Coat Colour Genetics in the Connemara Pony Population of New Zealand

Coat Colour Genetics in the

Connemara Pony Population

of New ZealandSheila Ramsay

29 June 2013

Coat Colour Genetics in the

Connemara Pony Population

of New ZealandSheila Ramsay

29 June 2013

Reasons for this StudyReasons for this Study• to look at whether there is a correlation between the development of Grey horse melanoma and birth coat colour.

• to determine the level of the chestnut allele in the New Zealand Connemara pony population.

• to determine the level of the chestnut allele in the New Zealand Connemara pony population.

• to determine the level of the cream dilution allele in the New Zealand Connemara pony population.

• to determine the level of the cream dilution allele in the New Zealand Connemara pony population.

Colour pigmentation isproduced by melanocytes

Colour pigmentation isproduced by melanocytes

Basic Colour Genetics of Equus caballus

the domestic horse/pony

• The genotype is the genetic code and controls every function in the body.

• The phenotype is what we see.

• The genotype and the phenotype can be at variance with each other.

Two base coats are coded for

in the horse:Black and Red

Two base coats are coded for

in the horse:Black and Red

ALL other colours aremodifications of the base

coat

The Extension locus ‘E’The Extension locus ‘E’

• ee = chestnut (recessive)• ee = chestnut (recessive)

• Determines black or red• Determines black or red• Black is dominant • Black is dominant

• EE or Ee = black• EE or Ee = black

Agouti locus ‘A’Agouti locus ‘A’

• Modifies black by changing black pigment to red on specific areas of the body.

• Modifies black by changing black pigment to red on specific areas of the body.• Produces bays and browns.• Produces bays and browns.

• Does not affect chestnut (red)• Does not affect chestnut (red)

• A is dominant A/A or A/a = bay or brown• A is dominant A/A or A/a = bay or brown• a/a = recessive• a/a = recessive

Cream Dilution CrCream Dilution Cr

Modifies the base coat colour by reducing the levelof pigmentation produced by the melanocytesModifies the base coat colour by reducing the levelof pigmentation produced by the melanocytes

Is dominant. If the mutation is present it HAS to be expressed.Is dominant. If the mutation is present it HAS to be expressed.

Works only on red pigment.Works only on red pigment.

One copy of the gene results in Palomino or Buckskin (dun) or Smokey Black. (single dilution)

One copy of the gene results in Palomino or Buckskin (dun) or Smokey Black. (single dilution)Two copies of the gene results in Cremello, Perlino or Smokey Cream (double dilution)

Two copies of the gene results in Cremello, Perlino or Smokey Cream (double dilution)

Grey Modification ‘G’Grey Modification ‘G’• Is a dominant gene – if present in the genotype it has to seen (expressed) in the phenotype. G/G, G/g

• The grey gene has a further mutation which causes melanoma .

• The grey mutation works on the cells in the neural crest of the developing embryo – well before cell differentiation (about day 12 after conception)

• Is a dominant gene – if present in the genotype it has to seen (expressed) in the phenotype. G/G, G/g

• The grey gene has a further mutation which causes melanoma .

• The grey mutation works on the cells in the neural crest of the developing embryo – well before cell differentiation (about day 12 after conception)

What does this all actually mean?

What does this all actually mean?

That what you see may just NOT be what you have actually got, and is this one reason why

many people colour test their ponies.

That what you see may just NOT be what you have actually got, and is this one reason why

many people colour test their ponies.

Black + Cream + GreyBlack + Cream + Grey

E+ a/a CCr G+

E+ a/a CCr G+

All three of these photographs are of the same pony

Black + Agouti = Bay or Brown + Cream + GreyBlack + Agouti = Bay or Brown + Cream + Grey

E+A+ CCr G+

Chestnut + Cream + GreyChestnut + Cream + Grey

ee (+/- Agouti) CCr G+

A grey pony can be any colour ‘under’ the grey.A grey pony can be any colour ‘under’ the grey.

Unless you bred the pony the only way to know for certain what is the birth coat colour of a grey is, is by colour testing.

Unless you bred the pony the only way to know for certain what is the birth coat colour of a grey is, is by colour testing.

A GG parent can ONLY produce grey progenyA GG parent can ONLY produce grey progenyGg/Gg matings have a 1:4 chance of producing a non grey foal.Gg/Gg matings have a 1:4 chance of producing a non grey foal.

This is why the progeny of grey to grey matings can surprise.This is why the progeny of grey to grey matings can surprise.

Like this surprisefrom two grey parents

Like this surprisefrom two grey parents

Colour Testing is a multi-step process

Colour Testing is a multi-step process

This work requires significant ‘bench time’ in the lab.

1. Preparation of the sample then DNA extraction using a thermocycler

2. Preparation of digest with the primers specific for each test, restriction enzymes and then PCR.

3. Where necessary, incubation maybe required

4. The product is then run on an electrophoresis gel

This work requires significant ‘bench time’ in the lab.

1. Preparation of the sample then DNA extraction using a thermocycler

2. Preparation of digest with the primers specific for each test, restriction enzymes and then PCR.

3. Where necessary, incubation maybe required

4. The product is then run on an electrophoresis gel

Electrophoresis

How were samples obtained?

How were samples obtained?

Some of the samples were collected at the same time as the blood samples for the Hoof Wall Separation Syndrome (HWSS) research at UC Davis. However there was also help from another source.

Some of the samples were collected at the same time as the blood samples for the Hoof Wall Separation Syndrome (HWSS) research at UC Davis. However there was also help from another source.

So what was looked at and what were the results?

So what was looked at and what were the results?

1. Relationship (if any) between birth coat colour and melanoma development.

2. Allele distribution within the New Zealand Connemara pony population

3. How this compares to that found in Ireland.

The Study PopulationThe Study Population

Of the 45 ponies in the study, nine ponies were imported from Australia since 2005. 13 ponies are

the progeny of parents imported from Australia since 2005. One pony is the product of frozen

semen from the UK. The balance of 22 ponies is derived from the original bloodlines which were imported to New Zealand from Australia, the UK

and Ireland in the 1970's and 80's.

Of the 45 ponies in the study, nine ponies were imported from Australia since 2005. 13 ponies are

the progeny of parents imported from Australia since 2005. One pony is the product of frozen

semen from the UK. The balance of 22 ponies is derived from the original bloodlines which were imported to New Zealand from Australia, the UK

and Ireland in the 1970's and 80's.

Of the estimated 88 mares considered to still be alive, 34 were enrolled into the study; therefore

this study covers 38.6% of the present estimated female Connemara pony population in this

country.

The number of stallions registered since 1970 stands at 20 of which nine have died, been gelded or exported, leaving 11 stallions standing at stud

at present. Of these 11 stallions, eight were enrolled in this study. Thus 72.7% of the present breeding stallions in this country are represented.

Of the estimated 88 mares considered to still be alive, 34 were enrolled into the study; therefore

this study covers 38.6% of the present estimated female Connemara pony population in this

country.

The number of stallions registered since 1970 stands at 20 of which nine have died, been gelded or exported, leaving 11 stallions standing at stud

at present. Of these 11 stallions, eight were enrolled in this study. Thus 72.7% of the present breeding stallions in this country are represented.

Bay Bucksk in Ches tnut Palom ino B lack0

5

10

15

Birth Coat Genotype of 45 Connemara Ponies in New Zealand

GreyNon-Grey

B a s e C o a t Ge n o typ e

Nu

mb

er

33/45 of the ponies were phenotypically grey; 73.3% of the study population. Of the 26.6% of non grey ponies

58.3% were bay, a third were bay dilutes (buckskin) with chestnut being the balance (8%)

The Grey phenotype in the New Zealand Connemara Pony

Population.

The Grey phenotype in the New Zealand Connemara Pony

Population.

In Ireland the prevalent colour in adult ponies is grey, with 70% of ponies exhibiting this phenotype. Followed by bay

(15%), dun (12%) [buckskin] with the remaining 3% comprised of brown, black, roan or chestnut.

77.7% of the population tested were either 'ee' (24.4% chestnut)

or carrying 'e' as a recessive

(53.3%).

77.7% of the population tested were either 'ee' (24.4% chestnut)

or carrying 'e' as a recessive

(53.3%).

Ee ee No e0

5

10

15

20

Distribution of the Chestnut ('e') Allele in a Population of Connemara ponies in New Zealand

GreyNon Grey

Alle le s

Nu

mb

er

The grey phenotype and melanoma

The grey phenotype and melanoma

1. Researchers in Sweden proposed that aggressive melanoma appeared to be associated with the true black genotype – E+ aa - homozygous or recessive for Agouti (carries no agouti allele).

1. Researchers in Sweden proposed that aggressive melanoma appeared to be associated with the true black genotype – E+ aa - homozygous or recessive for Agouti (carries no agouti allele).

2. Their study population was the Lipizzaner.2. Their study population was the Lipizzaner.

3. The chestnut allele is not present in the Lipizzaner breed.

3. The chestnut allele is not present in the Lipizzaner breed.

PROBLEM!!!!!PROBLEM!!!!!

How do you determine which genes might influence melanoma development if you do

not have them all??

Study another ‘grey breed’ which does have a full complement of colour alleles.

The results found in this study supports the work released by

University of MinnesotaJanuary 2013.

The results found in this study supports the work released by

University of MinnesotaJanuary 2013.

1. The extension locus E is not the determining factor in melanoma development.

1. The extension locus E is not the determining factor in melanoma development.

2. Agouti does appear to have a definite influence.2. Agouti does appear to have a definite influence.

3. Agouti changes black pigment to red. It may have functions in addition to colour change.

3. Agouti changes black pigment to red. It may have functions in addition to colour change.

4. Presence of Agouti appears to be beneficial in reducing the aggressiveness of melanoma development BUT

4. Presence of Agouti appears to be beneficial in reducing the aggressiveness of melanoma development BUT

It AppearsIt Appears

1. Homozygosity for grey is of the greatest influence in melanoma development.

1. Homozygosity for grey is of the greatest influence in melanoma development.

2. Multiple crossing of homozygous parents has an exponential effect on melanoma development.

2. Multiple crossing of homozygous parents has an exponential effect on melanoma development.

3. ‘aa’ increases the risk of melanoma development.3. ‘aa’ increases the risk of melanoma development.

B lack (EE /Ee aa)Ches tnut (ee/aa)

Ches tnut (ee/Aa)Ches tnut (ee/AA)

B lack (E+ /A a)B lack (E+ /A A)

0

2

4

6

8

10

12

Agouti status, Base Coat Colour and Reported Inc idence of Melanoma in a P opulation of Grey P henotype C onnem ara P onies in New Zealand

M elanom a +veM elanom a -ve

Ag o u ti S ta tu s a n d Ba s e C o a t C o lo u r

Num

ber

Take Home MessageTake Home Message

1. Homozygous greys have a higher incidence of melanoma.

2. AA ponies can NEVER produce a true black foal.

4. Smokey blacks (when they do not go grey) look brown (often a seal brown)

5. Buckskins can sometimes look bay.

3. EE ponies can NEVER produce a chestnut foal.