern, it has been named Sabino1 as it is not present in all sabino-patterned horses. More mutations will probably exist that account for other sabino patterns. The Coat Colour Sabino 1 test (P785) tests for the genetic status of the KIT gene. This gene has two variants (alleles). The allele SB1 is semi-dominant. One copy of the SB1 allele results in horses with broken Sabino markings and possibly only a small amount of white. Two copies of the SB1 allele result in at least 90% white, also referred to as Sabino-white. The allele N is recessive and does not have an effect on the basic colour.

The Coat Colour Sabino 1 test encloses the following results, in this scheme the results of the Coat Colour Sabino 1 test are shown in combination with the possible results for the tests that determine the basic Coat Colour (Coat Colour Chestnut and Coat Colour Agouti test):

The Fibroblast Growth Factor 5 (FGF5) determines the hair length. Four mutations in FGF5 have been identified that influence hair length in different breeds. There are breed specific mutations for Ragdolls, Norwegian Forest Cats and Main Coons and an different mutation has been found that influences hair length in all long-haired cat breeds. The Hair Length Norwegian Forest test (K462) tests for the Norwegian Forest cat specific mutation in the FGF5-gene and has two variants (alleles). The recessive allele results in long hair and the dominant allele results in short hair.

The Hair Length Norwegian Forest test encloses the following results:

The Fibroblast Growth Factor 5 (FGF5) determines the hair length. Four mutations in FGF5 have been identified that influence hair length in different breeds. There are breed specific mutations for Ragdolls, Norwegian Forest Cats and Main Coons and an different mutation has been found that influences hair length in all long-haired cat breeds. The Hair Length Ragdoll test (K463) tests for the Ragdoll specific mutation in the FGF5-gene and has two variants (alleles). The recessive allele results in long hair and the dominant allele results in short hair.

The Hair Length Ragdoll test encloses the following results:

Each horse has a basic coat colour, which can be black, bay/brown or chestnut. These basic coat colours are controlled by the Extension and Agouti genes. The Extension gene controls the production of black and red pigment while the distribution of black pigment is controlled by the agouti gene.

The rest of the colour genes act as modifiers (dilution or depigmentation) on the basic coat colour of the horse. There are at least five genes that dilute the coat colour of the horse: Cream, Champagne, Dun, Pearl, and Silver. The pattern genes modify the colour of the horse by deleting colour (depigmentation). These genes include Overo, Sabino, Tobiano, Grey, dominant white, white spotting, Appaloosa spotting and Pattern-1. For Appaloosa spotting VHLGenetics doesn’t offer a test yet.

Within the above described coat colour genes, three genes explain the major differences; the Agouti, Extension and Cream dilution genes. In the table below the possible combinations of the genes are indicated.

Each horse has a basic colour, which can be black, bay/brown or chestnut. These basic coat colours are controlled by the Extension and Agouti genes. The Extension gene (E-locus) controls the production of black and red pigment. The Coat Colour chestnut test (P904) tests for the genetic status of the Extension gene. The Extension gene has two variants (alleles). The dominant allele E produces black pigment and the recessive allele e produces red pigment. All horses, regardless of their coat colour do have the genetics for black or red pigment. Red horses (for example chestnut, sorrel, palomino and cremello) have two copies of the recessive allele e, also called homozygous ee. Black pigmented horses (for example black, bay, brown, smoky black, buckskin, smoky cream and perlino) have at least one copy of the allele E. They can be homozygous EE or heterozygous Ee.

The Coat Colour Chestnut test encloses the following results, in this scheme the results of the Coat Colour Chestnut test are shown in combination with the possible results for the Coat Colour Agouti test:

The dilute gene (MLPH gene) is responsible for the intensity of the coat colour by affecting the amount of pigments in the hair shaft. This gene is also known as the D-locus and dilutes all colours. The Coat Colour Dilution test (K760) tests for the genetic status of the D-locus. The D-locus has two variants (alleles). The allele D is dominant and does not have an effect on the coat colour. Only when the cat has two copies of the recessive allele d the coat colour is diluted. The dilution of black results in grey, called blue by cat breeders. Chocolate/brown dilutes into lilac, it is described as dove or light taupe gray, and is sometimes called frost or lavender. Cinnamon dilutes into Fawn, it is described as “coffee and cream” or caramel color. Some cat breeds are fixed for one of the alleles. The Egyptian Mau and Singapura are fixed for the dominant allele D. The breeds Chartreux, Korat and Russian Blue are fixed for the recessive allele d. Most other breeds can have both alleles.

The Coat Colour Dilution test encloses the following results, in this scheme the results of the Coat Colour Dilution test are shown in combination with the possible results for the B-locus):

The Fibroblast Growth Factor 5 (FGF5) determines the hair length. Four mutations in FGF5 have been identified that influence hair length in different breeds. There are breed specific mutations for Ragdolls, Norwegian Forest Cats and Main Coons and an different mutation has been found that influences hair length in all long-haired cat breeds. The Hair Length Maine Coon test (K461) tests for the Main Coon specific mutation in the FGF5-gene and has two variants (alleles). This mutation can also be present in Ragdolls. The recessive allele results in long hair and the dominant allele results in short hair.

The Hair Length Maine Coon test encloses the following results:

The Fibroblast Growth Factor 5 (FGF5) determines the hair length. Four mutations in FGF5 have been identified that influence hair length in different breeds. There are breed specific mutations for Ragdolls, Norwegian Forest Cats and Main Coons and an different mutation has been found that influences hair length in all long-haired cat breeds. The Hair Length all breeds test (K466) tests for the mutation that is not breed specific in the FGF5-gene and has two variants (alleles). The mutation can be present in all long-haired cat breeds. The recessive allele results in long hair and the dominant allele results in short hair.

The Hair Length all breeds test encloses the following results:

Cats display a wide variety of coat colours and patterns. Classification of these colours can be confusing sometimes because different registries or associations may use different names for the same colour. Several genes determine the coat colour of a cat. Unfortunately not for all genes involved the genetic background is known yet. For the genes known as A-, B-, D-, C-, and E-loci the genetic background is known and a DNA-test is offered at VHLGenetics to determine the genetic status of those genes. For the G-locus (White gloves), the S-locus (spotting) and the W-locus (albino) VHLGenetics doesn’t offer a DNA-test yet. For the O-locus (Orange), I-locus (Inhibitor) and the T-locus (Ticked) the genetic background is not known yet and therefore it is not possible to determine the genetic status of those genes with a DNA-test.

Within the above described coat colour genes, four genes explain the major differences; the B-, D-, C- and O-Locus genes. In the table below the possible combinations of these genes are indicated.

The Agouti gene (ASIP gene) is responsible for the production of a protein that regulates the distribution of black pigment (eumelanin) within the hair shaft. This gene is also known as the A-locus and is responsible for ticking and causes the individual hairs to have bands of light and heavy pigmentation. The agouti band can be seen in both black-based and red-based colours. The Coat Colour Agouti test (K757) tests for the genetic status of the A-locus. The A-locus has two variants (alleles). The allele A is dominant and produces ticked coat, hair shafts with alternating bands of yellow and black colour, ending with black tips (similar to the coat of a wild mouse or rabbit).The recessive allele produces a cat that is “self”coloured (solid). Only when the cat has two copies of the recessive allele a the coat colour is solid.

Another system of pigmentation in cats produces the tabby patterns of dark stripes interspersed with the lighter agouti tipped hairs. Hairs in the darker stripes do not have the shift between black and yellow pigment production and remain uniformly dark. The effect of the agouti protein on orange pigment is limited, thus tabby striping may still be seen on cats that are a/a for agouti.

The Coat Colour Agouti test (A-Locus) enclose the following results:

The coat colours black, chocolate/brown and cinnamon/red are controlled by the gene TYRP1 (tyrosinase-related protein 1) which is involved in the production of the black colour pigment eumelanin. This gene locus is also called the B-locus. The Coat Colour Cinnamon (K755) and Coat Colour Chocolate (K756) combined reveal the genetic status of the B-locus. The B-locus has three variants (alleles). The B allele is dominant over the alleles b and b’; allele b is dominant over allele b’. The dominant allele B results in a black coat colour. The allele b in a chocolate/brown colour and the allele b’ results in a cinnamon/red coat colour.

The Coat Colour Cinnamon and Coat Colour Chocolate tests (together B-locus) enclose the following results:

The Siamese and Burmese coat patterns are controlled by the gene TYR (tyrosinase) which produces an enzyme that is required for melanin production. The Burmese pattern is a result from reduced pigment production changing black pigment to sepia and orange to yellow. The Burmese points are darker than the body and the eyes are yellow-gray or yellow-green. The Siamese pattern shows reduced pigment production to the points and the eyes are blue. This gene is also known as Color gene or C-locus. The Coat Colour Siamese (K758) and Coat Colour Burmese (K759) combined reveal the genetic status of the C-locus. The C-locus has three variants (alleles). The C allele is dominant over the alleles cb and cs; allele cb is semi-dominant over allele cs. The dominant allele C does not have an effect on the coat colour. Two copies of the cb allele (homozygous cb/cb) results in a Burmese coat pattern. One copy of the allele cb and one copy of the allele cs (cb/cs) result in the intermediate mink colour. Two copies of the cs allele (cs/cs) results in a Siamese coat pattern.

The Coat Colour Siamese and Coat Colour Burmese tests (together C-locus) enclose the following results:

In the following scheme the results of the C-locus are shown in combination with the possible results for the B-locus and D-locus:

The Extension gene (MCR1 gene) controls the production of black and red pigment. In cats, shades of red color are determined by the dominant Orange gene (O-locus) located on the X chromosome. The genetic background of the O-Locus is still unknown. The Extension gene is also known as E-locus. The Coat Colour E locus, extension test (K639) tests for the genetic status of the E-locus. The E-locus has two variants (alleles). It is presumed that (almost) all cats are fixed for the dominant allele E, they have two copies of the dominant allele E and based on this gene alone could produce both red and black pigment. The recessive allele e results in kittens that are born with a black/brown tabby pattern (blue/apricot in dilute cats). As the kittens mature, the black/blue pigment is replaced by yellow resulting in the golden coat coloration seen in adult cats. Originally it was named X Colour, now it is called Amber. The recessive allele can be present in the Norwegian Forest cat and traces back to a single female ancestor from Norway born in 1981. Cats with two copies of the allele e only have the Amber Coat Colour when the dominant O allele at the O-locus is not present.

The Coat Colour E Locus, extension test encloses the following results, in this scheme the results of the Coat Colour E Locus, extension test are shown in combination with the possible results for the O-locus. For the O-locus no DNA test is available:

Dominant White and White Spotting are controlled by the KIT-gene. Dominant white is also described as the W-locus and White Spotting as the S-locus. The gene/genes controlling the pattern of White Spotting is still unknown. Additionally, not all white spots or patterns result from the KIT-gene as other genes can also have mutations that result in depigmentation phenotypes.

The KIT-gene has three variants (alleles). The DW allele is dominant over the alleles Ws and N; allele Ws is dominant over allele N. The dominant allele DW results in a white coat colour. The allele Ws in white spotting and the allele N has no effect on the coat colour.

Dominant White is distinct from albinism (C-locus) which results from a mutation in theTYR (tyrosinase) gene that has no known impact on hearing. One or two copies of the DW allele will result in a white cat with varying degrees of hearing impairment.

The Dominant White & White Spotting test encloses the following results:

The Fibroblast Growth Factor 5 (FGF5) determines the hair length. Four mutations in FGF5 have been identified that influence hair length in different breeds. There are breed specific mutations for Ragdolls, Norwegian Forest Cats and Main Coons and a different mutation has been found that influences hair length in all long-haired cat breeds. In the table below the possible combinations of these mutations are indicated.

The lysophosphatidic acid receptor 6 (LPAR6) gene influences the hair formation. The Cornish Rex, Curly/woolly coat test (K502) tests for the genetic status of the LPAR6 gene. The LPAR6 gene has two variants (alleles). The allele N is dominant and does not have an effect on the coat. Only when the cat has two copies of the recessive allele CC the coat is curly/woolly. The mutation is fixed in Cornish Rex cats. The test can be used in outcrossing programs, to help breeders select the cats that can produce curly coat in the next generation.

The Cornish Rex, Curly/woolly coat test encloses the following results:

Two different mutations in the Keratin 71 (KRT71) gene influence the hair formation in Devon Rex and Sphynx cats resulting in a curly coat or a nearly hairless coat. The Devon Rex, Curly Coat (K304) and Sphynx, Hairless Coat (K305) tests combined reveal the genetic status of the KRT71 gene. The KRT71 gene has three variants (alleles). The N allele is dominant over the alleles hr and dr; allele hr is dominant over allele dr. The dominant allele N does not have an effect on the coat type. Two copies of the hr allele (homozygous hr/hr) or one copy of the hr allele in combination with one copy of the dr allele (heterozygous hr/dr) results in a nearly hairless coat. Two copies of the dr allele (dr/dr) results in a curly coat. For Sphynx breeders, the tests identify hairless cats that carry the curly mutation and therefore, depending on the mate, might get offspring with a curly coat.

The Devon Rex, Curly Coat and Sphynx, Hairless Coat tests (together KRT71 gene) enclose the following results:

The Agouti gene (ASIP gene) is responsible for the production of a protein that regulates the distribution of black pigment (eumelanin) within the hair shaft. This gene is also known as the A-locus and determines whether an animal expresses an agouti appearance, and if so what type, by controlling the distribution of pigment in individual hairs. The agouti pattern can be seen in both black-based and red-based colours. The coat colour is further complicated by the interaction with the K-locus and the E-locus. The agouti pattern is only expressed if on the K-locus no copy of the KB allele is present in combination with at least one copy of the E or Em allele on the E-locus. The Coat Colour A-Locus test (H820) tests for the genetic status of the A-locus. The A-locus has four variants (alleles). The most dominant allele is Ay, followed by aw, then at, then a. The dominant Ay allele produces a sable or fawn coat colour. The allele aw produces a colour known as wild sable or wild type. With this colouration, the hairs switch pigmentation from black to reddish or fawn. This colour is sometimes seen in German Shepherds and other shepherd breeds. The allele at results in tan points (tan markings on a dark dog) and produces black-and-tan and tricolour dogs. A tricolour dog is black-and-tan plus white. The allele a is also called the recessive black allele and results in a solid black/brown/blue/lilac or bicolour dog. Some breeds are fixed for only one variant. The Norwegian Elkhound is fixed for the aw allele and the Beagle is fixed for the at allele. In many breeds 2 or 3 alleles are present.

The Coat Colour A-Locus test encloses the following results.

The MFSD12 gene, also known as I-Locus affects the expression of the pheomelanin (red) pigment. The MFSD12 gene has no effect on eumelanin (black) pigment, therefore the black coat and the black hair ends remain black. The mutation affects the pheomelanin in the entire coat resulting in a pure white or cream coat colour. The pigmentation of the nose, lips, eyes and skin remains unchanged. The intensity of the dilution may differ in various dog breeds. The Coat Colour I-Locus test (H821) tests for the genetic status of the I-Locus. The I-Locus has two variants (alleles). The allele I is dominant and does not have an effect on the coat colour. Only when the dog has two copies of the recessive allele i the coat colour is diluted.

The Coat Colour I-Locus test encloses the following results, in this scheme the results of the Coat Colour I-Locus test are shown in combination with the possible results for the E-Locus and B-Locus:

The dilute gene (MLPH gene) is responsible for the intensity of the coat colour by affecting the distribution of melanin-containing cells. This gene is also known as the D-Locus and dilutes all colours. Besides the hair colour also the colour of the nose is diluted and the colour of the eyes lightens to amber. The Coat Colour D-Locus Improved (MLPH) test (H847) tests for the genetic status of the D-Locus. The D-Locus has two variants (alleles). The allele D is dominant and does not have an effect on the coat colour. Only when the dog has two copies of the recessive allele d the coat colour is diluted. The dilution of black results in grey, also called blue or charcoal. The coat ranges from silver to almost black, but all have a blue nose. Chocolate/brown/liver dilutes into lilac/light tan/Isabella, their noses vary from pink, liver to isabella. Red/yellow/cream dilutes into champagne. In some breeds another, yet unidentified, mutation is present that causes coat colour dilution. This unidentified mutation is known to occur in Doberman Pinscher, French Bulldog, Italian Greyhound, Chow Chow and Shar-Pei.

The Coat Colour D-Locus Improved (MLPH) test encloses the following results, in this scheme the results of the Coat Colour D-Locus Improved (MLPH) test are shown in combination with the possible results for the E-Locus and B-Locus):

The Beta-defensin gene (CBD103 gene) produces dominant black vs. brindle vs. fawn coat colours. This gene is also known as the K-locus or Dominant black gene. The coat colour is further complicated by the interaction with the E-locus and the A-locus (agouti). The Coat Colour K-Locus (H819) tests for the genetic status of the K-Locus. The K-locus has three variants (alleles). The allele KB is dominant over the alleles kbr and ky; allele kbr is dominant over allele ky. The dominant allele KB, also called dominant black allele, does not allow the agouti gene to be expressed. A dog with at least one copy of the KB allele expresses a base colour, which is determined by the B- and E-Locus. The allele kbr results in brindling and allows the agouti to be expressed but causes brindling of the agouti patterns. The A-Locus (agouti) represents several different colours, such as fawn/sable, wild sable, tan points and recessive black. The allele ky allows the agouti to be expressed without brindling. When a dog has two copies of the ky allele (homozygous ky/ky) the agouti locus determines the dog’s coat colour. The test does not discriminate between the alleles kbr and ky.

The Coat Colour K-Locus test encloses the following results:

The Silver gene (SILV gene), also called premelanosome protein (PMEL17 gene) is responsible for Merle. This gene is also known as M-Locus. Merle only dilutes eumelanin (black) pigment; dogs with two copies of the allele e (homozygous e/e) at E-Locus have no black pigment, thus do not express merle. Merle is an incompletely dominant coat color pattern characterized by irregularly shaped patches of diluted pigment and solid color. Blue and partially blue eyes are typically seen with merle, and merle dogs often have a wide range of auditory and ophthalmologic defects. Breeds with merle coat pattern are Shetland Sheepdog, Collie, Border Collie, Australian Shepherd, Cardigan Welsh Corgi, Catahoula Leopard Dog, Dachshund, Great Dane, Bergamasco Sheepdog and Pyrenean Shepherd. The Coat Colour Merle test (H630) tests for the genetic status of the M-locus. The M-locus has three variants (alleles): M (merle, SINE with longer poly-A tail), Mc (cryptic merle, SINE with shorter poly-A tail) and N (non-merle, no SINE insertion. Dogs with cryptic merle (also called phantom or ghost merle), typically display little to no merling and some may be misclassified as non-merles.

The Coat Colour Merle test encloses the following results.

The hnRNP associated with lethal yellow gene (RALY gene) defines whether tan points or saddle tan is expressed in Basset Hounds and Pembroke Welsh Corgi dogs. Black and tan colour is characterized by light colour on the muzzle, above the eyes (tan points) and on the undersides of the dog on otherwise dark coat. Saddle tan resembles black and tan colour but the lighter areas are expanded leaving usually only the back to have dark patch. Saddled tan dogs are usually born black-and-tan and the black recedes as the dog grows. The coat colour is further complicated by the interaction with the E-locus, K-locus, A-locus and a yet unidentified gene. In order for the saddle tan pattern or tan points to be expressed, the dog needs to have at least one copy of the E or Em allele at the E-locus, two copies of the ky allele at the K-locus and one or two copies of the at allele at the A-locus. The Coat Colour Saddle tan vs black-and-tan test (H353) tests for the genetic status of the RALY gene. The RALY gene has two variants (alleles). The allele WT is dominant and causes the saddle tan coat colour. Only when the dog has two copies of the recessive allele dup the coat colour is black-and-tan. The saddle tan coat colour is present in a limited number of dog breeds including some of the terriers, scent hounds and herding dogs. In breeds that have only tan point dogs and no saddled tan dogs, the tan pointed dogs can have any genotype for the RALY gene. This suggests that more complex interactions are behind tan points in breeds that are not able to express saddle tan.

The Coat Colour Saddle tan vs black-and-tan test encloses the following results:

The Keratin 71 (KRT71) gene influences the hair formation. The Curly Coat test (H921) tests for the genetic status of the KRT71 gene. The KRT71 gene has two variants (alleles). The allele CC is dominant and results in a curly coat. Only when the dog has two copies of the recessive allele N the coat is of a non-curly type. Some breeds, such as the Irish Water Dog, are fixed for the dominant allele CC. Other breeds, such as Kuvasz, can have either curly or non-curly hair.

The Curly Coat test encloses the following results:

The R-spondin 2 (RSPO2) gene influences both the wiry texture and a growth pattern of the coat. The growth pattern of the coat, also known as “furnishings”, increases hair growth on the face and legs and is typified by the canine moustache and eyebrows. The term “furnishings” refers to the longer mustache and eyebrows seen in wire-haired dogs and other breeds. In breeds such as the Portuguese Water Dog, Labradoodle and Goldendoodles furnishings can be variable, but are the breed standard. Portuguese Water Dogs without furnishings are referred to as having an “Improper Coat” which is characterized by short hair on the head, face and legs. The Improper Coat/Furnishings test (H848) tests for the genetic status of the RSPO2 gene. The RSPO2 gene has two variants (alleles). The allele N is dominant and results in “furnishings”. Only when the dog has two copies of the recessive allele IC the dog does not have “furnishings”. Some breeds, such as the Airedale Terrier, are fixed for the dominant allele N.

The Improper Coat/Furnishings test encloses the following results:

The Fibroblast Growth Factor 5 (FGF5) determines the hair length. The Hair Length test (H765) tests for the genetic status of the FGF5-gene and has two variants (alleles). The allele S is dominant and results in short hair. Only when the dog has two copies of the recessive allele L the dog has long hair. Some breeds, such as Labradors, are fixed for the dominant allele S. Other breeds, such as Poodles, are fixed for the recessive allele L and some breeds, such as Dachshund, can have either long or short hair. In some breeds another, yet unidentified, mutation is present that influences hair length. This unidentified mutation is known to occur in Afghan Hounds, Yorkshire Terriers, and Silky Terriers.

The Hair Length test encloses the following results:

There are three variables involved in canine coat type: hair length, the presence of furnishings, and the presence of curly hair. When genotyping genetic variants on all three genes, there are a few coat patterns that can be discriminated. In the table below the possible combinations of these mutations are indicated.

The Hermansky-Pudlak syndrome 3 (HPS3) gene, also known as cocoa coat colour or co-locus is responsible for the brown colour in French Bulldogs. Mutations of the HPS3 gene interfere with the eumalin (black pigment) synthesis, which results in brown-pigmentation. The brown colour caused by the HPS3-variants is known to darken over age and to be slightly darker in adults that the brown colour caused by the TYRP1-related variants (B-locus). The co-locus is recessive and therefore needs two copies of the gene to present the phenotype. This co-locus can be present in French Bulldogs with various coat colours; brown, lilac, black, blue, cream, fawn or white, but the phenotype might be less visible in some cases. The complete phenotype of the coat, footpads and nose also depends on the A-, E-, K- and B-locus genes. Right now, no interaction in French Bulldogs between the co- and B-locus have been reported. Therefore it is not possible to predict what phenotype the combination of these variants would cause.

N/N = cocoa variant not present. The cocoa phenotype is not expressed, the offspring won’t inherit a copy of the co-locus.

N/co = carrier of the cocoa variant. Phenotype is not present. 50% of the offspring will inherit one copy of the co-locus.

co/co = the cocoa phenotype is present. The display of this phenotype depends on the interaction between other colour genes (loci). 100% of the offspring will get one copy of the co-locus. 

The white spotting patterns that occur in many dog breeds do not have a uniform genetic basis. The Microphthalmia Associated Transcription Factor gene (MITF gene) is associated with many white spotting patterns. This gene is also known as the S-Locus. There are three major white spotting patterns described. One pattern is called “Irish spotting” and is a symmetrical pattern with white markings on the undersides, collar and muzzle, and/or blaze as demonstrated by breeds such as the Boston Terrier, Corgi, Bernese Mountain dog and Basenji. Another pattern of less symmetrical white spotting in which random white spots occur on the body of the dog is often called piebald, parti or random white and is observed in several breeds, including the Beagle and Fox Terrier. The third major pattern is called extreme white and results in a dog that is almost entirely white but usually has at least some color on the head. Furthermore, there is a pattern called mantle, this pattern is similar to Irish spotting but with more white extending onto the thigh and up the torso, as seen in some Great Danes. Another pattern that is similar to Irish spotting is called flash or pseudo-Irish and occurs in Boxers. A mutation found in the MITF gene is associated with the piebald spotting pattern in more than 25 different dog breeds. The Coat Colour Piebald test (H326) tests for the genetic status of this mutation. It results in two variants (alleles). The allele N does not produce a piebald pattern, therefor dogs with two copies of the N allele do not display the piebald pattern. The allele S is associated with the piebald pattern, however the amount of white spotting expressed varies from breed to breed and among individuals within a breed. In many breeds such as Collie, Great Dane, Italian Greyhound, Shetland Sheepdog, Boxer and Bull Terrier, piebald behaves as a dosage-dependent trait. In those breeds the allele S is semi-dominant. One copy of the S allele (S/N) results in a limited white spotting pattern. Dogs with two copies of the  S allele (S/S) display more extreme white with colour only on the head and perhaps a body spot. In Boxers and Bull Terriers, dogs that have two copies of the S allele (S/S) are completely white while dogs that only have one copy of the S allele (N/S) display the mantle pattern (called flash in these breeds). However, additional mutations in MITF or other white-spotting genes that affect the amount of white being expressed appear to be present in these breeds. In some other breeds, the allele S is recessive and in those breeds two copies are needed to produce the piebald pattern.

The Coat Colour Piebald test encloses the following results:

The 20S proteasome β2 subunit (PSMB7) gene is responsible for the Harlequin coat pattern in Great Danes. This gene is also known as H-Locus. Harlequin is a pattern resulting from interaction of the Merle (M-locus) gene and the Harlequin (H-locus) gene on black pigment. The Harlequin gene can modify the Merle gene. The Harlequin pattern is only expressed if on the M-locus at least one copy of the M allele is present in combination with at least one copy of the E or Em allele on the E-locus. Dogs that are not merle, or only have red pigment, cannot express the Harlequin gene. The dominant Merle gene, by itself produces dark spots on a diluted background. If a Merle dog also inherits one copy of the Harlequin gene, the dark spots increase in size and the background pigment is removed (turns white). The Harlequin mutation in Great Danes is in homozygous state (two copies of the mutation) considered embryonic lethal as no live dogs with two copies of the mutation have been observed. This means that pups that are homozygous for the Harlequin mutation do not develop in the uterus and are reabsorbed very early in the development process. Therefore all Harlequin patterned dogs have only 1 copy of the Harlequin mutation. The Coat colour H-locus (Harlequin) test (H316) tests for the genetic status of the H-locus. This gene has two variants (alleles), H and N. The allele H is dominant. One copy of the H allele, together with at least one copy of both the M allele for the M-locus and the E allele for the E-locus results in dogs with the Harlequin pattern. Two copies of the H allele result in early embryonic death. The allele N does have no effect on the coat colour.

The Coat colour H-locus (Harlequin) test encloses the following results.

A mutation in the KIT-gene is associated with a white spotting pattern in German Shepherd Dogs, this pattern is  also called Panda White Spotting. The mutation is very recent, it appeared spontaneously in a female born in 2000. The gene for white-spotting is known as the S-locus (MITF-Gene), however this mutation in the German Shepherd dogs is in a different gene then the mutation causing white spotting in other dog breeds. The mutation causes white markings on the face, limbs, belly, neck, and tip of the tail, with the white being concentrated toward the front of the dog, similar to the irish spotting pattern. The amount of white can vary from dog to dog. The mutation that causes the Panda White pattern in German Shepherd dogs is in homozygous state (two copies of the mutation) considered embryonic lethal as no live dogs with the pattern and with two copies of the mutation have been observed. This means that pups that are homozygous for the Panda mutation do not develop in the uterus and are reabsorbed very early in the development process. Dogs that are heterozygous (one copy of the mutation) do not have any health defects associated with the Panda pattern. The Coat Colour Panda White Spotting test (H354) tests for the genetic status of the KIT-gene. This gene has two variants (alleles), P and N. The allele P is dominant. One copy of the P allele results in dogs with the Panda white pattern. Two copies of the P allele result in early embryonic death. The allele N does have no effect on the coat colour.

The Coat Colour Panda White Spotting test encloses the following results.