DNA-Testing for single gene traits: COAT COLOUR

1. DNA-Testing forsingle gene traits:COAT COLOUR Dr. Munro Marx and JoubertOosthuizen Unistel Medical Laboratories (Pty) Ltd.

2. From cell to genome Cells:The fundamental working units of a living organism. DNA:Found in the nucleus of cells All 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 genome Genomes sizes vary in different organisms.

3. DNA, Chromosomes, Genes and Proteins DNA is packaged in structures known as chromosomes (46 in humans, 46 in Sable antelope, 56 in Springbok, 60 in buffalo and cattle ). Several genes are located on each chromosome Genes are the functional and physical units of inheritance. Each genome has about 25 000 genes The basic structure of a gene supplies the code for the manufacture of Proteins. Proteins provide essential functions for life (digestion etc.) and form and structures (cells, hair etc.) Proteins consist of combinations of amino acids (20 different amino acids) The sequence of amino acids, the protein form and structure determines function.

4. Proteins and Proteome The total protein content of a cell is known as the proteome. Proteome is a dynamic system and reacts to both the internal and external environment. The chemistry and behaviour of a proteome is determined by the total gene function and expression in the same cell at the same time. Each cell has the genetic potential to manufacture and express all the proteins of the organism. Gene function and expression is selective and cell specific. Genes activated in a specific cell provide that cell with its unique function and characteristic e.g. liver cells, coat colour.

5. Where does coat colour fit in? • Animals evolved different skin and coat colour and patterns primarily as defence against predators or as an aid in predation. • An animal’s phenotype (what it looks like), is a result of a complex interaction between its inherited genetic makeup (genotype) and the environment in which it lives. • Genes involved in skin and coat colour are amazingly similar all over the animal kingdom.

6. Colour, Melanocytes and Melanin • The pigment melanin is the primary determinant of colour and is found in cells called melanocytes . • Melanin is produced in melanocytes in the eye, skin and hair (coat). • Skin and coat darkness / lightness is primarily determined by: • the amount of melanin in the melanocytes • the ratio between eumelamin (Black/Brown pigment) and pheomelamin (Red/Yellow). • Melanocytes originate in the neural crest and migrate during embryogenesis. This migration plays a role in colour patterning.

7. Genetic control of Skin and Coat Colour. • The skin and coat colour (pigmentation) of an animal is determined by GENES that code for different pigments. • Pigmentation caused by genes is constitutive pigmentation – an intrinsic property of the animal. • Two classes of genes affecting pigmentation have been identified: • Those affecting the pigment producing cells (melanocytes per se, especially on membrane). • Those affecting pigment synthesis (the inside workings of melanocytes).

8. Cause of colour and pattern diversity. • As with (virtualy) all diversity in the world of the living, colour and pattern diversity is caused by genes and their mutations. • The wide variety of colours and patterns observed is due to: • The number of genes involved, and • The number of mutations per gene. • Advantageous mutations are evolutionary retained, disadvantageous ones are lost. • In the wild disadvantageous mutations that inherit recessively may be retained for generations. Loss of the properties assisting survival prevent the recessive phenotype from surviving, thereby keeping the mutation’s frequency low.

9. Mutations that affect melanocytes. Melanocytes are responsible for eye, skin and coat colour, as well as patterning. Mutations may affect the following areas of melanocyte function: • Mutations affecting the melanocyte surface result in qualitative changes of pheomelanine or eumelanine . • These mutations influence what kind of pigment are produced and will influence the basic colour of the skin and coat. • Mutations affecting the inner workings of melanocytes. • These mutations result in quantitave changes in pigment production and may lead to changes in the basic colours (colour dilution). • Mutations affecting differentiation, proliferation and migration of melanocytes. • These mutations lead to changes in white spotting and MAY lead to changes in patterns

10. How do genes work to have the effect they have? Genes operate in pairs, with one of each inherited from each parent. • Some genes are dominant – if an animal have one of those genes, it will totally cancel the effect of the other gene. • Some are recessive – the gene’s effect will only be noticed if both copies are recessive. • Some are co-dominant – if two different genes are present, their effect will be a combination of the two. • Together with the above, a range of other effects may influence the inheritance and expression of a trait , eg: • Incomplete penetrance • multigene vs single gene inheritance • epistase • epigenetic factors • Etc, etc.

11. Other factors affecting skin and coat colour • Suplementary to genes are environmental factors and hormones (facultative pigmentation – inducable property). (A topic on it’s own, not for today.)

12. Colour genes illustrated – the horse as representative of the mammal (1) Horse colour genetics (constitutive pigmentation) is perhaps best understood. • The basic coat colours: Genes affecting the melanocyte surface (Extension and Agouti loci) determine the basic coat colours – Chestnut, Bay, Black • Chestnut: eumelanine (black/brown) in the skin, pheomelamine (red/yellow) in the hair, including mane and tail. • Black: eumelanine (black). In skin and hair (entire body). • Bay: pheomelanine (body) and eumelanine (mane and tail and lower leg) patterns. So far, so good, so simple!!

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

14. Colour and pattern genes – the mouse 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 give us hope that coat pattern will soon be as well understood as colour.

15. What about game coat colour? 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, but with slight sequence differences. 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, 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 specie in question should be used as starting point. A second phase would be the same procedure for the known genes that have a less dramatic effect (dilution, depigmentation, greying). Literature should be carefully monitored for any new developments. Finally breeding and breeding experiments might be required.

16. What about coat colour patterns? Is the basis for variation in coat colour pattern known? Hardly !!!. • Mammalian coat patterns (e.g., spots, stripes) are hypothesized to play important roles in camouflage and other relevant processes, yet the genetic and developmental bases for these phenotypes are completely unknown. (Eizirik, E. January 2010) • "The question of how color patterns are established in vertebrates has been a black box," says Marie Manceau, (Science, 2011). • They found that subtle changes in the Agouti gene's embryonic activity can also make a profound difference in the distribution of pigments across the entire body. Mary Manceau, Hopi E. Hoekstra et al: 2011)

17. The Beauty of 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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