white spotting patterns in mammals can be caused by mutations in the genes for the endothelin b receptor and c - kit , whose protein products are necessary for proper migration , differentiation or survival of the melanoblast population of cells .
although there are many different dog breeds that segregate white spotting patterns , no genes have been identified that are linked to these phenotypes .
an intercross was generated from a female newfoundland and a male border collie and the white spotting phenotypes of the intercross progeny were evaluated by measuring percentage surface area of white in the puppies .
the border collie markings segregated as a simple autosomal recessive ( 7/25 intercross progeny had the phenotype ) .
two candidate genes , for the endothelin b receptor ( ednrb ) and c - kit ( kit ) , were evaluated for segregation with the white spotting pattern .
polymorphisms between the border collie and newfoundland were identified for ednrb using southern analysis after a portion of the canine gene had been cloned .
polymorphisms for kit were identified using a microsatellite developed from a bacterial artificial chromosome containing the canine gene .
both ednrb and kit were excluded as a cause of the white spotting pattern in at least two of the intercross progeny .
although these genes have been implicated in white spotting in other mammals , including horses , pigs , cows , mice and rats , they do not appear to be responsible for the white spotting pattern found in the border collie breed of dog .
the genetics of coat color has been studied for many years in a variety of mammals , and the inheritance patterns of many of the relevant genes have been determined .
both scientists and breeders have studied the range of color and pattern that can be found in mammals .
dogs are uniquely suited for the investigation of the inheritance of coat color and patterns as the more than 200 different dog breeds are defined in part by a specific set of colors and patterns . '
white spotting ' in mice , rats , dogs and horses is characterized by irregular white patches of skin and hair that are devoid of pigment - producing melanocytes .
white spotting in domestic dog breeds has been postulated to be controlled by one locus , called s , which has at least three alleles .
the three alleles segregate in some breeds , while other breeds are homozygous and show a standard spotting pattern .
although the variation in coat colors and patterns in dogs is far greater than in other species , the genes responsible for coat color in dogs have yet to be identified at the molecular level , with the exception of that for the yellow coat color .
genes responsible for white spotting in mice and horses and for hypopigmentation defects in humans have been identified .
one of these , ednrb , encoding the endothelin b receptor , causes white spotting in the ednrb mouse .
some alleles of ednrb are associated with more severe defects , such as deafness and aganglionic megacolon .
mutations in the endothelin b receptor gene are also responsible for hirshsprung disease in humans .
this disease is characterized by intestinal aganglionosis , which is occasionally associated with hypopigmentation and/or deafness .
a similar syndrome in horses , called lethal white foal syndrome , is due to a mutation in the horse endothelin b receptor gene .
mutations in a second gene , ckit , which encodes a receptor tyrosine kinase , cause the piebald trait in humans .
piebaldism is characterized by white patches on skin and hair . in horses , a similar phenotype , called tobiano ,
as a resource for building a dog genetic map and as a tool to study the genes responsible for behavioral and morphological differences in the dog , an intercross was created between a male border collie and a female newfoundland .
the newfoundland parent had a small patch of white on the chest and was otherwise completely black ( figure 1a ) .
the border collie used in this cross had markings characteristic for the breed - black with white markings on the face , chest , neck , tail tip , ventral abdomen , all four digits , and extending up the front legs to the carpals ( figure 1b ) .
the border collie 's sire and dam had the same markings , consistent with homozygosity for the causative loci .
this cross provides the opportunity for analyzing the inheritance of the white spotting pattern exhibited by the border collie .
six f1 animals were produced which had medium - sized white patches on their chests .
these six dogs were intercrossed to produce 25 f2 progeny . in the f2 generation ,
7/25 had markings like the border collie parent , consistent with the phenotype being caused by a recessive allele of a single locus . to determine if ednrb was responsible for the white spotting pattern of the border collie , a portion of canine cdna was cloned .
the amino - acid sequence of canine endothelin b receptor was highly homologous to that of other mammalian endothelin b receptors ( figure 2 ) .
the canine cdna clone was used as a hybridization probe for dna hybridization blot experiments . a pvuii restriction fragment length polymorphism ( rflp )
spotted f2 progeny carried at least one allele of this polymorphism that was different from those carried by the border collie parent , indicating that this locus was not linked to white spotting pattern in this cross ( figure 3 ) .
to evaluate the segregation of the second candidate for white spotting , kit , in this cross , a polymorphic simple - sequence repeat was developed from bacterial artificial chromosome ( bac ) clones containing the kit gene .
three of the four bacs containing the kit gene also contained this simple - sequence repeat , as ascertained by pcr amplification of a product from the bacs using primers flanking the simple - sequence repeat .
in addition , positive pcr amplification of a product using both kit genomic primers and the simple - sequence repeat primers occurred in the same subset of radiation hybrid cell lines from a dog / hamster panel from research genetics , inc .
this simple - sequence repeat marker was polymorphic in the intercross and segregated independent of the white spotting phenotype ( figure 4 ) .
by analogy with mice and humans , mutations in both ednrb and kit were excellent candidates for white spotting in border collies ; however , these data taken together excluded both genes from being responsible for the distinctive border collie coat color markings .
although it has been postulated that all white spotting in dogs is due to the same major locus , allelism tests between the spotting phenotypes in all the different dog breeds have not been performed .
it is possible that either ednrb or kit could be responsible for white spotting in other breeds .
extreme white spotting , an additional allele of the s locus , segregates in the boxer breed .
the markers developed in this study were not polymorphic in available individuals of this breed , so we were unable to test for linkage to this presumptive allele of the s locus .
as more genes are placed on the canine genetic map , it will be possible to test other candidates for white spotting derived from studies of coat color genetics in mice .
white markings are often associated with deafness in dogs , cats , and even humans .
the link between white spotting and lack of innervation to the distal colon is the neural crest , from which melanocytes and enteric ganglia are both derived .
the exclusion of ednrb as a candidate gene for white spotting in border collies may explain why aganglionic megacolon is not associated with this trait in dogs .
the gene responsible for white spotting in dogs may shed light on new genes involved in the differentiation and survival of the neural crest .
seven of those 25 f2 progeny had white markings similar to the border collie parent .
to evaluate the white spotting , the percentage of white based on surface area was compared between parents , f1 and f2 progeny .
photographs of the dogs were taken and the weight of the white markings in the image was compared to the total weight of the dog image after cutting , on a percent basis .
a portion of the ednrb cdna was cloned as a 1,314 bp product using the tri - clone kit ( invitrogen corp . , california , usa ) using primers designed from mouse and human sequences ( e1(atg)f 5'-cag gta gca gca tgc agc-3 ' and e3 5'-gga acg gaa gtt gtc ata tcc-3 ' ) .
the clone was sequenced ( genbank af276427 ) and the deduced amino - acid sequence was compared to that of the published mammalian sequences using the ' pretty ' program ( gcg wisconsin package ; genetics computer group , wisconsin , usa ) . the 1,314 bp fragment
was radioactively labeled and used as a hybridization probe against genomic dna digested with pvuii .
segregation of ednrb was followed in the intercross progeny by this pvuii rflp ( figure 3 ) . to follow segregation of kit in this pedigree ,
a portion of kit was cloned by pcr using published primers   and used to screen a canine bac library ( bac - pac resources , bacpac resource center at the children 's hospital oakland research institute , california , usa ) .
a gaaa ( 18 ) repeat microsatellite marker was developed from one of the bacs ( kit forward 5'-gca tgg agc ctg ctt ctc-3 ' , kit reverse 5'-aga gca tcc ttg gtc tgt cc-3 ' ) .
pcr amplification of the simple - sequence repeat from the intercross animals is shown in figure 4 .
pcr amplification products of the kit microsatellite were separated by electrophoresis on a 10% polyacrylamide gel and stained with ethidium bromide before photography .
a newfoundland female ( a ) was bred to a border collie male ( b ) to produce animals for the intercross .
amino - acid sequence alignment of mammalian endothelin b receptors using the ' pretty ' function from the gcg wisconsin package .
accession numbers for sequences used for comparison : bovine ednrb , s63513 ; human ednrb , jq1042 ; rat ednrb , i57950 ; canine ednrb , af276427 . segregation of ednrb compared with the segregation of the white spotting phenotype .
genomic dna digested with pvuii was probed with a radioactively labeled fragment of ednrb dna which detected a pvuii restriction fragment length polymorphism ( rflp ) between the border collie and newfoundland grandparents .
bc , dna from the border collie grandparent ; n , dna from the newfoundland grandparent .
f1 , dna from the first generation hybrids of the newfoundland  border collie cross ; f2 , dna from the progeny of intercrosses between the f1 hybrids ; s , white spotted ; ns , not white spotted .
segregation of kit was followed by detection of a simple - sequence repeat polymorphic difference between the newfoundland and border collie grandparents .
bc , dna from the border collie grandparent ; n , dna from the newfoundland grandparent ; f1 , dna from the first generation hybrids ; f2 , dna from the second generation intercrosses .
we thank mark neff , sara okamura , ashild vik and michael bannasch for critical review of the manuscript .
this work was supported by a gift from martin and enid gleich and by the richard and rhoda e. goldman foundation .
 background : white spotting patterns in mammals can be caused by mutations in the genes for the endothelin b receptor and c - kit , whose protein products are necessary for proper migration , differentiation or survival of the melanoblast population of cells . 
 although there are many different dog breeds that segregate white spotting patterns , no genes have been identified that are linked to these phenotypes.results:an intercross was generated from a female newfoundland and a male border collie and the white spotting phenotypes of the intercross progeny were evaluated by measuring percentage surface area of white in the puppies 
 . the border collie markings segregated as a simple autosomal recessive ( 7/25 intercross progeny had the phenotype ) . 
 two candidate genes , for the endothelin b receptor ( ednrb ) and c - kit ( kit ) , were evaluated for segregation with the white spotting pattern . 
 polymorphisms between the border collie and newfoundland were identified for ednrb using southern analysis after a portion of the canine gene had been cloned . 
 polymorphisms for kit were identified using a microsatellite developed from a bacterial artificial chromosome containing the canine gene.conclusions:both ednrb and kit were excluded as a cause of the white spotting pattern in at least two of the intercross progeny . 
 although these genes have been implicated in white spotting in other mammals , including horses , pigs , cows , mice and rats , they do not appear to be responsible for the white spotting pattern found in the border collie breed of dog . 
Background:
Results:
Conclusions:
Background
Results and discussion
Materials and methods
Acknowledgements