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Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 428
Genetic Studies of
Pigmentation in Chicken
ULRIKA GUNNARSSON
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2009
ISSN 1651-6206
ISBN 978-91-554-7439-3
urn:nbn:se:uu:diva-98426
Dissertation presented at Uppsala University to be publicly examined in B42, BMC,
Husargatan 3 , Uppsala, Friday, April 3, 2009 at 09:15 for the degree of Doctor of Philosophy
(Faculty of Medicine). The examination will be conducted in English.
Abstract
Gunnarsson, U. 2009. Genetic Studies of Pigmentation in Chicken. Acta Universitatis
Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of
Medicine 428. 44 pp. Uppsala. 978-91-554-7439-3.
Domestic animals have been selected by humans for thousands of years, which have drastically
altered their genetic constitution and phenotypes. In this thesis, several of the most important
genes causing pigmentation differences between the wild red junglefowl (Gallus gallus) and
domestic chickens have been identified. Pigmentation phenotypes are easily scored, and the
genes underlying these phenotypes are valuable models to study gene function and gene
interaction.
Dominant white colour is widespread among domestic chickens. The Dominant white allele
specifically inhibits the expression of black (eumelanin) pigment and we identified several
insertion/deletion mutations in the PMEL17 gene causing the different phenotypes controlled
by this locus. The Silver allele on the other hand inhibits the expression of red (pheomelanin)
colour and is a genetic variant of the SLC45A2 gene. Silver is the first pheomelanin-specific
mutation(s) reported for this gene. An 8 kb deletion, including a conserved enhancer element,
14 kb upstream of the transcription factor SOX10 is causing the Dark brown phenotype. This
phenotype restricts the expression of eumelanin and enhances red pheomelanin in specific parts
of the plumage. These three gene identifications have extended the knowledge about genes
affecting melanocyte function.
Carotenoid-based pigmentation is of utmost importance in birds and other animals. The
yellow skin allele in chicken allows deposition of carotenoids in skin and explains why most
domestic chickens have yellow legs. We demonstrated that the yellow skin phenotype is caused
by a tissue specific regulatory mutation in the gene for the enzyme beta-caroten dioxygenase
2 (BCDO2). This was the first identification of a specific gene underlying carotenoid-based
pigmentation. Interestingly, the yellow skin haplotype was shown to originate from the grey
junglefowl (Gallus sonneratii) and not the red junglefowl as expected, thus presenting the first
conclusive evidence for a hybrid origin of the domestic chicken.
Keywords: chicken, pigmentation, eumelanin, pheomelanin, carotenoids, Dominant white,
PMEL17, Silver, SLC45A2/MATP, Dark brown, SOX10, yellow skin, BCDO2/CMO2,
domestication
Ulrika Gunnarsson, Department of Medical Biochemistry and Microbiology, Box 582,
Uppsala University, SE-75123 Uppsala, Sweden
© Ulrika Gunnarsson 2009
ISSN 1651-6206
ISBN 978-91-554-7439-3
urn:nbn:se:uu:diva-98426 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-98426)
Till min familj
List of papers
This thesis is based on the following papers, which are referred to in the text
by their roman numerals.
I.
Kerje S, Sharma P, Gunnarsson U, Kim H, Bagchi S,
Fredriksson R, Schütz K, Jensen P, von Heijne G,
Okimoto R, Andersson L. 2004. The Dominant white,
Dun and Smoky Color Variants in Chicken Are Associated With Insertion/Deletion Polymorphisms in the
PMEL17 Gene. Genetics. 168, 1507-1518.
II.
Gunnarsson U*, Hellström AR*, Tixier-Boichard M,
Minvielle F, Bed'hom B, Ito S, Jensen P, Rattink A,
Vereijken A, Andersson L. 2007. Mutations in
SLC45A2 Cause Plumage Color Variation in Chicken
and Japanese Quail. Genetics. 175, 867-77.
III.
Gunnarsson U, Kerje S, Bed'hom B, Sahlqvist A-S,
Ekwall O, Tixier-Boichard M, Kämpe O, Andersson
L. 2009. The Plumage Colour Dark Brown in
Chicken is Caused by an 8 kb Deletion Upstream of
SOX10. Manuscript.
IV.
Eriksson J, Larson G, Gunnarsson U, Bed'hom B,
Tixier-Boichard M, Strömstedt L, Wright D,
Jungerius A, Vereijken A, Randi E, Jensen P,
Andersson L. 2008. Identification of the Yellow Skin
Gene Reveals a Hybrid Origin of the Domestic
Chicken. PLoS Genet. 4(2):e1000010.
*These authors contributed equally to the work
Papers I, II and IV are reproduced by the permission of the journal concerned.
Contents
Introduction.....................................................................................................9
Genetic and genomic studies of phenotypic traits....................................10
Genetic variation..................................................................................10
Linkage analysis ..................................................................................11
Pedigrees..............................................................................................11
Positional cloning and identification of the causative mutation ..........12
Expression studies ...............................................................................13
Phylogenetic trees................................................................................13
Comparative genomics ........................................................................14
Domestic birds as model organisms.........................................................14
Domestication of the chicken ..............................................................14
Chicken as a model organism and the chicken genome ......................15
Japanese quail ......................................................................................16
The development of pigmentation............................................................17
Melanocyte development.....................................................................17
Eumelanosomes and pheomelanosomes..............................................18
Carotenoid pigment .............................................................................20
Present investigations....................................................................................22
Aims of this thesis ....................................................................................22
Background ..............................................................................................22
Pedigrees and animals..........................................................................22
Loci under investigation ......................................................................23
Results and Discussion.............................................................................25
Paper I..................................................................................................25
Paper II ................................................................................................28
Paper III ...............................................................................................30
Paper IV...............................................................................................31
Future prospects ............................................................................................33
Summary in Swedish ....................................................................................35
Genetiska studier av hönsens färgteckning ..............................................35
Acknowledgements.......................................................................................37
References.....................................................................................................40
Introduction
This year (2009) it is 200 years since the birth of Charles Darwin and 150
years since he published his famous book “The origin of species” or as it
was originally named “On the origin of species by means of natural selection”1. Darwin introduced the idea that all living organisms have evolved by
the course of natural selection, and he used domestic plants and animals as
examples of this, thereby questioning the ideas of the time that all domestic
races had possessed their own wild prototype1. The theories by Charles Darwin have been greatly questioned and even if the acceptance for his evolutionary theory is higher today there are still many non-believers in the world.
In a Gallup poll from 2008, only around 15% of the people in the United
States believed in evolution and that humans have developed over millions
of years2, 3. The evolutionary theory is much more accepted in Sweden and
other Nordic countries2.
Gregor Mendel (1822-1884) is the founder of genetic science, and ever
since Mendel’s pioneering studies of the mechanism behind inheritance in
peas, colour phenotypes have been studied4. It is now more than 100 years
since Mendel’s findings were rediscovered and the first studies of plumage
colour inheritance in chicken were performed5.
There are several advantages working with domestic animals when investigating phenotypic variation compared to more commonly used model organisms such as mice and rats. Domestic animals have been selected by humans for thousands of years and thus represent an abundant collection of
mutations that affect their phenotypic traits6, 7. Some of these traits are inherited according to Mendelian principles and caused by variation at a single
locus, i.e. monogenic traits. Examples of monogenic traits are for instance
the above mentioned colour phenotypes and simple familial heritable disorders. Other phenotypes are more complex and influenced by alleles at many
loci as well as environmental factors. These phenotypes are called polygenic,
complex or quantitative. Examples of complex traits are weight and behaviour.
In this thesis, phenotypic variation in plumage colour has been studied
with chicken as the main model organism. It has been shown that colour loci
in domestic animals have been selected for non-camouflaged patterns by
humans, with the purpose of making it easier for the early animal farmers to
keep control of their animals8. Colour phenotypes must also have been selected just because of the thrill of having something novel. With time some
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of these skin and plumage colours have become of economic significance to
the poultry industry. Different communities prefer different colours of the
skin of their chicken, and some plumage colours are used to determine the
sex of day old chicks for layer production9.
For a molecular geneticist the reason for studying pigmentation genetics
is the considerable significance it can have in understanding gene function
and gene interaction. Since some colour phenotypes are results of regulatory
mutations, these studies can shed light on transcriptional regulation. Much
more is to be learnt about the genetics underlying pigmentation and melanocyte development. In a broader perspective, studies like these could be of
importance for both human and veterinary medicine. As an example, the
Grey allele in horses predisposes for melanoma and the genes found to cause
the Grey phenotype are obvious candidates for human studies10.
Genetic and genomic studies of phenotypic traits
Genetic variation
In 1953 Watson and Crick published the structure of the DNA (deoxyribonucleic acid) double helix11. The building-blocks of DNA are the four nucleotides A (adenine), T (thymine), G (guanine) and C (cytosine). Triplets of
these four nucleotides constitute the genetic code that is a feature shared by
all living organisms. The size of the human genome is approximately 3 Gb
(3,000,000,000 bp) and a single bp variation at the wrong position in the
genome can be deleterious for the individual. Despite this fact, genetic variation is high and there is a lot of non-deleterious variation to be found. This
variation is used as a tool by geneticists to determine the degree of genetic
variation between individuals and classify them into groups depending on
their DNA sequence.
The most common (and commonly used) type of genetic variation is
SNPs (single nucleotide polymorphisms). SNPs are polymorphic nucleotides
in the DNA sequence, for example an A to G change. Other common variations are short insertion/deletions (indels) of a few bp, and polymorphic di-,
tri- and tetra-repeats (such as (AG)n) called microsatellites. CNVs (copy
number variations) are longer (>1 kb) segments of DNA variation, such as
insertion/deletions or duplications.
Today there are many methods for SNP scoring and the method of choice
depends on the number of markers used. When performing linkage studies, a
large number of SNPs are required. Up until a few years ago, microsatellites
were the genetic marker type chosen for this type of studies, but the fast
development of technology for cost effective and accurate typing of hundreds to thousands of SNPs has quickly made microsatellite typing a method
of the past. Today the cost for resequencing parts of or whole genomes is
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rapidly decreasing, and perhaps will we in a few years time also see SNP
typing as a historical method?
A phenotype is any observable trait of an individual and in this thesis
monogenic colour phenotypes have been studied. During sexual reproduction one haploid set of chromosomes is inherited from each parent. At each
polymorphic site in the genome the resulting individual can either become
homozygous or heterozygous.
Alleles causing a distinct phenotype can be acting in a recessive, dominant or co-dominant fashion. For a recessive trait locus, two copies of the
mutant allele are required to express the phenotype. For a dominant phenotype a single mutant allele inherited from one of the parents is sufficient.
Some traits show co-dominance, with the heterozygous individuals being an
intermediate between the two homozygous phenotypes.
When the phenotype is determined by a locus on the sex chromosomes
the pattern of inheritance depends on the sex of the individual. The heterogametic sex (the one having two different sex chromosomes, male humans are XY, female birds are ZW) will always express the phenotype associated with an allele regardless of its recessive or dominant nature since the
heterogametic sex only inherited one X (or Z) chromosome.
Linkage analysis
Linkage mapping can be used for the identification of chromosomal regions
harbouring a gene/genes underlying a certain phenotype. An informative
pedigree material and polymorphic genetic markers are required for linkage
analysis. When two markers show a tendency of co-segregation they are in
linkage with each other. A LOD (logarithm of the odds) score gives the odds
of linkage for a marker to the phenotype investigated, and is calculated as
the log (base 10) of the odds that two investigated loci are linked rather than
unlinked. A LOD score of 3 is usually considered as significant evidence of
linkage (odds in favour of linkage 1000:1). The recombination fraction (Θ)
is the number of recombinants divided by the total number of informative
meioses and ranges from Θ = 0 for loci that show complete linkage to a
maximum of Θ = 0.5 for loci showing independent segregation. A recombination fraction of 0.5 mean either that the loci are far apart on the same
chromosome, or that they are located on different chromosomes. The map
distance between two loci is given in centi-Morgan (cM). One cM is defined
as 1% recombination between two loci12.
Pedigrees
To study the inheritance of a phenotype, a family material or pedigree is
required. In human genetics several different families with the same disorder
is usually vital to obtain a significant LOD score and thereby linking the
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disease to a specific chromosomal region. When working with model organisms many more offspring can be generated (up to thousands if required). To
initiate a pedigree, parental animals (P) with different phenotypes are
crossed to generate a heterozygous F1 generation. These F1 individuals can
then be intercrossed to generate an F2 generation segregating for all loci explaining phenotypic differences between the parental populations. When
chromosomes are inherited from the F1 to F2 generation, recombination between the parental chromosomes occurs. The power of performing linkage
analysis in large F2 populations is seen by the highly significant LOD scores
generated. As an example the linkage between the Dominant white (I) locus
and the PMEL17 gene studied in paper I resulted in a LOD score of 107.2!
Another way to generate a pedigree is to backcross the F1 animals to one of
the parental populations.
Positional cloning and identification of the causative mutation
After linking the phenotype of interest to a chromosomal region the next step
is to narrow down the region and identify candidate genes and the causative
mutation. In fine-mapping experiments, more genetic markers are added in
the region of interest. The resolution of the region depends on the recombination frequency in the region and the size of the pedigree used for mapping.
When the region has been reduced to its limit by linkage analysis using a
pedigree material, no more recombination events can be found between the
closest markers and the investigated trait locus. The next step is to find the
minimum shared haplotype that is identical by descent (IBD) between individuals carrying the same ancestral mutation causing the phenotype. In an
experimental cross, intercrossing the F2 animals to generate subsequent generations, with recombination occurring in every generation, can generate
such a material. Another approach is to genotype or sequence animals from
other breeds sharing the phenotype of interest, under the assumption that
they carry the same causative mutation.
In paper III of this thesis we sequenced animals with different alleles at
the studied locus. This resulted in an IBD region among the animals with the
mutated Dark brown phenotype, but also gave us the causative mutation
since one of the wild-type individuals carried the ancestral haplotype. To
sequence animals from many different breeds with and without a causative
mutation has shown to be useful before. By sequencing several animals with
and without a QTL (quantitative trait locus) allele affecting muscle growth,
Van Laere et al. 2003 found a regulatory nucleotide substitution to be the
causative mutation, QTN (quantitative trait nucleotide) for a major QTL in
pigs13.
If there are many possible causative mutations in an IBD region several
web-based tools can be used to find candidate genes as well as regulatory
regions. Thanks to the many sequenced genomes, information about genes,
12
conservation and variation between species (and individuals), and much
more can be accessed with the help of several web browsers, for instance the
UCSC browser (www.genome.ucsc.edu/). To be functional, a causative mutation within a gene usually changes a well conserved amino acid, deletes an
exon(s) or generates a stop codon. Within regulatory regions functional mutations can be of several types14; single base pair mutations or insertion/deletions resulting in gain or loss of binding sites, insertion/deletions
can also affect the copy number of a given regulatory site. Regional duplications can result in novel regulatory regions and translocations can bring
genes into the near vicinity of new regulatory domains14. Sometimes new
regulatory target sites can be induced by a single base pair change in what
seems to be “non-functional DNA”. In the study of a QTL underlying muscle mass in sheep, a single base pair mutation in a non-conserved region was
shown to create a microRNA target site, which resulted in a significant phenotypic effect15.
Expression studies
If the phenotype is supposed to be caused by a regulatory mutation there are
many ways to analyze this further. An analysis of samples from individuals
with different phenotypes can show an expression difference at the mRNA
level by various methods. Some of the most commonly used methods are
Northern blot, qPCR or in situ hybridization.
In paper IV of this thesis a complement to these methods was used. The
pyrosequencing method was here used to quantify the expression of the
BCDO2 mRNA (cDNA) in liver and skin from individuals heterozygous for
a SNP in the BCDO2 gene. This is an efficient method to quantify the relative expression of a wild-type and mutant allele. Another useful method to
study the difference in expression is to perform direct sequencing on genomic DNA and cDNA from individuals heterozygous for the studied mutation, as seen in paper II of this thesis and also in the study of the Grey locus
in horses10.
Phylogenetic trees
A phylogenetic (evolutionary) tree attempts to reveal how different species
(or breeds) are related to each other. In 1859, Charles Darwin presented the
first “diagram” (tree) attempting to explain this1. To root a tree an out-group
is used. This is usually a species that is clearly more distantly related to the
species under analysis. To draw correct conclusions from a phylogenetic tree
analysis the input data must be well designed. For instance, hybridization
between species can confuse the output. To show a consistent result of high
quality, sequences from several regions of the genome (or whole genomes)
should be used. By studying sequences from different parts of the chicken
13
genome, paper IV shows that different parts of the domestic chicken genome
descend from at least two different species of wild junglefowls.
Comparative genomics
Comparative genomics is the study of sequence and genome similarities
found between species. Major methodological advances in sequencing technology and genome assembly during the last years have resulted in a rapid
increase of sequenced genomes. Comparison of genome sequences from
species that are closely or distantly related can answer different questions.
By comparing as distantly related species as human and chicken, conserved
elements important for essential biological functions can be identified. These
sequences called MSCs16 (multispecies conserved sequences) include both
genes and non-coding functional elements that are involved in controlling
gene expression. When comparing closely related species much more of the
sequence will be conserved and it can be difficult to find the functional elements specific for their short branch. To identify for example primate specific sequences numerous different species would be needed17. In conclusion, the shorter the branch length, the more species are required to be sequenced to find the MSCs specific for that branch, but to find some of the
MSCs important for basic vertebrate functions just a few sequences from
more distantly related species are required. The field of comparative genomics is rapidly growing and studies of functional non-coding elements will
probably be the next big advance in the understanding of biological functions and causes for disease.
A simple way to access information on conservation between species is to
use the publicly available genome browsers, for instance the UCSC Genome
Browser (www.genome.ucsc.edu).
The comparison of genome sequences within a species can be used to find
the functional variants involved in disease, but comparison to genome sequences from other species may be required to assess their biological significance.
In paper III of this thesis an element conserved between chicken, mouse
and rat was found to be deleted, causing the Dark brown phenotype.
Domestic birds as model organisms
Domestication of the chicken
Domestic chickens belong to the genus Gallus that includes four (sub) species; the red junglefowl (G. gallus), the Ceylon junglefowl (G. lafayettii), the
grey junglefowl (G. sonneratii) and the green junglefowl (G. varius).
14
Today the chicken is primarily used as a meat and egg producer but the
history of chicken goes back a long time, and initially the domesticated
chickens were used for religious purposes and cockfighting18. The red junglefowl was domesticated several thousand years ago and has by many been
thought to be the sole ancestor to the domestic chicken. Hutt19 wrote in 1949;
“…constant repetition of the familiar statement that all domestic fowls are
descended from the Red Jungle Fowl of India has apparently led some writers
to consider the question settled.”
In 1996 Fumihito et al. studied part of the mitochondrial (mt) DNA from
different junglefowl species and concluded a monophyletic origin of the
domestic chicken20. Nishibori et al. (2005) questioned this conclusion when
examination of the mtDNA and two segments of the nuclear genome from
the different species of junglefowl indicated interspecies hybridization between red and grey junglefowls, and between grey and Ceylon junglefowls21.
In his book from 1949, Hutt19 discussed the possibility that some of the
colour phenotypes seen in domestic chickens might have been inherited from
some other species than the red junglefowl, thus questioning the monophyletic origin of the domestic chicken. In paper IV we show that the yellow
skin (W*Y) allele originates from the grey junglefowl, confirming the hypothesis of Hutt.
Chicken as a model organism and the chicken genome
Chickens have been used as model animals to answer questions about development for thousands of years22. By opening eggs of chicken and studying
the progression of development at different stages, the Greek philosopher
Aristotle (384 BC-322 BC) funded the theory of epigenesis (the development from a simple to a more complex organism). These types of studies are
still an advantage with using chicken as a model animal in comparison to
mouse. The chicken embryos develop outside the body of the mother and are
therefore easily accessible, studied and manipulated.
Studies in chickens have among other things been important for studies of
cell migration. The neural crest (NC), that generate associations between
many different tissues and organs in the vertebrate body, is one of the structures of the embryo that has been studied in chicken during the last century,
to a large extent thanks to the quail-chick chimera system23. In these chimeras the heterochromatin is differently distributed in the nucleus depending on
its ancestry and can be stained by different methods to study the actions and
fate of the grafted cells. Studies in chicken have also contributed to various
other fields, for example immunology, virology, cancer and genetics22.
Genetic studies of chickens have been carried out for more than 100 years
and plumage colour in chicken was one of the first traits examined after
15
Mendel’s initial studies. The Dominant white (I) phenotype in chicken was
investigated already in 1902 and in the following years many more colour
phenotypes were studied, for example Silver (S) in 19125, 24.
The size of the chicken genome (1 x 109 bp) is approximately one third of
the human genome. Chickens have 38 pairs of autosomes and one pair of sex
chromosomes. Most bird karyotypes (including chicken) have chromosomes
of remarkably different lengths, referred to as macro- and microchromosomes. Females are the heterogametic sex (Z/W) while males are homogametic (Z/Z)25.
In 2004, the chicken genome sequence was published. It was a 6.6 x coverage draft sequence from a female red junglefowl25. In addition to the red
junglefowl genome sequence, 0.25 x coverage of the genome was also generated from a broiler, a layer and a silky26 (the sequenced layer was the hen
Agda from the SLU13 line, Uppsala27). From this study, 2.8 million SNPs in
the chicken genome were identified with an average rate of about five SNPs
per kb. The chicken recombination rate is high. In this investigation it was
found to be 2.5-21 cM per Mb, with a higher recombination rate on microchromosomes, compared with ~1cM per Mb in humans25.
Japanese quail
Japanese quail (Coturnix japonica) was domesticated around 1000 years
ago. It was first used as a songbird but the popularity of the bird for this
characteristic was reduced after World War II28. Since domestication it has
also been used for meat and egg production but never to the same extent as
the chicken.
Later it also became popular as a laboratory animal. Japanese quail is a
suitable experimental animal due to its small body size and short generation
time (four months). The quails are also easy to handle and it is possible to
keep many birds in a relatively small space29. The disadvantage is that there
are few breeds (varieties) of Japanese quail and the number of reported mutations in quail is small compared to chicken28, 29. Minimal selection has been
carried out in quail, resulting in much less focus on the bird itself with regard
to phenotypic anomalies and studies of mutants. Japanese quail is also very
susceptible to inbreeding depression28.
The plumage of wild-type adult Japanese quail is brown in variable
shades, including parts of other colours ranging from black to creamy
white28. Around 20-30 different colour mutants have been found in Japanese
quail, but only a few of them are available today. Some of the mutants reported in different studies are also alleles at the same locus and the same
mutant phenotype has occurred several times28, 30. In paper II of this thesis
two of these phenotypes were studied.
16
The development of pigmentation
The field of pigmentation biology is extensive. This summary will introduce
the genes and pathways relevant for this thesis.
Melanocyte development
Melanocytes (pigment cells) originate from the neural crest. The neural crest
consists of a group of multipotent embryonic cells that initially can be found
at the dorsal side of the neural tube31. After widespread migration in the developing embryo many different cell types (melanocytes, neurons and glial
cells, endocrine cells and mesenchymal cells) are derived from the neural
crest31. Many proteins and pathways are involved in melanocyte specification. Transcription factors such as TCF/LEF work together with PAX3 and
SOX10 to express the microphthalmia-associated transcription factor
(MITF) that has a crucial role in melanocyte development32-34. Other pathways involved in the melanocyte development can also regulate MITF expression33. MITF itself regulates the expression of many genes important for
melanocytes, among them TYR, TYRP1, TYRP2 (DCT), PMEL17, SLC45A2
(MATP, AIM-1) and MC1R33. An interspecies difference between mice and
zebrafish in the regulation of the melanocyte development has been seen.
The expression of TYR and DCT has in addition to the regulation by MITF,
been shown to be directly regulated by SOX10 in mice, but in zebrafish
there is a simple regulatory chain with SOX10 regulating MITF that in turn
regulates the downstream targets33, 35-37 (Figure 1).
Figure 1. A simplified illustration of the factors that work together to regulate the
expression of MITF and some of the genes that are under MITF regulation. The
illustration also shows the direct regulation of DCT and TYR by the SOX10 transcription factor that has been documented in mouse. (Figure modified from Goding
200032 and Hou and Pavan 200833).
17
SOX10
The SOX10 gene encodes a transcription factor belonging to the SOX (Sryrelated HMG box) family group E. The SOX proteins control a variety of
developmental processes including the melanocyte formation during neural
crest specification31, 34, 38. In 1998, a single base insertion resulting in a translation frameshift of SOX10 was found to be the causative mutation in the
Dom mice39, 40. Dom/Dom homozygous mice are embryonic lethal and heterozygous individuals have white spotting and defects in the colon41. Another mouse phenotype, the Hry mouse is associated with a 15.9 kb deletion
of highly conserved sequences upstream of SOX10. The homozygous Hry
mouse shows a loss of melanocytes and a constriction of the colon
(megacolon) resembling the Waardenburg-Shah syndrome (WS4) in humans42. A later study confirmed that this highly conserved region contains an
important cis-regulatory element for SOX10 expression during melanocyte
development43. The homologous element in chicken is studied in paper III.
Eumelanosomes and pheomelanosomes
Melanosomes are lysosome-related organelle structures located within
melanocytes (Figure 2). The early melanosome has been suggested to originate from unstructured and round vesicles appearing from the endoplasmic
reticulum (ER). This stage I melanosome is a vesicle which evolves to a
stage II (eu)melanosome (premelanosome) by developing into a fibrillar,
tyrosinase-positive organelle. Synthesis of melanin begins as soon as the
fibrillar matrix has been produced, and pigment is deposited on those fibrils
(now stage III (eu)melanosome). In highly pigmented cells the deposition of
melanin proceeds until the (eu)melanosome is packed and no fibre structure
is observable (stage IV (eu)melanosome)44, 45. The round pheomelanin
premelanosomes are less structured than the oval eumelanin premelanosomes and contain less melanin46 (Figure 2). Melanosomes are used as models for understanding the morphogenesis of lysosome-related organelles and
may help in understanding the etiology of disorders linked with lysosomerelated organelles47.
Pigmentation in birds and mammals results to a large extent from the
deposition and assembly of two different types of melanin, (brown-to-black)
eumelanin and (yellow-to-reddish-brown) pheomelanin. The melanin biosynthesis occurs in the melanosomes, and the rate-limiting enzyme of this
synthesis is tyrosinase (TYR). Tyrosinase and the tyrosinase-related proteins
TYRP1 and TYRP2 (DCT) are involved in the production of eumelanin. For
the assembly of pheomelanin, only cysteine (or glutathione) and some tyrosinase activity seem to be critical. When tyrosinase is expressed at low
levels, pheomelanin is produced by the addition of cysteine to dopaquinone
18
44, 48, 49
. Simplified, high tyrosinase activity results in eumelanin production
whereas low activity results in generation of pheomelanin.
Figure 2. A schematic drawing of the development of pheomelanosomes and eumelanosomes within the melanocyte. The round pheomelanin premelanosomes contain
less melanin and are less structured than the oval pre-eumelanosomes (stage II and
III). In the stage II and III premelanosomes developing into eumelanosomes the
PMEL17 protein results in a fibrillar matrix on which the eumelanin pigment can be
deposited in an organized fashion until no fibre structure is observable (stage IV).
High tyrosinase activity (fat arrow) together with TYRP1 and DCT results in eumelanin and low tyrosinase activity and some cysteine results in pheomelanin. MATP
is believed to direct TYR, TYRP1 and DCT from the trans-Golgi-network to stage II
premelanosomes. (Figure modified from Hearing 200545)
PMEL17
PMEL17 (pre-melanosomal protein 17) is an integral membrane protein,
also known as gp100, SILV and MMP115. Transcription of PMEL17 is
regulated by MITF50. PMEL17 is sufficient to drive the formation of the
fibrils found in the premelanosome and therefore is an essential component
of premelanosome biogenesis51. Critical for the development of these fibrils
is the cleavage of PMEL17 by a furin-like proprotein convertase47. PMEL17
polymerizes into fibrillar arrays (the eumelanosome backbone) upon which
the melanin pigment is assembled52(Figure 2). These arrays have been found
to be the first functional amyloid structures in nature and should be referred
to as amyloidin53.
The first mutation found in Pmel17 is causative for a recessive phenotype
first described in a black mouse strain that became progressively lighter
(more silvered) with age54. The silver (si) mice were found to have a G to A
transition that produce a nonsense mutation in the sequence encoding the C-
19
terminus of the protein, generating a premature stop codon, resulting in truncation of the last 25 amino acids55, 56.
SLC45A2/MATP
The membrane associated transporter protein (MATP) is also known as
AIM-1 and SLC45A2 and the function of this protein is not yet fully understood. MATP has 12 predicted transmembrane regions and shows sequence
and structural similarities to plant sucrose transporters57. Transcription of
MATP is also regulated by the MITF transcription factor and it is believed to
play a crucial role in directing tyrosinase and TYRP1 (and DCT?) from the
trans-Golgi network to stage II melanosomes. Mutations in MATP is thought
to disrupt this traffic, most probably by interrupting the sorting of vesicles
between trans-Golgi and the melanosomes44, 45, 58(Figure 2).
Mutations in MATP have been identified in many vertebrates. The first
mutations were detected in the medaka fish. In most of these mutants both
skin and eye defects are seen57. During the same time period, a series of alleles at the underwhite locus in mouse (Uwdbr > wild-type > uw > uwd) were
also found to represent mutations in MATP.
Mutations were also observed in human patients with oculocutaneous albinism type 4 (OCA4)58, 59. The first human MATP mutation was identified
in a patient with oculocutaneous albinism (generalized hypopigmentation of
skin, hair and eyes). This patient was found to be homozygous for a G to A
transition in the splice acceptor sequence of exon 2, resulting in the skipping
of this exon and thereby deleting the fourth transmembrane region, thus
changing the orientation of the following transmembrane regions59. After
this, many more mutations in MATP have been found in OCA patients by
genetic screenings of patient materials from different ethnic origins60, 61.
MATP polymorphisms have also been associated with normal human pigment variation62.
Carotenoid pigment
More than 1000 naturally occurring variants of carotenoids have been found
in plants and other photosynthetic organisms, but none of these can be synthesized by vertebrates and therefore needs to be derived from their foods63,
64
. In animals the most important function of carotenoids is their function as
vitamin A precursors64. Among mammals, particularly primates and ruminants are the ones accumulating carotenoids. As a consequence they can
develop yellow fat with age63. In both cattle and sheep, mutations resulting
in yellow fat among the animals represent a major and very costly problem
for the farmer, because of a marketing and consumer resistance65, 66. Birds
and fish use carotenoids in many external structures, for example the pink
colour of flamingos and salmon are due to carotenoids64. Among birds,
males with more colourful ornaments have been found to have a better im20
mune status due to the higher levels of circulating carotenoids, they are also
more attractive to females67. Carotenoids have therefore been said to be an
honest indication of an individual’s health status. Whether this is the reason
why humans in different regions of the world prefer to eat chickens with
yellow skin68 remains an unanswered question.
BCDO2/CMO2
Two different carotenoid-monooxygenases, CMO1 and CMO2, have been
identified in vertebrates64. CMO1 cleaves β-carotene by centric/symmetric
oxidative cleavage at the C15,C15’ double bond69. CMO2 (previously
known as BCDO2) cleaves β-carotene and lycopene (a carotenoid pigment,
for instance found in tomatoes) by an excentric/assymetric cleavage at the
C9’,C10’ double bond70. The cleavage of β-carotene is the key step in the
formation of vitamin A (retinol), CMO1 has been identified as the key enzyme in this process, and the physiological role of CMO2 is less understood69. In vertebrates vitamin A has multiple functions during development
and cell differentiation. Retinal and related compounds serve as the chromophores of rhodopsins (visual pigments) in animals. Tissue expression of both
CMO1 and CMO2 has been found to be ubiquitous, and vitamin A dependent processes may be tissue specifically regulated thanks to circulating carotenoids69-71. CMO1-/- mice accumulate large quantities of β-carotene in several tissues and have decreased vitamin A levels, this result makes the contribution from CMO2 for vitamin A production questionable69. The transcription of CMO1 has been shown to be regulated by PPARs and RXRs,
which indicates a regulatory link between carotenoid and fatty acid metabolism, and CMO1-/- mice also gain more weight than wt control animals69.
Lycopene has been shown to decrease the expression of CMO1 and PPARγ
(and to a small extent CMO2) in rats72, implying a role for this CMO2
cleaved carotenoid in the modulation of β-carotene and lipid metabolism.
21
Present investigations
Aims of this thesis
The objectives of this study have been:
•
to identify the genes for four major pigmentation phenotypes in
chicken (the I, S, Db and W loci), and thereby understand more
about the genetics underlying pigmentation variation.
•
to explore the chicken domestication process.
Background
Pedigrees and animals
The red junglefowl x White Leghorn (SLU13) pedigree
In 1998, a pedigree for gene mapping between one red junglefowl (RJF)
male and three White Leghorn (WL) females of the SLU13 line was generated 27, 73. The red junglefowl and White Leghorns are fixed for different
alleles at many loci controlling phenotypic traits and the genetic markers
used for linkage studies in this cross were tested for informativeness in the
parental generation. The cross was initiated by crossing the parental animals,
generating F1 animals. These animals were intercrossed and about 850 F2
individuals were generated. The initial linkage analysis was performed using
~100 evenly spaced markers73. After the initial scan, additional markers were
added in the regions of interest. To handle all the data and to perform linkage
analysis the CRIMAP software has been used74. A second set of about 350
informative SNPs has also been genotyped in the pedigree. Despite this,
many microchromosomes are still not covered. Digital pictures of 814 F2
animals were used for phenotypic classification75. This pedigree was used in
papers I and II.
The red junglefowl x White Leghorn (OS) pedigree
In 2005, an intercross between a second White Leghorn line, the Obese
strain (OS) chicken76, and the red junglefowl (RJF) was generated. The
Obese strain is a unique model for spontaneous autoimmune thyroiditis, and
22
this cross was generated to perform genetic analysis of this autoimmune
disorder. The cross was initiated by crossing two RJF males with eight OS
females and one OS male with two RJF females. From the F1 generation
eight males and 35 females were selected to generate the F2 generation of
about 800 individuals (Sahlqvist A-S et al. in prep). 356 informative SNPs
and some extra markers in interesting regions have been analyzed in the
cross using the CRIMAP74 software (Sahlqvist A-S et al. in prep). Also in
this cross markers on many of the microchromosomes are missing. All but
one batch of the F2 animals got Levaxin (thyroxin) supplemented to their
food due to the hypothyroidism in the OS parental line. Digital pictures of
high quality were taken of all the F2 birds every third week of their life (at 1,
3, 6, 9, 12, 15, 18, 21, 24 and 28 weeks of age). All pictures were sorted by
individual in Extensis Portfolio 6 (Extensis, Inc, Portland, OR, USA) and
used for phenotypic classification of the birds. This pedigree was used in
paper III. Samples from the RJF and OS line were also used in papers II and
IV.
Samples from international collaborators
From collaborators at the INRA GFA experimental unit in France, we have
received both DNA samples and tissues for RNA extraction. Many of the
DNA samples from different breeds were collected by the AvianDiv project77. Samples received from INRA have been used in papers II, III and IV.
From collaborators at Nutreco/Hendrix Genetics in The Netherlands, we
received two experimental crosses segregating for the Silver and yellow skin
phenotypes, studied in papers II and IV respectively.
Loci under investigation
The I locus in chicken (paper I)
The Dominant white (I) colour is typical for the White Leghorn breed and it
was one of the first traits examined after the rediscovery of Mendel’s laws of
inheritance5. This incompletely dominant allele drastically reduces the eumelanin pigment9. The melanosomes of Dominant white chickens have also
been found to have an irregular shape and assembly78. The I locus was previously mapped to chicken linkage group E22C19W28 which shows conserved synteny with mouse chromosome 10 and human chromosome 1279, 80.
Dun (ID) is another allele at the I locus. The Dun allele was identified in a
Pit-Gamecock bird and gives a brownish colour in heterozygotes and a whitish colour in homozygotes9. A third allele at this locus is causing the greyish
Smoky phenotype. Smoky is allelic to I and arose as a partial phenotype revertant in a White Leghorn line fixed for Dominant white (R. Okimoto, B.
Payne and D. Salter, unpublished results).
23
The S and Al loci in Chicken and Japanese quail (paper II)
In 1912 the sex-linked locus for the Silver (S) plumage colour was identified
as an inhibitor of red colour in chicken24. S is allelic to the recessive
red/brown colour (gold, s+) found in the red junglefowl. The mode of inheritance of the Silver phenotype is complex because the phenotype seems to be
strongly influenced by modifying genes9.
A third allele at the S locus is sex-linked imperfect albinism (sal). This is
the bottom recessive allele at this locus81, 82. The sal birds have a white plumage colour with a ghost patterning that is dependent on genetic background.
The eyes are red at hatch, but except for the red pupils they darken with
age81, 83-85. In his book from 1949, Hutt stated that sex-linked albinism has
also been found in many other domestic bird species, such as turkey, budgerigar and canary19. The possibility of studying this phenotype as part of a
comparative approach made us interested in this phenotype in Japanese quail
(Coturnix japonica).
Imperfect albinism (al) has been identified in at least four populations of
Japanese quail28. One of the first to report this phenotype was Lauber in
196486. The chicks have bright pink eyes and yellow to white plumage colour and adult birds have white plumage with buff ghost-barring, lacking
melanin granules28, 86. Melanoblast cells from albino quail differentiate to
functional melanocytes containing melanosomes, but without melanin pigment or tyrosinase activity in these melanosomes. However, the albino quail
melanocytes still have tyrosinase activity in the Golgi-endoplasmic reticulum-lysosome and in the Golgi vesicles. These results indicate that the al
mutation affects the tyrosinase transport from the Golgi to the melanosomes87. The imperfect albino phenotype (al) is recessive to wild-type (Al+).
By crossing male chickens homozygous for S, s+ or sal to female Japanese
quail hemizygous for Al+ or al it was found that the S and Al loci in chicken
and quail are orthologous. All offspring generated from the interspecies
cross between albinos were albino88.
The cinnamon phenotype (alC) is caused by a second mutant allele at the
Al locus in quail and considered to be indistinguishable from the dark-eyed
dilute (alD) phenotype that has also been found in Japanese quail and been
linked to the Al locus28. The eyes of the chicks are red and have subnormal
melanin pigmentation but darkens with age, and the brown pigments of the
feathers are diluted without affecting the wild-type plumage pattern (all phenotypic measurements were described for the alD phenotype)28. The alC allele
is recessive to wild-type (Al) but dominant to imperfect albinism (al)30.
The chicken Db locus (paper III)
In 1972, the Dark Brown (Db) phenotype was described in autosomally
barred Fayoumi chickens89. In females the pheomelanin appears as an orange-tan or burnt orange colour and males have completely red-brown
24
breasts89, 90. The Db locus is linked to the autosomal barring (Ab/Pg) locus.
In a map of chicken chromosome 1, Bitgood and Somes 1990 mapped Db in
between P (pea comb) and Pg (pattern/(lacing/autosomal barring/pencilling)), P – 33 cM – Db - 20 cM - Pg. Previous studies have shown
that the expression of the Db allele is modified by alleles at the Extension
(E) locus89-92. The E locus encodes the melanocortin 1-receptor (MC1R) and
affects the relative distribution of the eumelanin and pheomelanin pigments75. The breasts of females with the wild-type allele at MC1R (e+) show
a salmon-brown colour and the Db allele has previously been found to have
limited effect on these salmon-coloured breast feathers9, 90. Db has on some
genetic backgrounds been found to act as a sex-influenced phenotype, expressed as dominant in males and recessive in females91.
The W locus in chicken (paper IV)
The W locus in chicken was first described by Bateson in 19025. The yellow
skin (W*Y) allele has been considered to be the mutant form because the red
junglefowl carry the white skin (W*W) allele. The phenotype cannot be easily scored until the chicks are 10-12 weeks of age since the W locus controls
the amount of xanthophylls in the skin and is dependent on its deposition.
Food high in carotenoids (such as yellow corn) enhances the yellow pigmentation of the skin. During lay, carotenoids are deposited in the egg-yolk,
resulting in less pigmented skin of the females during periods of extensive
egg laying9. In 1949 Hutt launched the idea that the yellow skin phenotype
should have been inherited from the grey junglefowl, suggesting a polyphyletic origin of the domestic chicken19.
Results and Discussion
Paper I
The Dominant white, Dun and Smoky Color Variants in Chicken Are
Associated With Insertion/Deletion Polymorphisms in the PMEL17
Gene
In this study, linkage mapping was carried out in the red junglefowl x White
Leghorn SLU13 pedigree to confirm the mapping of the Dominant white (I)
locus to linkage group E22C19W2879. The segregation at the I locus did not
deviate significantly from the expected 3:1 ratio in the F2 individuals from
the intercross. A sexual dimorphism in plumage colour was noted, this is due
to the sexual dimorphism in chicken phenotype and the sex-linked Barred
and Silver loci also segregating in this cross. The candidate gene PMEL17
was found by comparative mapping with human (chr12) and mouse (chr10)
which show conserved synteny with E22C19W2880. A recombination frac25
tion of zero and a LOD score of 107.2 between I and PMEL17, strongly indicated that PMEL17 could be the gene causing the different phenotypes
found at the I locus in chicken.
Apart from the Dominant white colour that is characteristic of White Leghorns, the Dun (ID) and Smoky (IS) phenotypes were also examined in this
study. The complete PMEL17 gene was sequenced beginning 32 bp before
the start codon and including 111 bp of the 3’ UTR. A total of 56 SNPs and
eight insertion/deletion polymorphisms (indels) were found across populations. Dominant white, Smoky and Dun were all found to be caused by indels
disrupting fairly conserved regions in the PMEL17 protein (Figure 3). A 9bp insertion in exon 10 (723insWAP) was found to be associated with the
Dominant white and Smoky phenotypes. The pigmentation defect in Dominant white birds was partially restored in the Smoky birds by a unique 12-bp
deletion in exon 6 (280delPTVT), and our interpretation is that this deletion
thus reverts part of the protein function. The Dun genotype was clearly distinct from the others with several unique changes, a 15-bp deletion in exon
10 (731delLGTAA) and three amino acid changes (A35V, G105S and
R740C) as results of missense mutations. The Dominant white (I) allele was
later shown to protect the birds from feather-pecking compared to birds carrying the wild-type (i) allele at this locus93.
Since this study was performed, mutations have been found in PMEL17
in other species. In dogs, the incompletely dominant merle phenotype (characterized by patches of diluted pigment) has been found to be associated
with an insertion of a SINE element at the intron 10/exon 11 boundary in
several breeds94. Some of these dogs also have auditory and ophthalmologic
anomalies. In the zebrafish mutant fading vision (fdv) a point mutation has
been found to give a premature stop codon and thereby a truncated protein
(lacking the transmembrane domain, the proteolytic cleavage site and the AP
binding motif). These mutants have hypopigmentation in skin melanocytes
and the retinal pigment epithelium, resulting in a visual defect in the larvae
that is restored as the development proceeds53. In Silver horses the causative
allele specifically reduce the production of eumelanin. Interestingly, the
most probable causative mutation results in the same amino acid change
(R618C) as one of the missense mutations found in Dun chickens (R740C)95.
The same mutation could also be causative for the Multiple Congenital Ocular Anomalies (MCOA) in horses, but this phenotype might also be due to a
mutation in close proximity to PMEL1796.
Figure 3. (next page) Alignment of the PMEL17 amino acid sequence associated
with the wild type (i) allele present in the red junglefowl, and the Dominant white
(I), Smoky (IS) and Dun (ID) alleles in comparison with human (S73003) and mouse
(NM_021882) sequences including the mouse silver allele (AF119092). Sequence
identities are indicated by dashes and insertion/deletion differences are indicated by
dots. The signal sequence, the four copies of the 24-amino acid repeat in chicken,
the transmembrane, and the cytoplasmic region are indicated. The arrow indicates
the proteolytic cleavage site that generates an aminoterminal Mα and a carboxyter-
26
minal Mβ fragment. The insertion/deletion polymorphisms associated with I, IS and
ID are boxed.
Signal sequence
chicken_i
MRLHGAIVLL AALLALVTAQ QRGGGRSRGG VKGSAWGGRP APFRSWDTAR
chicken_I
---------- ---------- ---------- ---------- ---------chicken_IS ---------- ---------- ---------- ---------- ---------chicken_ID ---------- ---------- ---------- ----V----- ---------human
MDLVLKRC LLH--VIG-L LAV-ATKVPR NQDWLGVS-Q LRTKA-NRQL
mouse
MV-VQRRS FLPVLVLS-L LAV-ALEGSR NQDWLGVP-Q LVTKT-NRQL
mouse_silv
MV-VQRRS FLPVLVLS-L LAV-ALEGSR NQDWLGVP-Q LVTKT-NRQL
YRPWQEGTAR
----------------------------PE-T-..-Q
-PE-T-..VQ
-PE-T-..VQ
QNDCWRGGDV
---------------------------RL------QGSN-----QGSN-----Q-
TFDISNDAPT
---------------------------SLKV---G-SLRVI--G-SLRVI--G--
chicken_i
LVGARATFSI ALRFPGTQTV LPDGRVVWSQ NCTVNGTRML QGDPVYPEQL
chicken_I
---------- ---------- ---------- ---------- ---------chicken_IS ---------- ---------- ---------- ---------- ---------chicken_ID ---------- -----S---- ---------- ---------- ---------human
- I--N-S--- --N---S-K- ----Q-I-VN -TII--SQVW G-Q----QET
mouse
- ---N-S--- --H---S-K- ----Q-I-AN -TII--SQVW G-Q----QEP
mouse_silv ----N-S--- --H---S-K- ----Q-I-AN -TII--SQVW G-Q----QEP
AEGSDGVFPD
---------------------------DDAC..I--DDAC..---DDAC..----
GQPFPRSAWG
----------------------------G-C-SGS-S
-G-C-SGPKP
-G-C-SGPKP
KRGRFVYVWW
---------------------------QKRS-----K
PKRS-----K
PKRS-----K
chicken_i
TWGRYWQVVD GATSQLTVGT DGVALGSYTM EVVVYHYRGR QRFIPIGHAS
chicken_I
---------- ---------- ---------- ---------- ---------chicken_IS ---------- ---------- ---------- ---------- ---------chicken_ID ---------- ---------- ---------- ---------- ---------human
- --Q----LG -PV-G-SI-- GRAM--TH-- --T---R--S RSYV-LA-Smouse
- --K----LG -PV-RSSIA- RHAK--TH-- --T---R--S -SYV-LA--mouse_silv ---K----LG -PV-R-SIA- GHAK--TH-- --T---R--S -SYV-LA---
TQFSITDQVP
---------------------------SA-T-----ST-T-----ST-T------
IAVDVTQLEV
---------------------------FS-S-S--RA
FS-S-S--QA
FS-S-S--QA
AAGDGGSFVR
---------------------------LD-GNKH-LLD-ETKH-LLD-ETKH-L-
chicken_i
NRPVAFNVRL HDPSHYLRDA DISYSWDFGD QSGTLISRSP TVTHTYLQAG
chicken_I
---------- ---------- ---------- ---------- ---------chicken_IS ---------- ---------- ---------- ---------. ... ------D
---------- ---------- ---------- ---------- ---------chicken_I
human
- Q-LT-ALQ- ----G--AE- -L--T----- S-------AL V------EPmouse
- H-LI-ALQ- ----G--AE- -L--T----- GT------AL D------ESmouse_silv -H-LI-ALQ- ----G--AE- -L--T----- GT------AL D------ES-
SFAARLVLQA
---------------------------PVT-QV----VT-QV----VT-QV----
AIPLSSCGTS
-------------------------------T---S----V---S----V---S-
APPVVDPTTG
---------------------------PV-.......
PV-.......
PV-.......
chicken_i
PVPSLGPTAT QPVGPTGSGT ATAPSNLTGS GTAAAPGTTA APRASGAPAE
chicken_I
---------- ---------- ---------- ---------- ---------chicken_IS ---------- ---------- ---------- ---------- ---------chicken_ID ---------- ---------- ---------- ---------- ---------human
. ......... ...-T-DGHR P--EAPN-TA -QVPTTEVVG TTPGQAPT-mouse
. ......... ...-T-DGYM P--EAPG-T- RQGTTTKVVG TTPGQMPTTQ
mouse_silv .......... ...-T-DGYM P--EAPG-T- RQGTTTKVVG TTPGQMPTTQ
PTGVSVAVLS
----------------------------S-TTSVQVP
-S-TT-VQMP
-S-TT-VQMP
PVLSTAVANA
--------D--------D---------QMPTAESTGM
QM-T......
QM-T......
chicken_i
AAGTDPTADP LPPTSVSSGG DAPGTVAPTA VEGSVAAGVG TAEDVAAATP
chicken_I
---------- ---------- ---------- ---------- ---------chicken_IS ---------- ---------- ---------- ---------- ---------chicken_ID ---------- ---------- ---------- ---------- ---------human
T PEKV-VSEV MGT-LAEMST PEATGMT-AE -SIV-LS-TT A-QVTTTEWV
mouse
. ......SAV IDT-LAEVST TEGTGTT--R .....PS-TT V-QATTTE..
mouse_silv .......SAV IDT-LAEVST TEGTGTT--R .....PS-TT V-QATTTE..
Repeat 2
Repeat 3
chicken_i
IADPTAGATD GDAVGPTAAA TAESIADPTA GATDGDAVGP TAAATAESIA
chicken_I
---------- -----....- ---------- ---------- ---------chicken_IS ---------- -----....- ---------- ---------- ---------chicken_ID ---------- ---------- ---------- ---------- ---------human
G PLLDGT--. .......... .......... .......... ..........
mouse
S PLLDDTD-. .......... .......... .......... ..........
mouse_silv SPLLDDTD-. .......... .......... .......... ..........
GATAADVAVD
---------------------------ET--RELPIP
..........
..........
Repeat 4
DPIVGATDGD
--........
--........
---------..........
..........
..........
DSAATEPLPD
---------------------------TTEVISTA-V
TTEV-ATTSE
TTEV-ATTSE
Repeat 1
TAGATDGDAV
---------------------------EPEGP-ASSI
...GP-ASPL
...GP-ASPL
AVGPTAAATA
..........
..........
---------..........
..........
..........
ESIADPTAGA
......---......------------..........
..........
..........
chicken_i
TAVSSGSATA GATAEPLLLV KRQAPEAEPT GCVLYRYGTF STELNIVQGI
chicken_I
---------- ---------- ---------- ---------- ---------chicken_IS ---------- ---------- ---------- ---------- ---------chicken_ID ---------- ---------- ---------- ---------- ---------human
. ......... ......-R-- ---V-LD... .-------S- -VT-D----mouse
. ......... ......IM-- ---V-LD... .-------S- -LA-D----mouse_silv .......... ......IM-- ---V-LD... .-------S- -LA-D-----
ESVAIVQVVP
-----------------------------AE-L-A---AE-L-A---AE-L-A--
AAPEGSGNSV
---------------------------S...-E-DAF
F...SE-DAF
F...SE-DAF
ELTVTCEGSL
-------------------------------S-Q-G----S-Q-G----S-Q-GTransPAASGTTLTV
---------------------------AGLGQVP-IGGLGQAP-LGGLGQAP-L-
chicken_i
PEEVCTVVAD AECRTAQMQT CSAVAPAPGC QLVLRQDFNQ .SGLYCLNVS LANGNGLAVA STHVAVGGAS
chicken_I
---------- ---------- ---------- ---------- .--------- ---------- ---------chicken_IS ---------- ---------- ---------- ---------- .--------- ---------- ---------chicken_ID ---------- ---------- ---------- ---------- ---------- ---------- ---------human
- K-A-MEISS PG-QPPAQRL -QP-L-S-A- ----H-ILKG G--T------ --DT-S---V --QLIMP-QE
mouse
- K-A-MDISS PG-QPPAQRL -QS-P-S-D- ----H-VLKG G--T------ --DA-S---- --QLV-P-QD
mouse_silv -K-A-MDISS PG-QPPAQRL -QS-P-S-D- ----H-VLKG G--T------ --DA-S---- --QLV-P-QD
membrane region
Cytoplasmic region
chicken_i
GLL...LIAA ALGTAAYTYR RVKYSPLLPT APTAPRPHSW LPPGATLRLL LRQAFGGAPS GESSPLLRAN
chicken_I
--- WAP ---- ---------- ---------- ---------- ---------- ---------- ---------chicken_IS --- WAP ---- ---------- ---------- ---------- ---------- ---------- ---------chicken_ID ---------- -.....---- C--------- ---------- ---------- ---------- ---------human
- I-...-VLM -VVL-SLI-- -RLMKQDFSV PQLPHSSSH- -RLPRIFCSC ........-I --N----SGQ
mouse
- I-...-VLV -VVL-SLILG IDLR-RAQF. .-KCHMVALT AA-ASG--AR ........GL --N----SGQ
mouse_silv -I-...-VLV -VVL-SLIH- HRLKKQGS.V SQMPHGSTH
GPTAAATAES
---------------------------MS-ESI-GSL
L--QSS-GSI
L--QSS-GSI
AV*
------Q-Q--
Several PMEL17 mutations have been identified in various species, but
no complete loss-of-function has been found in vertebrates and the first hu27
man mutation is still to be found. PMEL17 is known to be involved in eumelanin formation but the protein may have other functions as well. The fact
that no mutation that completely inactivates the function of PMEL17 has yet
been discovered suggests that PMEL17 has a crucial but unknown function
besides its role in melanogenesis.
Paper II
Mutations in SLC45A2 Cause Plumage Color Variation in Chicken and
Japanese Quail
This study demonstrates that mutations in SLC45A2/MATP are associated
with the Silver (S), cinnamon (alC) and sex-linked imperfect albinism (sal)
and (Al+) phenotypes in chicken and Japanese quail (Figure 4).
By linkage analysis the MATP gene was mapped to the upper part of
chicken chromosome Z where the sex-linked Silver locus is known to be
located. The nucleotide sequence of all seven exons was determined from
genomic DNA of both chicken and Japanese quail. The Silver allele in
chicken was found to be associated with a C to A transversion, causing a
missense mutation in exon 4 (Leu347Met) in all tested breeds with the Silver
phenotype except White Leghorn. This amino acid change is affecting a
highly conserved part of the seventh transmembrane region. The S allele
found in White Leghorns is associated with an A to G transition, resulting in
a missense mutation in exon three (Tyr277Cys) affecting a loop region. This
allele was also found in several breeds with the wild-type phenotype. We
have been unable to map the Silver phenotype in our RJFxWL(SLU13)
pedigree and thus, the phenotypic consequence of this mutation might be
highly influenced by the genetic background. Perhaps this mutation shows
its effect in pure White Leghorns due to their whole package of pigmentation
alleles (I, E, B and S). A one bp deletion (106delT) was found in the chickens with sex-linked imperfect albinism resulting in a frameshift at codon 36
and translation stop in exon one.
In Japanese quail the mutation associated with sex-linked imperfect albinism was found to be a G to T transversion in the last nucleotide of intron
three. cDNA sequencing confirmed that exon four is not present in the transcript. The cinnamon allele in Japanese quail is causing a more severe phenotype than the Silver alleles found in chicken, and was found to be due to a
C to A transversion in exon one. This results in a nonconservative Ala72Asp
amino acid change at a conserved position of the first transmembrane domain.
In summary, the mapping of the contig harbouring MATP to the top of the
Z chromosome and the five mutations found, show beyond doubts that mutations in the membrane associated transporter protein (MATP) cause phenotypic variation in chicken and Japanese quail. The incompletely dominant S
28
phenotype in chicken only inhibits the red (pheomelanin) colour of the plumage. No pheomelanin-specific mutations in MATP have been reported earlier. The semidominant D153N mutation found in horse has a stronger effect
on pheomelanin production but also affects eumelanin in homozygous
form97. Our hypothesis is that one of the functions of MATP is to transport
the cysteine essential for pheomelanin production into the melanosome, and
that this specific function is disrupted in the birds with the Silver allele. In
the albino birds with loss-of-function mutations all functions of MATP is
disrupted and neither eumelanin nor pheomelanin pigment is produced.
Figure 4. Membrane topology prediction of the MATP protein using TMHMM (v.
2.0). The location of the frameshift mutation (S36fs) associated with the sal allele
and the two missense mutations Y277C and L347M associated with the Silver allele
in chicken are indicated by black arrowheads. The A72D mutation associated with
the cinnamon allele in Japanese quail is marked with a grey arrowhead. The amino
acids missing in the MATP protein encoded by the albino allele in Japanese quail is
shaded in grey.
29
Paper III
The Plumage Colour Dark Brown in Chicken is Caused by an 8 kb
Deletion Upstream of SOX10
In this study we show that an 8 kb deletion located 14 kb upstream of SOX10
results in the Dark brown (Db) phenotype in chicken (Figure 5). The Db
phenotype was scored by examining pictures from each of the 765 F2 individuals in the OSxRJF intercross. 161 individuals were scored as having the
Db phenotype (41 females, 62 type 1 males and 58 type 2 males). In females
the phenotype was seen as an orange tan over the entire plumage except for
the tail feathers. In males two different categories of the phenotype was
scored. The type 1 males had a bright brown/red/orange breast, but no
pheomelanin was seen in the tail, the type 2 males had a similar but a less
apparent phenotype. A two-point linkage study including the 41 Db females
confirmed linkage of the locus to chicken chromosome 192. No causative
mutation was found within the protein encoding parts of SOX10, but when
examining conserved regions around the gene the same region as the one
found to be deleted in the Hry mice42 was found to be missing in the Dark
brown individuals.
Resequencing of two db+ (RJF) birds and six birds from different lines
exhibiting the Db phenotype resulted in a 12.8 kb haplotype (IBD region)
shared among all sequenced birds with the Db phenotype, thus excluding all
polymorphisms found outside this area as causative mutations (Figure 5).
Unexpectedly, one of the sequenced RJF individuals carried a haplotype that
was identical to the Db haplotype except for the deletion and its associated
10 bp insertion. This result strongly supports the proposition that the deleted
region is causative for the Db phenotype, as the 8 kb deletion is the only
unique sequence difference between the Db and db+ chromosomes. In addition to this result the deletion was present in 20 chickens known to carry the
Db allele, but in none of the 43 tested birds not expected to carry the Db
allele.
Among the type 1 males 49 were found to be homozygous for the deletion
and 13 were heterozygous. In the type two group only three were found to be
homozygous Db. In this group most individuals (49/58) were instead heterozygous for the deletion, explaining the Db locus as co-dominant, with a
more pronounced phenotype in the homozygous individuals. In the type 2
group, six males were also found to be homozygous wt (db+), this result is
most probably due to a misclassification when scoring the phenotype.
All 41 females phenotyped as Dark brown were found to be homozygous
for the deletion. This implies, in agreement with previous studies91, that the
expression of the phenotype also is influenced by the sex of the bird.
The Hry mouse strain has as previously mentioned a 15.9 kb deletion of a
non-coding conserved sequence 47 kb upstream the Sox10 transcription start
site. The deleted conserved element is corresponding to the one found in this
30
study, and it has also been further examined as a segment likely to play a
role in Sox10 expression during the migration and development of melanocytes43.
Db shows a stronger pheomelanistic expression in the breast (ventral)
than in the tail (dorsal) part of the birds. One idea is that SOX10 is differently expressed in breast and tail another hypothesis is that the pattern is a
result of a dose relationship between agouti (ASIP) and MC1R. Both these
hypothesis are at present under study, as we attempt to confirm that the 8 kb
deletion is a cis-acting regulatory mutation.
Figure 5. The location of an ~8 kb deletion upstream of SOX10 associated with the
Dark brown phenotype (Db_DEL). The borders of the 12.8 kb haplotype showing
complete association with the Db allele across populations are also indicated
(Db_HAP). The figure also shows the coding part of SOX10 and sequence conservation between species. The figure was generated using the chicken genome assembly
as presented on the UCSC Genome Browser (www.genome.ucsc.edu, Chicken May
2006 Assembly).
Paper IV
Identification of the Yellow Skin Gene Reveals a Hybrid Origin of the
Domestic Chicken
In this study we show that the yellow skin phenotype in chicken is caused by
a cis-acting and tissue specific regulatory mutation(s) that inhibit the expression of BCDO2/CMO2 in skin. We also conclude that there must be a hybrid
origin of the domestic chicken, given that the yellow skin allele is derived
from the grey junglefowl.
The yellow skin (W) locus had previously been assigned to chicken chromosome 2480. In this study the W locus was initially mapped close to the
APOA1 gene (Θ = 0.07, LOD = 16.4) at the distal end of chromosome 24.
This was done by the use of a backcross pedigree (Y/WxY/Y) comprising 91
individuals. Partial sequencing of the candidate gene BCDO2 resulted in a
SNP fixed in all tested breeds with the yellow skin phenotype. Further sequencing resulted in a 23.8 kb IBD region where all these breeds show the
exact same haplotype (Figure 6).
31
No obvious causative mutation was found within the coding sequence and
thus the effect was supposed to be a regulatory mutation acting on the
BCDO2 gene. A pyrosequencing test on cDNA from six heterozygous individuals showed that the BCDO2 expression was clearly affected in skin
(90% of the transcripts expressed in skin originated from the white skin allele), but not in liver (Figure 6).
In comparison with chickens carrying the white skin allele (including red
junglefowls) a higher sequence divergence (0.81%) than the expected
0.5%26, was seen between the haplotypes at the yellow skin locus. This indicated that the yellow skin allele might in fact be descendant from some of the
other junglefowl species as proposed by Hutt19. For the BCDO2 haplotype
region the yellow skinned individuals clustered with the grey junglefowl,
whereas for all other tested regions of the genome they clustered with the red
junglefowl. This implies that the biggest part of the chicken genome is descendant from the red junglefowl, but that small selected parts have been
inherited from the grey junglefowl. Whether or not the other two junglefowl
species have also contributed to the domestic chicken remains to be shown.
Since this study, research groups in Australia and New Zealand has found
a stop codon in BCDO2 to have a large effect on fat colour in cattle65.
Figure 6. (A) Gene content of the yellow skin interval and the 23.8 kb yellow skin IBD
region indicated by a box. The annotation is based on the chicken genome assembly
(www.genome.ucsc.edu, Chicken May 2006 Assembly). (B) Differential expression of
the BCDO2 transcript in skin but not liver from yellow skin heterozygotes using genomic
DNA (gDNA) as control. The polymorphic position chr24:6,268,434 bp was used to
monitor differential expression using pyrosequencing. T and C at this position correspond to the white and yellow skin alleles respectively. (C) Summary of the examination
of differential expression in skin and liver from six heterozygous (W/Y) birds. Genomic
DNA from the three different genotypes were used as controls.
32
Future prospects
In this thesis I have presented data from genetic studies of domestic chickens. During the domestication, humans have selected numerous mutations
affecting the phenotype of the birds. The same is true for all of our domestic
animals (and plants). By the fast evolving sequencing technologies, sequencing and resequencing of many more species and breeds will be seen in the
near future. From these studies we will have the possibility to learn more
about the evolution of different species, but also more about genome structures, the functional elements in the genomes, and be able to pin-point selective sweeps and mutations underlying phenotypic diversity. These sequencing efforts will also give us an almost unlimited number of genetic markers
to test in breeds and pedigrees for the fine-mapping of trait loci. The possibility of sequence capture technology, to select a candidate region of up to a
Mb for resequencing, will tremendously speed up the process of finding the
causative mutation98.
No PMEL17 mutation has yet been found in humans and there is not yet
any knockout mouse for this gene. If PMEL17 is only affecting pigmentation
a “spontaneous” knockout would have been expected to be found and studied by now, given the extensive screening for coat colour mutations in the
mouse. The fact that a knockout has not been seen in any species suggests
that PMEL17 has other functions that are essential for survival, and that a
complete loss-of-function mutation is lethal. Since PMEL17 primarily affect
the assembly of eumelanin, a study of red-haired-humans lacking eumelanin
pigmentation could reveal the first human mutations in this gene.
Given that no pheomelanin-specific mutations in MATP/SLC45A2 have
been reported earlier it would be of interest to learn more about the function
of MATP, especially its respective effect on pheomelanin and eumelanin
production. Why do some mutations in this gene affect the synthesis of both
eumelanin and pheomelanin, whereas other inhibits the production of
pheomelanin but leaves the production of eumelanin unaffected?
Hutt19 claimed that the Silver allele in chicken may have been derived
from the grey junglefowl. By sequencing exon 3 and 4 of the MATP gene in
Grey junglefowl we can exclude this statement, since the grey junglefowl
did not have any of the two Silver mutations found in paper II (unpublished
results). The grey phenotype in this species might still be a result of a MATP
mutation and sequencing the rest of the exons could perhaps answer this.
Since the turkey, budgerigar and canary are suspected to have sex-linked
33
albinism caused by mutations in the same gene as chicken and Japanese
quail, MATP is an obvious candidate gene for these phenotypes as well9.
With respect to the yellow skin locus (paper IV), Hutt19 was correct about
the phenotype deriving from the grey junglefowl. By whole genome resequencing of the three additional junglefowl species (the Ceylon, grey and
green junglefowl) we could learn if more of the grey junglefowl genome has
been selected for in our domestic breeds and then perhaps find some more
causative mutations for traits deriving from this species. If the Ceylon and
green junglefowl have somehow contributed to the domestic chicken is currently unknown and resequencing of these genomes could therefore give us
more knowledge about the domestication process.
A knockout mouse has been created for the CMO1 gene69, resulting in an
accumulation of β-carotene and prevention of vitamin A production. This
knockout also suffered from damaged lipid homeostasis. No knockout
mouse has been generated for the CMO2/BCDO2 gene and the function of
this enzyme has been questioned69. At a recent conference a stop codon in
CMO2 was described in cattle65. I do not know where in the gene this stop
codon is located or if there are more phenotypes in these animals than the
yellow fat. Until this has been carefully studied I think it could be of interest
to knock out the CMO2 gene in mice to determine how it resembles/differs
from the phenotype of the CMO1-/- mice. Perhaps it could also be interesting
to generate CMO1/CMO2 double knockouts to investigate whether these
enzymes have overlapping functions that are totally disrupted and results in a
more severe phenotype if both genes are inactivated. It would also be of
interest to understand which of the polymorphic sites within the 23.8 kb
haplotype region for yellow skin that is causative for the phenotype and what
factor that binds there. This would improve our knowledge about the regulation of this gene.
Expression studies of the Dark brown allele (paper III) should be performed. How does this deletion affect the eumelanin distribution and give an
expression of pheomelanin instead? How is this deletion different from that
of the Hry mouse42 (which has a more severe phenotype, affecting both
melanocyte migration and resulting in megacolon)?
34
Summary in Swedish
Genetiska studier av hönsens färgteckning
I min avhandling har jag studerat gener som reglerar pigmentering. Färgfenotyper är enkla att avläsa visuellt för varje enskild individ och följer ofta en
enkel monogen nedärving. Detta gör att generna som styr pigmentering är
värdefulla modeller för att förstå hur gener fungerar och integrerar med
andra gener. Jag har under min doktorandtid till stor del studerat dessa fenotyper i två unika korsningar mellan den röda djungelhönan (Gallus gallus)
och två olika linjer av vit Leghorn.
I den första artikeln studerades det lokus (kromosomregion) som ger dominant vit färg hos höns. Här fann vi koppling mellan den vita färgen och
PMEL17 genen. Efter karakterisering av genen hos höns med olika fjäderfärg fann vi tre mutationer som ger tre olika fenotyper (egenskaper, i detta
fall fjäderfärger). Två av mutationerna (dominant vit färg och Dun mutationen) hindrar utrycket av eumelanin (svart pigment) och orsakas av en insertion på tre aminosyror respektive en deletion på fem aminosyror i transmembranregionen av PMEL17. Smoky mutationen uppstod som en partiell reversion i en linje som är homozygot för mutationen som ger dominant vit färg
och har därför både insertionen på tre aminosyror i transmembranregionen,
samt en deletion av fyra aminosyror i den del av proteinet som kodas av
exon sex.
I det andra delarbetet studerades ett könsbundet lokus (Silver) som associerats med inhibering av feomelanin (rött pigment) samt ofullständig albinism i både höns och vaktel. Genen SLC45A2 (tidigare känd som MATP)
identifierades som kandidatgen på grund av en tidigare känd koppling till en
retrovirusinsertion i hönsgenomet. Genetisk analys i vår F2 korsning bekräftade en lokalisering av SLC45A2 till den region högt upp på kromosom Z dit
Silver också lokaliserats. Både Silver i höns och cinnamon i vaktel är mutanter där uttrycket av feomelanin hämmas och vi visar att detta beror på två
olika mutationer i konserverade transmembranregioner av SLC45A2. Ytterligare en mutation associerad med Silver fenotypen i vit Leghorn höns har
identifierats i en icke konserverad region. De två mutationerna som orsakar
ofullständig albinism (sal och al) ger båda upphov till icke-funktionella protein. Hos höns med sal allelen har en nukleotid i exon 1 deleterats och läsramsförändringen gör att inget funktionellt protein kan bildas. I al allelen
hos vaktel fann vi en mutation i den sista nukleotiden av intron tre. Detta
35
leder till att hela exon fyra klyvs ut med resultatet att proteinets struktur
drastiskt förändras.
I den tredje artikeln fann vi koppling mellan fenotypen Dark brown (Db)
och SOX10 genen hos höns. Denna fenotyp nedärvs dominant i tupparna och
recessivt i hönorna i en korsning mellan röd djungelhöna och vit Leghorn. Vi
visar att ett ~8 kb stort fragment av ett konserverat och tidigare studerat regulatoriskt element för reglering av pigmentceller saknas hos alla individer
med Db fenotypen. Alla andra möjliga polymorfier mellan Db individernas
haplotyp och vildtypen kunde uteslutas.
I det fjärde och sista delarbetet studerades det lokus som ger upphov till
gul hud hos många domesticerade hönsraser. Denna fenotyp är orsakad av en
recessiv allel (W*Y) som tillåter deponering av gula karotenoider i huden. I
detta arbete visar vi att den gula färgen orsakas av en vävnadsspecifik regulatorisk mutation som inhiberar uttrycket av BCDO2 genen i huden. I höns
av vildtyp (W*W) som saknar mutationen bryter BCDO2 ner karotenoider
som tagits upp genom födan, men denna funktion har gått förlorad i individer med gul hud. En annan mycket spännande upptäckt under detta arbete
var att allelen för gul hud härrör från den grå djungelhönan (Gallus sonneratii) och därmed kullkastades den tidigare allmänna uppfattningen att den
röda djungelhönan var den enda ursprungsarten till dagens tamhöns.
Arbete tre och fyra visar på betydelsen av regulatoriska mutationer som
orsak för fenotypisk diversitet.
36
Acknowledgements
I would like to express my gratitude to ALL people that I have spent time
and collaborated with during my PhD studies. You have made this thesis
possible!
Especially I would like to thank:
Min huvudhandledare Leif Andersson, tack för att du med ditt lugn och din
passion för forskningen väglett mig och ökat min förståelse för vetenskapliga frågeställningar. För mig har dessa år varit väldigt lärorika och något av
en känslomässig berg-och-dalbana i vissa perioder. Tack för att du tagit dig
tid när jag behövt det. Lycka till med alla projekt i framtiden! 
Susanne Kerje, min biträdande handledare och vän. Jag tror aldrig att du sagt
att du inte har tid? Tack för att du fick in mig i gruppen! Jag hoppas vi
kommer att träffas och diskutera allt mellan himmel och jord även i framtiden.
Min biträdande handledare Göran Andersson, tack för kommentarer på min
avhandling och andra texter under dessa år. Jag uppskattar också ditt intresse
för mina projekt och allmän uppmuntran under min doktorandtid!
Frida, Lina, Ellen & Sus! Jag tycker att människorna är minst lika viktiga på
en arbetsplats som arbetsuppgifterna och ni har verkligen bidragit till en
trevlig arbetsmiljö!  Kul att vi fortsätter att träffas, snacka och äta då och
då nu när vi börjar spridas för vinden.
Per, Anders & Jonas, det har varit kul att jobba med er! Ni har alla mycket
spännande på gång, lycka till med allt pojkar (eller män menar jag såklart,
sorry)!
Min rumskompis Freyja, det har varit roligt och intressant att dela rum med
dig!
Ulla, du är en klippa!  Tack för allt du lärt mig under åren och för fint
samarbete med alla pyrokörningar!
37
Gudrun, jag saknar dig! Du har varit ett enormt stöd. Hoppas du trivs med
livet som pensionär och farmor. Tur att det är många disputationer nu i vår
så att man får chans att träffa dig lite!
Göran H & Erik B-R, tack för allt ni hjälpt till och fixat med!
Alf, det var underbart att jobba med dig! Du fixade allt och var jättetrevlig
när man kom och hälsade på dig och hönsen i Skara!
Stina, alla bilder du tog på hönsen har varit guld värda!
Jen, thanks for helping out with the pictures!
I would also like to take this opportunity to thank past and present members
of the ”chicken group”. It has been interesting to discuss science with you at
our Tuesday meetings, and I enjoyed getting to know some of you a bit better during the trips to Skara! I would also like to thank all our Swedish and
international collaborators for good team-work during these years. I hope it
will continue!
And again, all past, present and new members of the lab! You have all contributed to the atmosphere and I will really miss Fredagsfika! Hopefully I
can sneak in now and then during the autumn as well to meet you all! Will
this Café ever become a reality, as a spin-off from our labs love for cake?
E-spik för all hjälp med avhandlingens utformning.
Gijs Afink, thanks for introducing me to the world of science during my
”forskningspraktik”! You were a really good teacher and I still perform
many procedures in the lab exactly the way you taught me. 
Alla personer som jag lärde känna och fick jobba med under UGSBR-året!
Det var jätteskoj, skulle gärna köra en runda till! Också stort tack till alla
som var med och gjorde SNiB-konferensen i Uppsala möjlig. Många trevliga
middagar blev det!
Julia, Erika, Therese & Veronica ni gjorde grundutbildningen till en dans på
rosor! Jag har haft så mycket skoj med er. Hoppas jag ska bli lite bättre på att
hålla kontakten när den här hektiska perioden är över.
Jonas, Eva, Linus & Malin, stort tack för att ni tagit hand om min familj på
bästa sätt under alla helger som jag häckat på BMC. De har haft jätteroligt
varje gång och jag har varit så avundsjuk… Hoppas vi kan hitta på fler roliga
saker i vår!
38
Katarina, Thomas & David, tack för mycket trevligt resesällskap! Jag hoppas
på en ny resa 2010 eller så, kan det passa? Skulle vara superkul!
Eva N, det är skönt att alltid ha någon att ringa när man behöver prata av sig
lite. Hoppas vi ses snart!
Britta med familj, jag hoppas på många trevliga lekstunder i vår/sommar!
Alla trevliga barnfamiljer, förskolefröknar & grannar vi lärt känna de senaste
åren, känns jättekul att bli lite riktig Uppsalabo efter sisådär 10 år i stan. Nu
vill jag stanna här hos er för ni är så himla trevliga!
Släkt & vänner i norr (och på andra sidan jordklotet) samt den ingifta släkten
i söder! Det är superskönt att få komma och hänga med er under sommarsemester och julledighet. Tack för att ni finns!
Mian, Christophe, Hugo, Alex & Edwin! Längtar till fjällresan i påsk, ska bli
så kul att träffa er alla!
Birgitta & Anna, vad skulle vi göra utan er? I höst måste vi nog komma och
plocka några hundra liter lingon, så mycket Anna-sylt som vi gör av med.
Birgitta du ska ha stort tack för att du alltid ställer upp med stort som smått!
Mamma, Pappa & småbrorsorna Anders & Stig, tack för att ni peppat och
trott på mig! Ska bli mycket spännande att se vad som händer när det här är
klart… kan inte svara på det just nu, men först blir det lite paus med utökning av släkten i alla fall!
Martin & Vilgot, mina grabbar, ni är verkligen toppen!  Vad skulle jag
göra utan er? Nu när det här är färdigt hoppas jag att vi ska få mycket mer tid
att mysa och hitta på roliga saker tillsammans! Jag älskar er ♥.
39
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