Mapping of the silver coat colour locus.pdf

Institutionen för husdjursgenetik

Mapping of the silver coat colour locus

in the horse

Emma Brunberg

Supervisors:

Gabriella Lindgren, UU Examensarbete 279

Sofia Mikko, SLU 2006

Examensarbete ingår som en obligatorisk del i utbildningen och syftar till att under handledning ge de

studerande träning i att självständigt och på ett vetenskapligt sätt lösa en uppgift. Föreliggande uppsats

är således ett elevarbete och dess innehåll, resultat och slutsatser bör bedömas mot denna bakgrund.

Examensarbete på D-nivå i ämnet husdjursgenetik, 20 p (30 ECTS).

Institutionen för husdjursgenetik

Mapping of the silver coat colour locus

in the horse

Emma Brunberg

Agrovoc: Horse coat colour, silver dapple, PMEL17, genetics

Supervisors:

Gabriella Lindgren, UU Examensarbete 279

Sofia Mikko, SLU 2006

Examensarbete ingår som en obligatorisk del i utbildningen och syftar till att under handledning ge de

studerande träning i att självständigt och på ett vetenskapligt sätt lösa en uppgift. Föreliggande uppsats är således

ett elevarbete och dess innehåll, resultat och slutsatser bör bedömas mot denna bakgrund. Examensarbete på D-

nivå i ämnet husdjursgenetik, 20 p (30 ECTS).

Contents

bstract

Introduction

The silver dapple colour

Linkage analysis

SILV as a candidate gene

The present thesis

Materials and methods

Genotyping of genomic DNA using microsatellite markers

Linkage analysis

Sequencing of SILV

SNP analysis using pyrosequencing

Results

Genetic markers and genotyping

Linkage analysis

Polymorphism discovery by DNA sequencing

Association of DNA polymorphism and the silver phenotype

iscussion

Acknowledgements

References

Pictures

Figures

Tables

Abstract

The silver dappled colour in horses is controlled by a dominant allele that dilutes the black

pigment eumelanin. A black or brown horse that carries the allele becomes diluted in mainly

mane and tail, while the hair of the body remains dark. The silver dapple colour is common in

the Icelandic horse population and has also been observed in for example Shetland pony,

Norwegian nordland, Rocky Mountain pony and Ardenne.

The purpose of this project has been to try to map and characterise the silver dapple coat

colour locus in the horse genome. This has been done by performing a linkage analysis with

markers throughout the genome and a marker near a candidate gene; SILV at Equus Caballus

chromosome 6 (ECA6). SILV is coding for a protein called Pmel17 that is involved in the

production of eumelanin. The gene has been shown to control colour-diluting processes in

mouse, chicken and dog. A half-sib family consisting of one Icelandic stallion, 34 of his

offspring and 29 of their mothers were used (five offspring shared mother with another

offspring). Seventeen of the offspring had the silver colour. Significant linkage was found

between the silver phenotype and the marker TKY284 near the SILV gene. The six last exons

and introns were sequenced and SNPs were found in intron 9/10 and in exon 11. The

nucleotide substitution in exon 11 changes the second amino acid in the cytoplasmic region

from an arginine to a cysteine and the same mutation is found in one type of hypopigmented

chicken. Twenty-five silver horses from three breeds and 55 non-silver individuals were

genotyped for the mutation and this showed a full association with the phenotype. These data

shows that SILV probably is responsible for the silver dapple phenotype in horses. The

cytoplasmic region of the protein is well conserved among vertebrates. The remaining parts of

the SILV gene will be sequenced to search for more mutations and the mutation in exon 11

will be transfected into cell-lines to see how it affects the protein function. Of the pigment

genes that earlier have been cloned, a majority is associated with human hereditary pigment

diseases. Identifying genes and mutations for a pigmentary disease or a pigment phenotype

can increase the understanding of the function of the protein and the molecular mechanisms

behind the process of pigmentation and pigmentary changes.

Introduction

Coat colour is a trait with a great variation among our domesticated animals. Breeders and

animal owners have always been affected by and paid attention to coat colour variation. The

colours have been surrounded by rumours that a certain quality is associated with a colour and

these animals can be more or less economically valuable. The knowledge of how certain

genes and their gene products affect coat colour is of great interest for basic research. In

mouse, around 125 known loci are known to affect pigmentation if mutated. Some coat

colours are associated with diseases, for which it is necessary to know the underlying

functions of the coat colour gene to be able to treat these pigmentary disorders. Some coat

colours are difficult to distinguish from others and molecular genetics can provide DNA-tests

for these colours.

Melanin is the pigment that is essential for the colour of skin, hair and eyes. The

pigment is synthesised by melanocytes, which are cells originating from the neural crest

(Passeron et al., 2005). The pigment is synthesised in special organelles, the melanosomes.

Melanins are derivates of L-DOPA that are derived from α-tyrosine or α-phenylalanin, a

reaction that can be catalysed by an integral membrane melanosome constituent, tyrosinase

(Slominski et al., 2004). There are two types of melanin, eumelanin and pheomelanin. The

two reactions that produce the two pigment types are different. The production of

pheomelanins (red pigment) involves reduction reactions and the production of eumelanins or

black pigment, involves oxidation and hydroxylation reactions that will result in reactive

indoles. If these indoles are exposed to components in the cells they could be deleterious and

that is why the reactions require melanosomes (Raposo and Marks, 2002).

The silver dapple coat colour

In horses there are three basic colours; Black, bay and chestnut: The bay and chestnut horses

can vary from relatively light to darker shades of the same colour. For the black colour, the

horse has to have the E-allele at the extension locus. This is a dominant allele and therefore all

E- -horses will be able to produce black pigment (Furugren, 2002). Horses that are black or

bay as a basic colour will be E- at the extension locus (Furugren, 2002). Another locus, A

(agouti), will control the distribution of black and red areas on horses that can produce black

pigment and can restrict the black pigment to certain parts of the body (Sponenberg, 2003).

This means that a black horse will carry the recessive alleles aa and a brown or bay horse will

be A- at the Agouti locus . A chestnut can carry any allele at this locus, but will still show a

real phenotype because of the lack of black pigment (i.e. a chestnut horse will always be ee at

the extension locus) (Furugren, 2002).

Many of the colour variants that derive from the basic colours are diluted. Those colours

often fascinate breeders and are often very popular. There are four categories of the diluted

colours: linebacked duns, cream-related colours, champagne and silver dapple (Sponenberg,

2003). The silver dappled colour in horses is controlled by a dominant allele that dilutes the

black pigment eumelanin. A black or brown horse that carries the allele becomes diluted in

mainly mane and tail, while the hair of the body remains darker. The genetically black horses

are diluted to dark brown or almost black colour with silver grey or white mane and tail. The

genetically bay or brown individuals are diluted to a lighter brown or almost chestnut-like

colour with silver grey or white mane and tail. The silver brown individuals can be hard to

distinguish from a chestnut horse with flaxen mane and tail, but it often has a darker shade on

the legs (Bowling, 1996) and lighter eyelashes (See Picture 1 and 2). In some countries and

some breeds one distinguishes between a large variety of silver variants that are believed to

depend on the basic colour of the horse. For example, the bay individuals are thought to be the

ones that gives the typical “red silvers” while the brown silvers are believed to have a darker

brown shade as a basic colour (Sponenberg, 2003). Chestnut horses can carry the silver allele

and inherit it to the offspring, but will not be affected in colour because the gene only affects

the black pigment. This means that the silver allele will only change the phenotype on E- -

individuals. The silver allele is assumed to be fully dominant, i.e. silver heterozygotes and

homozygotes are indistinguishable (Furugren, 2002).

The silver dapple colour is common in the Icelandic horse population and has also been

observed in for example Shetland pony, Norwegian nordland, Rocky Mountain pony and

Ardenne. The reason for the presence of the silver coat colour in Icelandic horse, Shetland

pony, Norwegian Nordland and Rocky Mountain is probably due to connections between

Norway, Iceland and Great Britain during the colonisation of Iceland. The silver locus in the

Swedish Ardenne horse comes from Belgium and therefore it is possible that the mutation

causing the silver colour has arised more than once (Furugren, 2002). The colour has also

been registered in Mountain Pleasure Horse, Kentucky Mountain Saddle Horse and Arabians.

Silver dappled horses could also be present in several other breeds, but are likely to be

inaccurate identified and therefore not recorded (Sponenberg, 2003).

In some breeds, several silver horses have ocular abnormalities, varying from minimal to

quite severe eye defects. The defect is not properly documented but some researchers believe

that it is a part of the gene action at this locus and that homozygotes are more severely

affected than the heterozygotes (Sponenberg, 2003). In many breeds, however, there is no

problem with eye defects among the silver dappled individuals. The ocular defect could

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