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Review Article Advancements in Life Sciences – International Quarterly Journal of Biological Sciences

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Date Received: 02/05/2020; Date Revised: 20/08/2020; Coat Colour Genetics: A Review Date Published Online: 31/08/2020; 1,2 1 1 3 Rashid Saif *, Ali Iftekhar , Fatima Asif , Mohammad Suliman Alghanem

Authors’ Affiliation: 1. Institute of Abstract Biotechnology, Gulab Devi Educational anis lupus familiaris is one of the most beloved species with hundreds of world-wide recognized Complex, Lahore - Pakistan , which can be differentiated from each other by specific morphological, behavioral and adoptive 2. Decode Genomics, traits. Morphological characteristics of dog breeds get more attention which can be defined mostly by 323-D, Town II, coat color and its texture, and considered to be incredibly lucrative traits in this valued species. Although Punjab University C Employees Housing the genetic foundation of coat color has been well stated in the literature, but still very little is known about the Scheme, Lahore - growth pattern, length and curly coat trait . Skin pigmentation is determined by eumelanin and Pakistan 3. Department of pheomelanin switching phenomenon which is under the control of Melanocortin 1 Receptor and Agouti Signaling Biology, Tabuk genes. Genetic variations in the genes involved in pigmentation pathway provide basic understanding of University - Kingdom melanocortin physiology and evolutionary adaptation of this trait. So in this review, we highlighted, gathered and of Saudi Arabia comprehend the genetic mutations, associated and likely to be associated variants in the genes involved in the coat color and texture trait along with their . Moreover, genetic diversity of other associated genes Corresponding Author: Rashid Saif were also pointed out to understand this phenomena in detail along with their genotypes for better understanding Email: the expression and mode of inheritance of this trait for describing dog breeds with more accuracy. [email protected]

How to Cite: Saif R, Iftekhar A, Asif F, Alghanem MS (2020). Colour Genetics: A Review. Adv. Life Sci. 7(4): 215-224.

Keywords: Dog breeds; Dog color variants; Dog texture genetics; Dog differentiation

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from the pool and actively searched for Introduction unique and attractive features [5,9,10]. Coat color and texture are important characteristics of domestic dog breeds. Though the genetic groundwork of Variation in Canine genome coat color has been well characterized, incredibly little is Variation in canine genome results from artificial known about the genes persuading coat pattern. In selection and domestication of the dogs. Possible domestic dogs there is a wide range of coat colors, genome variation mechanisms in dog include: mutational patterns and textures. Conceivably, throughout evolution hotspots, copy number variants (CNVs), chromosomal of the canine from their wild ancestors, coat color in fission, and gene duplications, or simple dog breeds have been the unintentional effect of some sequence repeats, high levels of repeat purity and the different selective process [1]. Divergence of the canine plentifulness of SINEC_Cf (carnivore-specific short- into a different type and ultimately separate breeds interspersed nuclear elements) elements in the canine accelerated color variation as aspects such as genome [8,11]. camouflage and visibility aided the dogs’ functionality. Variation in certain genes are associated with the coat Coat color and texture of the are important texture and color in dogs. These variations are of great characteristics of dog breeds. Studying these coat interest for breeders and pet owners. For example, two variations in dog breeds can reveal how the genes variants of KRT71 (-71) gene exists and are found responsible for coat colors and texture are transferred to be associated with curly coat in certain dog species and evolved over time giving rise to such great [12,13] and variations in the SGK3 gene are highly morphological diversity. associated with hairlessness in dogs and American hairless [14,15]. Genetics of dog breeds In the early 1990s, the dog genome project was initiated, motivated by the desire of researchers to discover the Methods genes that led to many of the illnesses that Literature search strategy and selection criteria dogs endured. Some dog breeds have been in nature for A systematic search was carried out from PubMed, only a couple hundred years. Most display reduced Google Scholar and Google Web Browser by providing genetic diversity owing to the common descent of dog key terms “dog, dog coat genetics, dog coat color, dog breeds from a small number of owners, produced by coat texture, dog genetics, dog coat inheritance etc.”. crossing closely related individuals [2]. In addition, According to the particular contents further literature was breeds often experience population obstacles as breed screened and analyzed. In this study 40 research articles popularity waxes and wanes. Modern breeds of dogs were selected to make a comprehensive review. have a broad range of colors, patterns and textures. Knowledge of the genetics of canine coat coloring and Discussion patterning and coat texturing and length has improved a Dog coat texture great deal in recent years. The dog has 39 pairs of The appearance of a canine coat can be determined not coming from each parent [3] and a pair of only by the color and distribution of a pigment, but also these determines the sex of dog and all other may by the features of the coat such as length, curls or possess the coat color and texture genes. presence or absence of coat hair. Some of the most fascinating advancements in recognizing the evolution of Canine genetic diversity dog variability are the dynamic features of the texture The closest relative to the dog is undeniably the and color of the fur. Coat texture and coat color are most grey wolf. The dog differs by only 0.04% in nuclear DNA important features of domestic dogs. Canine fur texture, coding sequence from its ancestor wolf [4,5]. pattern, length and curl are controlled by variation in only Mitochondrial DNA (mtDNA) phylogenetics studies three genes and account for almost all coat phenotypes between domestic dogs and show multiple in domestic dogs. These three genes include: RSPO2 backcrossing events that took place between 15,000 and (R-spondin–2), FGF5 (fibroblast growth factor–5), and 100,000 years ago [6,7]. One may wonder if today's grey KRT71 (keratin-71) [16]. wolf comprises all the variability needed to create the variance found in modern domestic dogs. Genes associated with dog coat texture The artificial selection for breed standards started Three genes RSPO2, FGF5 and KRT71 are mostly during the Victorian era, indicating that astute breeders, responsible for phenotypic variations in dog coat. A 167 novel genes and effective breed selection have all led to deletion at the 3′ end of RSPO2 is responsible the morphological variability found in dogs in the past for wiry hair and furnishings (increased hair growth on 200 years [8]. face and legs) as seen in Portuguese and There are over 400 domestic dog species which sho [16,17]. KRT71, on the other hand, is w distinctive differences in morphology, physiology and associated with curly hair in dogs [13], however, the behavior [9]. The main reason for phenotypic diversity in variant of the gene is not present in all curly coated hair dogs is because of two reasons: 1) Initial wolf dogs like curly coated (CCRs) and later a domestication more than 15,000 years ago [6,7,9] and 2) second variant of KRT71 gene was studied that is found The development of distinct breeds in the last few in CCRs associated with curly and wavy hair and can be hundred years in which humans picked small groups of possibly associated with follicular dysplasia in dogs [12].

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FGF5 gene is associated with long or short hair coat in Hair length in dogs is usually classified both as long or dog. Examples of FGF5 carrier dogs include Afghan short and is controlled by two alleles: L > l. L represents , and many other breeds [8,18]. short hair and l represents long hair. Hair length is a very Apart from these three genes there are some other important trait and varies among different dog breeds. genes associated with hairlessness seen in many dog Hair length variation is mainly controlled by variation in breeds. SGK3 and FOX13 genes are associated with FGF5 gene [8,16,20]. lack of coat or hairlessness. FOX13 is studied in Chinese Crested, Peruvian Inca Orchid (Peruvian FGF5 gene (L Locus) Hairless) and Mexican breeds [19] and Coat appearance is the main morphological distinctions SGK3 is studied in Scottish Deerhound dogs [15] and among various dog breeds. FGF5 gene is associated American hairless terriers [14]. with short or long fur in many dog breeds [8,16,18]. Long coat is inherited in autosomal recessive way [18,20], Gene variants associated with dog coat texture When two FGF5 carriers are mated, theoretically, 25% Genetic variation is seen is the genes of dog associated long coated offspring will born. Five FGF5 variants with coat and fur texture and most of these variants have studied in different dog breeds, responsible for long/short similar characteristics. Variants of the genes associated hair phenotype, are discussed in Table 2 [18]. with dog coat texture with respect to their location, mutation and role in dog coat genetics are FGF5 Gene variants Protein variants Breed described below. c.284G>T Cys95Phe Many g.8193T>A Block Exon 2 splicing c.556_571del16 p.(Ala186ThrfsX69) Eurasier Curly coat in dogs c.559_560dupGG p.(Arg188AlafsX73) Afghan Hound, Eurasier Classification of dogs’ breed on the basis of curly hair is c.578C>T p.(Ala193Val) , Siberian challenging. Large variability of curl types is present. Husky, Samoyed Curly hair coat is a multifarious trait. Dog breeds can be Table 2: Different variants of FGF5 (fibroblast growth factor–5) classified into different classes on the basis of coat seen in different dog species associated with long/short fur texture. The large Curly Coated is a superlative characteristics. dog who holds all drive and determination that all retrievers retain. Their coat is made of dense mass of Wiry hair and furnishings small, tight, crisp black and reddish-brown curls. Coat Wire coats in canine are rough and spiky. It is not soft as texture in curly hair varies from soft waves to tight curls. like other coat textures. Shedding capability is lost in wire coat texture breeds due to allergy. Dogs with this texture KRT71 gene (R or Cu Locus) have found another feature that is beard. This additional Curly coat type in domestic animals is a relatively comm feature is considered as furnishing. Mutation in RSPO2 on phenotype. KRT71 gene in dogs is found to be signaling protein gene will lead to wiry hair. The insertion associated with curly hair phenotype. Two variants are of 167 base pairs in this signaling protein gene results in found in dogs that are associated with curly and wavy the production of more keratin which ultimately produce hair coat. The variant c.451 C>T is a missense variant in more hair. Exon 2 [12] of KRT71 gene and it results in p.Arg151Trp substitution [13,16]. This variant is highly associated with RSPO2 gene curly coat in Irish Water and Standard At the 3′ end UTR of the R-spondin-2 (RSPO2) [13]. chromosome (position 11,634,766), a deletion of 167 bp However, during the testing of Curly Coated is strongly associated with wiry hair and the furnishings; Retrievers, none of them carried this variant of gene the latter is indicative of the moustache and eyebrows in suggesting that there is a second variant of KRT71 gene many dog breeds [8,17]. This mutation has no impact on that is associated with curly coat in CCRs [13]. A 2018 the RSPO2 gene protein coding region, but the 3′UTR studies found a second variant of KRT71 gene from often codes for elements that affect the stability of mRNA whole-genome sequencing of Curly Coated Retrievers. [16,18]. In 2009, Cadieu investigated whether the This is an indel variant, c.1266-1273 del-in ACA, in Exon insertion was linked to a change in RSPO2 expression 7 and cause frameshift and it results in frequency. In muzzle skin biopsies of dogs with p.(Ser422ArgfsTer?) (Table 1). This variant is fixed in furnishings, they found triple spike in RSPO2 Curly Coated Retrievers [12]. transcriptions [16].

KRT7 Gene Protein variants Breed Reference Furnishings (Wh “wire hair” Locus or F Locus) variants Furnishing is inherited as an autosomal dominant trait. c.451 C>T Arg151Trp Irish Water [13] Dog Furnishing refers to distinct eyebrows and moustaches c.1266-1273 Ser422ArgfsTer? Curly Coated [12] in certain dog species. It also represents increased hair del-in ACA Retrievers growth on face and legs. RSPO2 gene is closely Table 1: Different known variants of KRT71 (keratin-71) gene in associated with furnishing in dogs. Cadieu tested dogs associated with curly coat. RSPO2 gene in seven dog breeds with distinct eyebrows and moustaches and found a 167 base-pair insertion in Hair Length (Long/Short) untranslated region of the gene. A dog lacking furnishing

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You’re reading Dog Coat Colour Genetics: A Review carries two wild type alleles without any insertion in mutation in FOX13 gene. The whole body of the dog, in RSPO2 gene [16]. Furnishing is seen in the following this case, is covered with long silky coat and no notable breeds: Brussels Griffon, , abnormalities are seen [19]. , German Shorthaired , Irish Soft- Coated Wheaten SGK3 gene SGK3 is another gene strongly associated with Improper coat (IC Locus) hairlessness in a few dog breeds. Variants of these Insertion in the RSPO2 gene can sometimes result in genes were first studied in American Hairless Terrier [14] improper or non-standard coat phenotype in some dog and later a new variant of the gene was found to be breeds as seen in Portuguese Water Dogs (PWD), the associated with hairlessness in hairless Scottish wild type allele results in Improper Coat [17]. Improper Deerhound (Table 3) [15]. Coat is inherited as an autosomal recessive trait. A non- standard fur of short hair on the head, face, and legs American Hairless Terriers characterizes the Improper coat phenotype. There is Hairlessness in Hairless American Terrier is smooth, straight or moderately wavy hair on the entire inherited as an autosomal recessive trait and the dog body. Dogs with improper coat phenotype are needs to inherit it from both parents. For American homozygous for wt-allele whereas, dogs with typical coat Hairless Terriers, homozygosity is not fatal as in other carries 1 or 2 alleles with insertion. species of hair. This hairlessness mutation is not related Wild type allele rate was estimated from randomly to any medical problems affecting the appearance of selected genotypic samples in the American population teeth or fertility of the dog as seen in the case of mutation of PWD to be 12% [17]. Although Improper coat in FOX13 gene. The dogs with Mutation in SKG13 gene phenotype do not affect the health of dogs but their are born healthy and with a light/scattered coat that is breeding and potential of selling offspring is difficult. lost within the first month of their conception, this condition may be referred to as hypotrichosis [14]. Lack of coat / hairlessness A mutation in Serum and Glucocorticoids (SGK3) Hairlessness or lack of coat is a very common phenotype resulting in 4 base pair deletion and frame-shift seen in several dog species and the best examples c.283_286delttag was identified in 2017 which results in include American hairless terrier, Peruvian Inca Orchid Hairlessness in American Terrier dog species [14]. and Chinese crested. Hairlessness in dogs mainly results from mutation in two genes: FOX13 and SGK3 Hairless Scottish Deerhounds [7,14,15,19]. Mutations in these genes result in different Recently a new variant of SKG13 gene has been levels of hairlessness, most of the times the whole body discovered associated with hairlessness in some dog is hairless. Based on the species, there can be hair tufts breeds specially in Scottish Deerhounds [15]. Scottish on the distal limbs as well as the top of the head. Deerhound dogs are known to have a 3 to 4 inches long Mutation in these genes may also result in abnormal and wiry coat and hairless Scottish Deerhound dogs are teeth patterns and missing teeth. However, some seem extremely rare. In 2019, Parker et.al found a new variant only to have minor anomalies like fragile toenails [19]. of the gene SGK3 in hairless Scottish Deerhounds after carrying out whole genome analysis. They predicted the FOX13 gene (Hr Locus) inheritance pattern of this mutation to be recessive and A mutation in FOX13 results in canine ectodermal both parents should carry the variant allele. The variant dysplasia which is characterized by a lack of hair coat is a single base pair insertion in exon 2 of the gene and and abnormal teeth. It is a frameshift mutation resulting results in frame-shift c.137_138insT. This mutation leads from a 7-bp duplication in exon 1 of FOX13 gene [19]. to early termination of protein and is predicted to be Mutations in this gene results in different levels of associated with hairlessness in Scottish Deerhound [15]. hairlessness, most of the times the whole body is hairless. Based on the species, there can be hair tufts on SGK3 Gene Protein variants Breed Reference the distal limbs as well as the top of the head. Mutation variants in these genes may also result in abnormal teeth patterns c.283_286delttag Val96GlyfsTer50 American [14] hairless terrier and missing teeth. However, some seem only to have c.137_138insT Glu47GlyfsTer3 Scottish [15] minor anomalies like fragile toenails [19]. Deerhound Hairlessness is inherited as an autosomal dominant Table 3: Different known variants of SGK3 (Serum and trait and this trait phenotype will only appear in dogs Glucocorticoids) gene in dogs associated with Hairlessness in when they carry only 1 mutant allele. The dogs carrying American Terriers and Scottish Deerhound. 2 mutant alleles i.e. from both parents die in prenatal stage. Dogs carrying FOX13 gene may have two Association of genes with dog coat phenotypes phenotypes: 1) Hairless Dogs 2) Powder-Puff or Coated There are four main coat characteristics seen in different [19]. dog breeds: 1) Hair length (short or long), 2) The Hairless dogs have a clean smooth body appearance presence or absence of curls, 3) Furnishings and with no hair on body except a crest on head feet or tail. Improper coat and 4) Lack of coat (hairlessness). With Hairless Dogs carry 1 copy of mutation in FOX13 gene. regard to hair types, various variations of the genes They have weak and abnormal teeth and neonatal RSPO2 (R-spondin-2), FGF5 (fibroblast growth factor-5), deaths are very common. Powder-Puff carries no and KRT71 (Keratin 71) were found to be representing,

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together, the majority of the phenotypes of purebred MC5R gene impacts the level of shedding in different dog dogs (Table 4) [16]. breeds [21]. It is very closely linked with the RSPO2 gene. It is a complex inheritance trait and found in all dog Phenotype FGF5 RSPO2 KRT71 FOX13 SGK3 Breed breeds. Short Absent Absent Absent Absent Absent Basset MC5R gene is normally expressed in Hound Wire Absent Present Absent Absent Absent Australian glands where it produces a waxy substance known as Terrier sebum. Sebaceous glands secretions keep the hair Wire and Curly Absent Present Present Absent Absent Airedale flexible and supports lubrication, thermal control and Terrier Long Present Absent Absent Absent Absent Golden water repellence. Mutation in MC5R gene, which is a Retriever point mutation, disrupts the sebum structure resulting in Long + Present Present Absent Absent Absent Bearded Furnishing increased shedding [21]. This gene exists in two variants: Curly Present Absent Present Absent Absent Irish Water A and G. G variant is the original natural variant and a Spaniel Curly + Present Present Present Absent Absent result of evolution. Dogs with 1 copy of G variant have Furnishing Frisé high level of shedding whereas dogs carrying 2 copies Hairless torso, Present Present Present/ Present/ Absent Chinese long hair on Absent Absent crested have much higher levels of shedding. head, feet and tail Locus/Gene Mutation Genotype Phenotype Inheritance Hairless torso, Absent Absent Absent Present/ Absent Peruvian Pattern short hair on Absent Inca Orchid head Cuc/Cuc Curly coat Autosomal Hairless Absent Absent Absent Absent Present American Cu (KRT71) Point CuC/C Wavy coat Incomplete hairless Mutation C/C Straight coat Dominant terrier L/L Short haired Autosomal Table 4: Combination of different alleles produce different coat phenotypes. L (FGF5) Point L/l Short haired Recessive Mutation FGF5 gene variants are responsible for long/short coat hair phenotype. (carrier) l/l Long haired RSPO2 gene is associated with wiry hair and furnishings. KRT71 variants are F/F Furnishings Autosomal seen in curly coat dog breeds. FOX13 and SGK3, on the other hand are IC (RSPO2) Insertion F/IC Furnishings Recessive IC/IC Improper (Improper responsible for hairlessness in dogs. coat coat), Autosomal Dominant (Furnishing) Hr/Hr Embryonic Hr (FOX13) Duplication lethal Autosomal hr/Hr Hairless Dominant hr/hr Powder-puff SD/SD High SD (MC5R) Point shedding Complex Mutation sd/SD Moderate Inheritance sd/sd Low shedding Table 5: Inheritance pattern of coat texture in dog breeds.

Dog coat color Canine coat color is substantiated by the eight genes found in their genome. Variation in these specific genes Figure 1: Combination of different alleles produce different coat alter its expression which results in different coat colors. texture. Dog (A) is an example of short hair phenotype, (B) have a A precise alteration at a single locus of the particular wiry coat, (C) dog breed shows wiry and curly hair, (D) is an gene affects the coat color which in results classify dog example of long hair coat, (E) shows long hair with characteristic breeds in various classes. furnishing, (F) is a standard curly coat, (G) represents furnishing Dog coat color is ruled by their parent genes. There on curly hair type, (H) have hairless torso with long hair on head, are about 19,000 genes in a dog genome [22]. A limited feet and tail, whereas, (I) is hairless with short hair on head, feet number of genes control the phenotypic appearance of and tail, (J) is an example of . (All individual dog dogs such as color, patterns and textures. There are pictures are taken from American www.akc.org). several coat colors. Eumelanin: which is black, chocolate brown and greyish in color, Phaeomelanin: Shedding containing tan pigment with all shades of red and pink Shedding is a very common and natural phenomena and White: there is no pigment (lack of pigment). seen in different dog breeds. However, all dogs do not Coat color is systematized by pigment. There shed the same. Some dogs shed more hair while others are only two basic pigments which result in the color less or not at all. are known to shed very little. variations in canines. These include Eumelanin – black The degree of shedding, as in many instances, is not and Phaeomelanin – red [22]. Deviations in these strictly controlled by one gene but rather by a pigments are responsible for different colors (Figure 3; combination of genes. There is a close relationship A-F). between shedding MC5R (SD locus) and furnishing Melanocytes production occur by two types of melanin RSPO2 (F locus). the one is black/brown photo-protective eumelanin pigment, and the other is red-yellow cytotoxic MC5R gene (SD locus) phaeomelanin pigment. Many melanogenic pathways

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You’re reading Dog Coat Colour Genetics: A Review are controlled by numerous paracrine factors secreted E) [23]. White hair on dogs tends to occur when the cells by surrounding keratinocytes by stimulating the do not produce any pigment (Figure 3; F). This impacts alteration among phaeomelanin and eumelanin. In this some areas of the dog's coat most often. When pathway, microphthalmia transcription factor (MITF) eumelanin is not produced in the nose, the dog may end plays a vital role by regulating the expression of up with a pink nose or if it is not present in eyes it results the TYR (), TRP-1 (Tyrosine Related Protein) in blue eyes. In rare cases, the whole body of dog is and DCT () genes that code affected which results in albino dogs [24]. enzymes involved in pigment production. Gene variants associated with Canine coat color Pigment-type switching diversity Eumelanin and pheomelanin synthesis is regulated There are 8 major genes and their variants responsible through an intercellular signaling pathway in the hair for huge diversity in dog coat color. Dog coat color genes follicle at a specific location and time. The pigment-type can be characterized into Pigment Dilution genes (C switching gene MC1R and Agouti signaling protein Locus, B Locus, D Locus, M Locus and G Locus) and the (ASIP) gene are the contributors of this pathway. MC1R Pigment switching genes (A Locus, E Locus and the K is a G-protein coupled receptor expressed on the Locus). Some of the genes associated with some Loci surfaces of melanocytes and it promotes eumelanin (like C Locus or G Locus) are not completely investigated synthesis. This receptor is regulated by ASIP, which yet. Dog coat color genes and their variants are inhibits the MC1R signaling pathway promoting the discussed further: synthesis of pheomelanin. Mutations in ASIP or MC1R genes lead to altered distribution of eumelanin and ASIP gene (A or Agouti locus) pheomelanin in dogs. MC1R gain of function mutation ASIP (Agouti signaling protein) gene is found in results in excessive eumelanin production, whereas chromosome 24 of dogs. There are four known alleles of ASIP gain of function mutation is responsible for this gene and in the following hierarchy Ay > aw > at > a excessive pheomelanin production. As a consequence, [25]. Locus E (MC1R) and Locus K (CBD103) affect the ASIP has dominant yellow/red allele and recessive black expression of locus A (Figure 2). In the presence of e/e allele and MC1R has a dominant black allele genotype and KB allele of aforementioned Locus/genes respectively, A Locus is not expressed [25-28]. If allele ay is present in dog then the dog will have coat color and the presence of other alleles does not matter. This suggests that the allele ay is dominant over other alleles of the gene. If aw allele is present in combination with aw/aw, aw/at or aw/a the dog will have a wild sable coat color suggesting that the allele aw is dominant in this case over a and at. In the case of at/a or at/at the dog will have a tan or black or tricolor phenotype. The allele a is recessive and dogs with genotype a/a have black coat color (Table 4). The aw allele is the wild type allele and can be traced in wild wolfs and coyote [25,28] and causes the eumelanin to change in phaeomelanin which is

Figure 2: Canine coat pigment-switching mechanism in a responsible for agouti (like the color of grey wolf) color. melanocyte. Melanocytes produce eumelanin and pheomelanin in A SINE insertion has been identified to be present in specialized organelles called melanosomes, Eumelanosomes have intron 1 of ASIP gene at allele and the a allele and absent a more regular structure as compared to Pheomelanosomes. in aw and ay alleles. The ay allele developed Eumelanogenesis is depicted on top portion of the figure and independently and two mutations c.246G>T (A82S) a Pheomelanogenesis on the bottom portion. MC1R (a G-protein c.250G>A (R83H) in this allele differentiate it from aw coupled receptor expressed on the surface of melanocytes) and its allele [25]. The at allele is only different from the aw allele second messenger cAMP regulate the type of pigment synthesized because of the presence of the SINE insertion and the by melanocytes. Tyrosinase is an enzyme involved in conversation recessive allele a is caused as a result of SINE insertion of tyrosine to melanin. High MC1R signaling triggers the expression of TYR, TYRP1, OCA2 and PMEL, leading to enhanced as well as c.286C>T (p.R96C) in exon 4 [27] (Figure 3; eumelanin production. Whereas, low levels of cAMP triggers G-J). SLC7A11 cysteine transporters which lead to enhanced pheomelanin production. CBD103 is a MC1R ligand produced by K Genotype Coat Color Genotype Coat Color locus, it prevents the ASIP from inhibiting MC1R and hence Ay/Ay Fawn/Sable aw/at Wild sable promotes eumelanin production. When the melanosomes mature, Ay/aw Fawn/Sable aw/a Wild sable they’re transported to dendritic. MATP membrane Ay/at Fawn/Sable at/at Tricolor y t regulate the internal melanocyte environment. A /a Fawn/Sable a /a Tricolor aw/aw Wild sable a/a Black Table 6: Associated genotype with coat color phenotype in dogs Generation of huge range of dog coat colors carrying Agouti gene. Eumelanin and phaeomelanin produce a wide variety of dog coat colors in all various combinations (Figure 3; A- TYRP1 gene (B or brown locus)

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TYRP1 gene (Tyrosine related protein 1) is mapped in many dog breeds. Dog breeds showing Em allele the chromosome 11 of the canine genome and it is characteristics include: , Afghan and involved in the production of brown or black eumelanin. Staffordshire . EG allele can result in grizzle Locus B is also referred to as Live gene because of coloration in dog breed as well as domino color in brown color phenotypic characteristics. Locus B has 2 Afghan . This allele results as a mutation known alleles: allele B and allele b which controls the c.233G>T in the gene MC1R and this mutation is seen in black color of coat and nose and the brown color of coat both the dogs. The EG allele grizzle and domino and nose respectively (Figure 3; K-L). The color mutation coloration only appear if the dog is carrying two at alleles of Locus B is only effective on dogs that carry E/E or E/e and stays hidden if the dog carries ay allele. The Eh allele on Locus E (Extension) [29]. In some dog breeds, like results from c.250G>A mutation and inhibits eumelanin the French , the exact mutation responsible for and it is studied in the dogs with sable phenotype. The chocolate brown color is not exactly known yet. hierarchy of Locus E is: Em > Eg > E > E> e (Table 5). The wild type allele B is responsible for the black coat color and it is dominant over allele b (Figure 2). Dogs with Genotype Coat Color Genotype Coat Color allele B will have black coat color and black nose. The E/E Black Eg/ Eg Grizzle/Domino brown eumelanin is only produced when two b alleles are E/e Black Eg /E Grizzle/Domino present (Figure 3; K-L). For brown coat color three e/e Red/Yellow Eg/e Grizzle/Domino s pigment variants of the TYRP1 gene are discovered: b Em/Em Black Mask Eh/Eh Sable color (c.991C>T), bc (c.121T>A) and bd (c.1033_1035del) and Em/E Black Mask Eh/E Black m h aus E /e Black Mask E /e Sable color the fourth variant b c.555T>G.c is only found in Em /Eg Black Mask Eh/Em Black Mask breeds. Table 7: Associated genotypes with the coat color phenotype in dogs carrying MC1R gene alleles. MLPH gene (D or dilution locus) MLPH gene (Melanophilin protein) is mapped in the CBD103 gene (K or dominant black locus) chromosome 25 of the dogs and the mutation is The CBD103 (Beta-defensin gene) is mapped in the responsible for lighter or diluted coat colors (Figure 3; M- chromosome 16 of canine genome and is responsible for N). The recessive alleles of this gene are represented black coat color [33]. Three alleles in the K locus are with d. Variations in MLPH gene affects the discovered: KB (Dominant Black), Kbr () and Ky melanosomes transport in melanocytes resulting in a (Recessive yellow or non-solid black). The K Locus is lighter coat color or dilution (Figure 3; M,N) [29]. linked with the E Locus and is not expressed if the E There are two allelic variants of D locus: d1 (c.-22G>A) Locus genotype is e/e and requires at least one wild type and a recently discovered allele d2 ( c.705G>C) [30]. E allele (Figure 2) [34,35]. KB is a dominant allele and it Diluted color is inherited as an autosomal recessive trait. inhibits the Agouti (A Locus) and results in solid black The d1 variant is seen in the following breeds: Labrador coat color phenotype. Kbr is another mutation in the K Retriever, Australian shepherd and German dog etc. and locus gene, however, it allows the expression of Agouti the variant of d2 is studied in Chow-Chow, Sloughi, (A Locus) and results in brindling. This allele is dominant , Papillon and . The non- over Ky allele but recessive with KB. Ky allele is the third diluted allele, in case of dogs with no dilution, is the allele of K Locus and allows the expression of Agouti dominant D allele. The genotype of dog carrying D locus locus too but without brindling. In homozygous state i.e. allele could be homozygous as d1/d1 or d2/d2 or it can be Ky/Ky locus A is responsible for dog coat color but since compound heterozygous d1/d2 [3]. this allele is recessive over other two alleles, in any other genotypic form the A locus is not fully expressed as it is. MC1R gene (E or extension locus) MC1R gene (Melanocortin 1 receptor) is mapped in the TYR and SLC45A2 genes (C or colored/chinchilla chromosome 5 of canine genome [29,31] and controls locus) the production of pigment in melanocytes (Figure 3; O- The TYR (Tyrosinase) gene mapped in chromosome. 21 S). Melanocytes control dog coat color. The wild type of canine causes in many animal species and dominant allele of the gene is E which produces is thought to be responsible for white coat phenotype in eumelanin (black pigment). Other alleles in the E locus dogs. The mutations in this gene is not completely series include Em (black mask on face), e (recessive studied yet. Five alleles of C locus are theorized: C allele) and the newly discovered alleles Eg and Eh. (normal wild type allele expressing phaeomelanin, The allele e is created by a mutation in the gene results in deep intense coat), cch allele causes partial c.916C>T that turns the eumelanin to phaeomelanin in inhibition of phaeomelanin production in coat, ce the coat [31,32] this recessive form of allele results in completely inhibits phaeomelanin production and results bright red or yellow coat color in many dog breeds. The in a white coat color, cb, cd and ca alleles results in albino Em allele is a result of point mutation c.790A>G and it is like appearance in dogs. None of these alleles are responsible for melanistic mask. The expression of this completely studied yet and some experimental works allele depends on the alleles on Locus A and Locus K. (cDNA study of albino and white coated dogs) suggest Dogs that are black or any dark colored dogs have a no polymorphism in this gene [36]. melanistic mask that is hard to distinguish and the A second gene SLC45A2 that is found to be involved melanistic mask may not always be black as seen in in albinism in humans is thought to the gene resulting in

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C locus in dogs. Experimental works show a Length of PolyT variation Phenotype sequence homozygous deletion in white Dobermans dogs in this 35-62 T Cryptic Merle (Mc) Only small merle gene [36]. breed shows the characteristics patches of Albinism but does not carry mutation in TYR and 63-73 T Dilution Merle (Md) Diluted merle patches SLC45A2 genes but a second mutation in SLC45A2 74-85 T Merle (M) Regular merle pattern 85 T or more Harlequin (Mh) Black patches gene leaving a room open for much experimental work Table 8: Length of PolyT insertion and the merle patterns type. to be done on C locus. PSMB7 gene (H or harlequin locus) The PSMB7 gene is a modifier gene of merle SILV, together these two loci control the harlequin phenotype (white coats with black spots or patches all over) in some dog species i.e. (Figure 3; X). H Locus gene has been mapped in the chromosome. 9 of canine. H locus results from a point mutation in the 20S proteasome β2 subunit at the H locus [37-39]. There are two alleles of H locus: H and h, H allele results from the point mutation and the h allele is the wild type allele. Dogs will not have harlequin coat phenotype if they do not carry the merle mutation. H/H genotype is lethal for dogs and the dog will die in embryonic stage.

MITF gene (S or white spotting locus) The MITF gene (Microphthalmia Associated Transcription Factor) is responsible for irregular white Figure 3: Relationship between the E Locus, A Locus, K Locus, B patches or white spotting on dog body. White coloration Locus and the D Locus. Expression of E Locus is important for the in dogs is not caused by lack of eumelanin or expression of K, A and B Locus. e/e genotype results in recessive phaeomelanin pigments but white dogs lack any br red coat color phenotype, KK, Kk and Kk produce solid black coat pigment. The MITF gene is responsible for the color whereas kbrkbr and kbrk are responsible for brindle coats. Expression of Locus A is affected by E and K Loci and Locus A is development of melanocytes and the white pigmentation not expressed in case of e/e genotype or Kbr allele. A Locus results from the lack of melanocytes. There are 4 known produce Fawn, Wolf grey Tricolor or Recessive black coat alleles of the S locus: S Locus (Solid color with no white phenotypes. In case of b/b genotype the eumelanin changes from patterns), si (Irish spotting, white spots often seen as black into chocolate or liver brown. d/d genotype results in coat white collar, belly and legs), sw (Extreme white spotting) color dilution. and sp (). S allele is the wild type and results in no white pigmentation whereas a SINE insertion SILV or PMEL17 gene (M or merle locus) mutation is responsible for other allele variants. There SILV gene (Silver Locus protein) formerly known as the are two MITF gene promoters that carry mutations, the PMEL17 gene (Pre-melanosome protein) is linked with MTIF-M promoter has three mutations (SINE insertion, some health issues in dogs and is responsible for SNP and LP-Variable Length Polymorphism) and the irregular diluted or solid colored patches on dog coat MITF-1B promoter has one mutation which is the 12bp (Figure 3; T-W). Merle gene has been identified in many deletion in exon 1B. Mutations in several MITF gene dog species but this specific coat pattern phenotype associated regions results in white spotting. Piebald occurs in only few dog breeds like Australian Shepherd means irregular spotting and the amount of white a dog and etc. Merle gene only dilutes eumelanin expresses as phenotype depends on the number of pigment. A black coated dog showing merle patterns is allele variants a dog carry and Piebald allele is recessive known as a Blue Merle and brown dog having merle to the S allele. g.21,836,232_218,364,27ins>del pattern is known as Red Merle [37]. mutation in the MITF gene results in the insertion of There are two alleles of merle gene: M and m whereas, SINE-Cf element that results in extremely white spots m is the normal wild type allele. Merle mutation results sw/sw or piebald sp/sp. This mutation is not seen in the as a SINE insertion in the SILV gene this insertion is a case of S and si alleles [40]. PolyT insertion of a variable length and the length of this insertion determines the merle phenotype in dogs (Table Locus Inheritance Locus Inheritance 6). Merle is inherited as an autosomal, incomplete trait. Pattern Pattern A Locus Ay > aw > at > a Em (Melanistic Autosomal In the case of Mx allele the x represents the length of mask) Dominant PolyT sequence insertion. There are 25% chances of a B Locus Autosomal H Locus Autosomal Recessive Dominant M/M dog to be born when two M carries are bred, this is D Locus Autosomal K Locus Autosomal “Double Merle” and the dogs will have hearing or vision Recessive Dominant E Locus Autosomal M Locus Autosomal deficiencies. Hidden merle is when the merle is not (Red/Yellow) Recessive Dominant recognizable phenotypically but the dog is genetically Eg Locus Complex S Locus Autosomal Co- Mx. (Grizzle) Inheritance dominant Table 9: Inheritance pattern of coat color in dog breeds.

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CONCLUSION Breeding for a particular trait in dogs would be quite expensive and a tedious job. The progeny needs to reach their maturity level only after which the determination of the success of the cross could be carried out. If the trait is very complex, a lot of time and effort would be needed to achieve a desirable outcome. Marker-Assisted Selection (MAS) is used to help reduce the amount of time required to determine whether the progeny have the trait or not. Another advantage is that it’s a cost-effective technique with reference to screening of traits. By detecting the unique trait at the level of DNA, Figure 4: Schematic diagram of coat color diversity in dogs. (A-D) represent different shades of Eumelanin, (E) represents identification of positive selection can occur quite early. Phaeomelanin and (F) is White. (G-J) dog breeds are carrying Agouti Another advantage of this technique is that these gene (Locus A), and (K-L) dog species are examples of black coat markers are not affected by environmental changes. color and brown coat color resulting from the (B Locus). (M-N) dog species have diluted coat color followed by (O-S) dog breeds carrying the (E Locus). (T-X) are dog breeds with different types of Author’s Contribution Merle and Harlequin patterns. All authors contributed equally to this review.

Coat color and texture variability in Pakistani dog breeds Conflict of interest Pakistani dog breeds are found to be different from The authors declare no conflict of interest relevant to this foreign dog breeds not only in their coat color but also in review article. some other characteristics. Pakistani is black, brown, fawn and brindle. It requires minimal grooming. References Seasonal flea treatment is necessary. This is one of the 1. Hédan B, Corre S, Hitte C, Dréano S, Vilboux T, et al. Coat colour in superlative selections if there is no time, skill or money dogs: identification of the merle locus in the Australian shepherd to take care of a high maintenance dog. CNVs and SNPs breed. (2006); 2(1): 9. 2. Ostrander EAJAS. Genetics and the Shape of Dogs: Studying the leads to the variety of coat color, patterns and texture in new sequence of the canine genome shows how tiny genetic dog breeds of Pakistani and other countries due to the changes can create enormous variation within a single species. environmental and physiological conditions. (2007); 95(5): 406-413. Bakharwal Dog, an indigenous Pakistani dog breed 3. Kaelin CB, Barsh GSJTGotD. Colour, Texture and Length in the Dog. (2012); 57. has dense straight hair and supposedly carries l/l 4. Bardeleben C, Moore RL, Wayne RKJMp, evolution. A molecular genotype of FGF5 gene. Whereas and Gaddi phylogeny of the Canidae based on six nuclear loci. (2005); 37(3): Dog, 2 Pakistani dog breeds with straight medium-length 815-831. 5. Lindblad-Toh K, Wade CM, Mikkelsen TS, Karlsson EK, Jaffe DB, et coat hair. Most of the dog breeds in Pakistan have al. Genome sequence, comparative analysis and haplotype straight coat hair as a result of natural breeding and since structure of the domestic dog. (2005); 438(7069): 803. it is more suitable for Pakistani environmental conditions 6. Savolainen P, Zhang Y-p, Luo J, Lundeberg J, Leitner TJS. Genetic and it also saves grooming costs. evidence for an East Asian origin of domestic dogs. (2002); 298(5598): 1610-1613. In Pakistan there is a large number of street dogs 7. Wayne RK, Leonard JA, Vilà CJDdNg, paradigms a. Genetic (free-ranging urban dogs) including Pakistani Mastiff, analysis of dog domestication. (2006); 279-293. Gaddi Kutta and Indian Pariah dogs. The major reason 8. Shearin AL, Ostrander EAJPb. Canine morphology: hunting for genes and tracking mutations. (2010); 8(3): e1000310. for existence of huge number of stray dogs in Pakistan is 9. Vaysse A, Ratnakumar A, Derrien T, Axelsson E, Pielberg GR, et al. unplanned breeding. While this leads to a huge variability Identification of genomic regions associated with phenotypic in coat texture and color among dog breeds but variation between dog breeds using selection mapping. (2011); mutations may lead to various other genetic defects. 7(10): e1002316. 10. Vilà C, Savolainen P, Maldonado JE, Amorim IR, Rice JE, et al. Large amount of garbage exposed on streets and Multiple and ancient origins of the domestic dog. (1997); 276(5319): different areas enable stray dogs to survive and 1687-1689. reproduce. People would also feed stray dogs and keep 11. Minnick MF, Stillwell LC, Heineman JM, Stiegler GLJG. A highly repetitive DNA sequence possibly unique to canids. (1992); 110(2): them as “free roaming ”. These factors contribute to 235-238. huge coat variability among Pakistani dog breeds. 12. Bauer A, Hadji Rasouliha S, Brunner M, Jagannathan V, Bucher I, Adopting a dog in Pakistan depends on a large number et al. A second KRT 71 allele in curly coated dogs. (2019); 50(1): 97- of factors. Most often people would keep dogs to guard 100. 13. Salmela E, Niskanen J, Arumilli M, Donner J, Lohi H, et al. A novel their livestock like the Gaddi Kutta. Temperature is a KRT71 variant in curly‐coated dogs. (2019); 50(1): 101-104. huge factor that is considered before adopting any dog 14. Parker HG, Harris A, Dreger DL, Davis BW, Ostrander breed. Dogs that are easy to maintain and don’t require EAJPTotRSBBS. The bald and the beautiful: hairlessness in domestic dog breeds. (2017); 372(1713): 20150488. grooming very often are preferred because of economic 15. Parker HG, Whitaker DT, Harris AC, Ostrander EAJGG, Genomes, state of people and availability of services. Genetics. Whole Genome Analysis of a Single

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