DNA-Testing forDNA-Testing forsingle gene traits:single gene traits:

COAT COLOURCOAT COLOURDr. Munro Marx and Joubert OosthuizenDr. Munro Marx and Joubert OosthuizenUnistel Medical Laboratories (Pty) Ltd.Unistel Medical Laboratories (Pty) Ltd.

From cell to genomeFrom cell to genome

Cells:The fundamental working units of a living organism.

DNA:Found in the nucleus of cellsAll instructions to regulate the activities of cells are tucked up in the DNA. The DNA molecule is a side by side arrangement of nucleotides (e.g. ATTCCGGA).

The Genome;The organism’s total DNA content is known as the genomeGenomes sizes vary in different organisms.

DNA, Chromosomes, Genes and ProteinsDNA, Chromosomes, Genes and Proteins DNADNA is packaged in structures known as is packaged in structures known as

chromosomes (46 in humans, 46 in Sable chromosomes (46 in humans, 46 in Sable antelope, 56 in Springbok, 60 in buffalo and antelope, 56 in Springbok, 60 in buffalo and cattle ). cattle ).

Several genes are located on each Several genes are located on each chromosomechromosome

Genes are the functional and physical units of Genes are the functional and physical units of inheritance. Each genome has about 25 000 inheritance. Each genome has about 25 000 genes genes

The basic structure of a gene supplies the The basic structure of a gene supplies the code for the manufacture of code for the manufacture of Proteins. .

Proteins Proteins provide essential functions for life provide essential functions for life (digestion etc.) and form and structures (cells, (digestion etc.) and form and structures (cells, hair etc.) hair etc.)

Proteins consist of combinations of Proteins consist of combinations of amino acids (20 different amino acids) (20 different amino acids)

The sequence of amino acids, the protein form The sequence of amino acids, the protein form and structure determines function. and structure determines function.

Proteins and ProteomeProteins and Proteome The total protein content of a cell is known as the The total protein content of a cell is known as the

proteome. . Proteome is a dynamic system and reacts to both Proteome is a dynamic system and reacts to both

the internal and external environment. the internal and external environment. The chemistry and behaviour of a proteome is The chemistry and behaviour of a proteome is

determined by the total gene function and determined by the total gene function and expression in the same cell at the same time. expression in the same cell at the same time.

Each cell has the genetic potential to manufacture Each cell has the genetic potential to manufacture and express all the proteins of the organism. and express all the proteins of the organism.

Gene function and expression is selective and cell Gene function and expression is selective and cell specific. specific.

Genes activated in a specific cell provide that cell Genes activated in a specific cell provide that cell with its unique function and characteristic e.g. with its unique function and characteristic e.g. liver cells, coat colour. liver cells, coat colour.

Where does coat colour fit in?

Animals evolved different skin and coat Animals evolved different skin and coat colour and patterns primarily as defence colour and patterns primarily as defence against predators or as an aid in predation.against predators or as an aid in predation.

An animal’s phenotype (what it looks like), An animal’s phenotype (what it looks like), is a result of a complex interaction between is a result of a complex interaction between its inherited genetic makeup (genotype) and its inherited genetic makeup (genotype) and the environment in which it lives.the environment in which it lives.

Genes involved in skin and coat colour are Genes involved in skin and coat colour are amazingly similar all over the animal amazingly similar all over the animal kingdom.kingdom.

Colour, Melanocytes and MelaninColour, Melanocytes and Melanin The pigment The pigment melaninmelanin is the primary determinant of is the primary determinant of

colour and is found in cells called colour and is found in cells called melanocytes . . Melanin is produced in melanocytes in the eye, skin and Melanin is produced in melanocytes in the eye, skin and

hair (coat).hair (coat). Skin and coat darkness / lightness is primarily Skin and coat darkness / lightness is primarily

determined by: determined by: the the amount of melaninamount of melanin in the melanocytes in the melanocytes the the ratio betweenratio between eumelamin (Black/Brown pigment) and eumelamin (Black/Brown pigment) and

pheomelamin (Red/Yellow).pheomelamin (Red/Yellow).

Melanocytes originate in the neural crest and migrate Melanocytes originate in the neural crest and migrate during embryogenesis. This during embryogenesis. This migrationmigration plays a role in plays a role in colour patterningcolour patterning..

Genetic control of Skin and Coat Colour.

The skin and coat colour (pigmentation) of an The skin and coat colour (pigmentation) of an animal is determined by GENES that code for animal is determined by GENES that code for different pigments. different pigments.

Pigmentation caused by genes is constitutive Pigmentation caused by genes is constitutive pigmentation – an intrinsic property of the animal. pigmentation – an intrinsic property of the animal.

Two classes of genes affecting pigmentation have Two classes of genes affecting pigmentation have been identified:been identified:

Those affecting the pigment producing cells Those affecting the pigment producing cells (melanocytes per se, especially on membrane).(melanocytes per se, especially on membrane).

Those affecting pigment synthesis (the inside Those affecting pigment synthesis (the inside workings of melanocytes).workings of melanocytes).

Cause of colour and pattern Cause of colour and pattern diversity.diversity.

As with (virtualy) all diversity in the world of the living, colour and As with (virtualy) all diversity in the world of the living, colour and pattern diversity is caused by genes and their mutations.pattern diversity is caused by genes and their mutations.

The wide variety of colours and patterns observed is due to:The wide variety of colours and patterns observed is due to: The number of genes involved, andThe number of genes involved, and The number of mutations per gene.The number of mutations per gene.

Advantageous mutations are evolutionary retained, Advantageous mutations are evolutionary retained, disadvantageous ones are lost.disadvantageous ones are lost.

In the wild disadvantageous mutations that inherit recessively may In the wild disadvantageous mutations that inherit recessively may be retained for generations. Loss of the properties assisting be retained for generations. Loss of the properties assisting survival prevent the recessive phenotype from surviving, thereby survival prevent the recessive phenotype from surviving, thereby keeping the mutation’s frequency low.keeping the mutation’s frequency low.

Mutations that affect melanocytes.Mutations that affect melanocytes.

Mutations affecting the melanocyte surface result in Mutations affecting the melanocyte surface result in qualitative changes qualitative changes of pheomelanine or eumelanine .of pheomelanine or eumelanine .

These mutations influence what kind of pigment are These mutations influence what kind of pigment are produced and will influence the basic colour of the skin and produced and will influence the basic colour of the skin and coat.coat.

Mutations affecting the inner workings of Mutations affecting the inner workings of melanocytesmelanocytes. .

These mutations result in These mutations result in quantitave changes quantitave changes in pigment in pigment production and may lead to changes in the basic colours production and may lead to changes in the basic colours (colour dilution).(colour dilution).

Mutations affecting differentiation, proliferation and Mutations affecting differentiation, proliferation and migration of melanocytes.migration of melanocytes.

These mutations lead to changes in white spotting These mutations lead to changes in white spotting and MAY and MAY lead to lead to changes in patterns changes in patterns

Melanocytes are responsible for eye, skin and coat colour, as Melanocytes are responsible for eye, skin and coat colour, as well as patterning. Mutations may affect the following areas well as patterning. Mutations may affect the following areas of melanocyte function:of melanocyte function:

How do genes work to have the effect they have?

Genes operate in pairs, with one of Genes operate in pairs, with one of each inherited from each parent.each inherited from each parent.

Some genes are dominant – if an animal have Some genes are dominant – if an animal have one of those genes, it will totally cancel the one of those genes, it will totally cancel the effect of the other gene.effect of the other gene. Some are recessive – the gene’s effect will Some are recessive – the gene’s effect will only be noticed if both copies are recessive.only be noticed if both copies are recessive. Some are co-dominant – if two different genes Some are co-dominant – if two different genes are present, their effect will be a combination of are present, their effect will be a combination of the two.the two. Together with the above, a range of other Together with the above, a range of other effects may influence the inheritance and effects may influence the inheritance and expression of a trait , eg:expression of a trait , eg:

Incomplete penetranceIncomplete penetrance multigene vs single gene inheritancemultigene vs single gene inheritance epistaseepistase epigenetic factors epigenetic factors Etc, etc.Etc, etc.

Other factors affectingOther factors affecting skin and coat colour skin and coat colour

Suplementary to genes are environmental factors Suplementary to genes are environmental factors and hormones (facultative pigmentation – inducable and hormones (facultative pigmentation – inducable property). (A topic on it’s own, not for today.)property). (A topic on it’s own, not for today.)

Colour genes illustrated – the horse Colour genes illustrated – the horse as representative of the mammal (1)as representative of the mammal (1)

Horse colour genetics (constitutive pigmentation) is Horse colour genetics (constitutive pigmentation) is perhaps best understood.perhaps best understood.

The basic coat colours: Genes affecting the melanocyte surface (Extension and Agouti loci) determine the basic coat colours – Chestnut, Bay, Black ChestnutChestnut: eumelanine (black/brown) in the skin, pheomelamine : eumelanine (black/brown) in the skin, pheomelamine

(red/yellow) in the hair, including mane and tail.(red/yellow) in the hair, including mane and tail. BlackBlack: eumelanine (black). In skin and hair (entire body).: eumelanine (black). In skin and hair (entire body). BayBay: pheomelanine : pheomelanine (body) and eumelanine (mane and tail and

lower leg) patterns.

So far, so good, so simple!!

Colour genes illustrated – the horse Colour genes illustrated – the horse as representative of the mammal (2)as representative of the mammal (2)

PhenotypeLocus (symbol) gene abbrev

PalominolBuckskin Cream (CR) MATP

Cremello/Perlino - creamy white coat colour with blue eyes

Cream (CR) MATP

Dun - diluted basic colours and primitive markings (c.g dorsal stripe, zebra stripes on legs)

Dun (D) Not known

Silver dapple - chocolate-to-reddish body with whitelgrey mane and tail

Silver (Z) PMEL 17

Champagne - metallic sheen (eye and skin colour may change with age)

Champagne (CH) SLC36Al

GreyingProgressive greying with age; associated with melanoma Grey (G) STX17 Roan - interspersed white hairs in basic colour

Roan (RN) Associated to KIT

Overo-spotting - irregular white spotting, often horizontal distribution Overo (0) EDNRB Tobiano-spotting - regular white spotting, often vertical distribution Tobiano (TO) KIT

Depigmen-tation

Dilution coat colours

16 Genes influencing coat colour has so far been found. Two are responsible for primary colour, while the rest, some singly, some in combination, modify the basic colour a varying degree.

For each of these genes, mutations have been described.

Mutations within these genes can multiply the effects on colour

Colour and pattern genes – the mouse Colour and pattern genes – the mouse The mouse is the most studied mammal, also as far as pigmentation The mouse is the most studied mammal, also as far as pigmentation is concerned. Some sobering data is given in the table below. It also is concerned. Some sobering data is given in the table below. It also

give us hope that coat pattern will soon be as well understood as give us hope that coat pattern will soon be as well understood as colour.colour.

302 Mouse genes and variants involved in pigmentation have been identified. The variants are 302 Mouse genes and variants involved in pigmentation have been identified. The variants are mutated genes - the total number of genes is less than 302, but still many times the 16 of the mutated genes - the total number of genes is less than 302, but still many times the 16 of the

horse.horse.

Of these 171 have been cloned and studied in detail.Of these 171 have been cloned and studied in detail.

For each of these171 genes a human homologue has been found. There seems to be a For each of these171 genes a human homologue has been found. There seems to be a large degree of similarity in mammalian pigmentation genetics over species.large degree of similarity in mammalian pigmentation genetics over species.

For 149 of the 171 genes homologues have been found in the Zebra-fish. This bodes well for For 149 of the 171 genes homologues have been found in the Zebra-fish. This bodes well for utilization of knowledge across species.utilization of knowledge across species.

Coat colour - Coat colour - GeneralGeneral

Pattern - eg. Pattern - eg. spot, belt, stripe, spot, belt, stripe,

toe, etc.toe, etc.

Coat, Hair, Fur - Coat, Hair, Fur - Other effectsOther effects

Skin - Dark, Skin - Dark, Light, Darken, Light, Darken,

LightenLighten

Skin - Other Skin - Other EffectsEffects

Pigmentation - Pigmentation - hyper, hypo, hyper, hypo, depigment.depigment.

OtherOther

22 (7 cloned)22 (7 cloned) 97 (24 cloned)97 (24 cloned) 52 (28 cloned)52 (28 cloned) 19 (11 cloned)19 (11 cloned) 7 (6 cloned)7 (6 cloned) 17 (16 cloned)17 (16 cloned) 88 (79 cloned)88 (79 cloned)

These same genes have been found in many mammals, even though they may These same genes have been found in many mammals, even though they may not have exactly the same effect. Most will be present in most game species, not have exactly the same effect. Most will be present in most game species, but with slight sequence differences.but with slight sequence differences.

A starting point would be to determine the DNA sequences of the known primary A starting point would be to determine the DNA sequences of the known primary colour genes (Extension and Agouti loci) of both standard and other phenotypes, colour genes (Extension and Agouti loci) of both standard and other phenotypes, analyse for differences and investigate the genetic effect, if any, of each. analyse for differences and investigate the genetic effect, if any, of each.

Species for which the gene sequences are known that are closest to the game Species for which the gene sequences are known that are closest to the game specie in question should be used as starting point.specie in question should be used as starting point.

A second phase would be the same procedure for the known genes that have a A second phase would be the same procedure for the known genes that have a less dramatic effect (dilution, depigmentation, greying).less dramatic effect (dilution, depigmentation, greying).

Literature should be carefully monitored for any new developments.Literature should be carefully monitored for any new developments. Finally breeding and breeding experiments might be required.Finally breeding and breeding experiments might be required.

What about game coat colour?What about game coat colour?

What about coat colour patterns?

Is the basis for variation in coat colour Is the basis for variation in coat colour pattern known? Hardly !!!.pattern known? Hardly !!!.

Mammalian coat patterns (e.g., spots, stripes) are hypothesized to Mammalian coat patterns (e.g., spots, stripes) are hypothesized to play important roles in camouflage and other relevant processes, play important roles in camouflage and other relevant processes, yet the genetic and developmental bases for these phenotypes yet the genetic and developmental bases for these phenotypes are completely unknownare completely unknown. . (Eizirik, E. January 2010)(Eizirik, E. January 2010)

"The question of how color patterns are established in vertebrates "The question of how color patterns are established in vertebrates has been a black box," has been a black box," says Marie Manceau, (says Marie Manceau, (Science, 2011)Science, 2011)..

They found that subtle changes in the They found that subtle changes in the Agouti gene's embryonic Agouti gene's embryonic activity can also make a profound difference in the distribution of activity can also make a profound difference in the distribution of pigmentspigments across the entire body. across the entire body. Mary Manceau, Hopi E. Hoekstra Mary Manceau, Hopi E. Hoekstra et al: 2011)et al: 2011)

The Beauty of MutationsThe Beauty of MutationsWhy mutations?Why mutations?

Our environment constantly changes, the Earth and its ecosystems change.

Populations must change to survive Evolutionary change requires variation, the

raw material on which natural selection works

One mechanism for variation and change is at the DNA level.

Mutations can be beneficial and enable the organism to adapt to a changing environment.

However, most mutations are deleterious, and cause varied genetic problems

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