AU2004249943B8

AU2004249943B8 - Genotype test

Info

Publication number: AU2004249943B8
Application number: AU2004249943A
Authority: AU
Inventors: Neale Fretwell; Paul Glyn Jones
Original assignee: Mars Inc
Current assignee: Mars Inc
Priority date: 2003-06-16
Filing date: 2004-06-16
Publication date: 2008-07-10
Anticipated expiration: 2024-06-16
Also published as: WO2004113570A3; GB0313964D0; AU2004249943A1; EP1636377A2; WO2004113570A2; JP2007528205A; AU2004249943B2

Description

I

00 GENOTYPE TEST ;Field of the invention The invention relates to a method for determining the nutritional, medical or behavioural needs of a dog.

C Background of the invention The domestic dog (Canisfamiliaris) species comprises a large number of distinct breeds. Hundreds of different dog breeds have been established. Popular dog breeds include Labrador retrievers, Golden retrievers, German shepherds, Dachshunds, Shih Tzu, Yorkshire terriers, Poodles, Rottweilers, Boxers and Cocker spaniels.

Dog breeds typically differ in size, conformation, behaviour and physiology.

Different breeds can vary in size by as much as two orders of magnitude, and have differing metabolic and nutritional requirements. Particular dog breeds also have food sensitivities or predisposition to disease, which require preventative treatments and/or diets. Differences also exist in the digestibility of nutrients among breeds.

Summary of the invention The invention allows the determination of the needs and characteristics of a dog, based on detection of SNPs (single nucleotide polymorphisms) in the dog. The invention takes advantage of the breed structure of the dog population to provide a genetic test for determining the nutritional, medical and behavioural needs of a dog by detecting particular SNPs in the dog. These needs may be ones for which the underlying genetic basis is unknown. The genetic test of the invention thus allows 2 00 knowledge of breed-specific characteristics to be applied to addressing the specific N, needs of a dog. The invention additionally provides SNP sequences that can be used Sin the genetic test.

Accordingly the invention provides: a method of determining the genetic breed inheritance of a dog, the method comprising: S(a) detecting the presence or absence of one or more breed-specific SNP markers in the dog; and identifying therefrom the genetic breed inheritance of the dog; use of one or more breed-specific SNP markers for identifying the genetic breed inheritance of a dog; a method for identifying the genetic breed inheritance of a dog, the method comprising: inputting genetic data from the dog to a computer system; (ii) comparing the data to a computer database, which database comprises information relating to breed-specific genomic SNPs; and (iii) determining on the basis of the comparison the presence or absence of one or more breed-specific SNP markers, thereby identifying the breed inheritance of the dog; a computer program comprising program code means that, when executed on a computer system, instruct the computer system to perform a method according to the invention; a computer system arranged to perform a method according to the invention comprising: means for receiving genetic data from the dog; j 00 (ii) a module for comparing the data with a database comprising N information relating to breed-specific genomic SNPs; and ;Z(iii) means for determining on the basis of said comparison the breed inheritance of the dog; a method of preparing customised food for a dog, comprising: determining the genetic breed inheritance of the dog by a N method according to the invention; generating a customised dog food formulation comprising components suitable for the breed(s) which contributed to the genetic breed inheritance of the dog, and which does not comprise components that are not suitable for the breed(s) which contributed to the genetic breed inheritance of the dog; and preparing a dog food according to the customised dog food formulation; a method of providing a customised dog food, the method comprising providing to the dog's owner, the person responsible for feeding the dog or a vet a food which contains components suitable for the breed(s) which have contributed to the genetic breed inheritance of the dog, and which does not contain components that are not suitable for the breed(s) which have contributed to the genetic breed inheritance of the dog, wherein the breed inheritance of the dog has been identified by detecting the presence or absence of one or more breed-specific SNP markers in the dog genome; and use of a computer system according to the present invention to make a customised dog food product.

Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps 4 0 0 or components or groups thereof, but do not preclude the presence or addition of one 0 or more other features, integers, steps, components or groups thereof.

Brief description of the Figure WO 2004/113570 PCT/GB2004/002559 Figure 1 illustrates schematically an embodiment of functional components arranged to carry out the present invention.

Brief description of the sequence listing SEQ ID NO: 1 sets out the nucleic acid sequence of the English Mastiff mast cell chymase gene containing the C5375T SNP.

SEQ ID NO: 2 sets out the sequence of the forward primer used to amplify the English Mastiff mast cell chymase gene sequence containing the C5375T SNP.

SEQ ID NO: 3 sets out the sequence of the reverse primer used to amplify the English Mastiff mast cell chymase gene sequence containing the C5375T SNP.

SEQ ID NO: 4 sets out the RAGE_8Kb_6000 contig nucleic acid sequence.

SEQ ID NO: 5 sets out the RAGE_8Kb_6002 contig nucleic acid sequence.

SEQ ID NO: 6 sets out the RAGE_8Kb_5959 contig nucleic acid sequence.

SEQ ID NO: 7 sets out the FCGR3B_7.42Kb_5238 contig nucleic acid sequence.

SEQ ID NO: 8 sets out the PAI1_10Kb_2979 contig nucleic acid sequence.

SEQ ID NO: 9 sets out the RAGE_8Kb_6006 contig nucleic acid sequence.

SEQ ID NO: 10 sets out the FCGR3B_7.42Kb_5264 contig nucleic acid sequence.

SEQ ID NO: 11 sets out the FCGR3B_7.42Kb_5137 contig nucleic acid sequence.

SEQ ID NO: 12 sets out the FCGR3B_7.42Kb_5002 contig nucleic acid sequence.

SEQ ID NO: 13 sets out the FCGR3B_7.42Kb_5167 contig nucleic acid sequence.

SEQ ID NO: 14 sets out the RAGE_8Kb_5820 contig nucleic acid sequence.

SEQ ID NO: 15 sets out the FCGR2A_9.86Kb_8708 contig nucleic acid sequence.

SEQ ID NO: 16 sets out the RAGE_8Kb_5847 contig nucleic acid sequence.

SEQ ID NO: 17 sets out the RAGE_5Kb_4329 contig nucleic acid sequence.

SEQ ID NO: 18 sets out the RAGE_5Kb_4766 contig nucleic acid sequence.

SEQ ID NO: 19 sets out the RAGE_8Kb_6182 contig nucleic acid sequence.

SEQ ID NO: 20 sets out the FCGR3B_7.42Kb_5239 contig nucleic acid sequence.

WO 2004/113570 PCT/GB2004/002559 6 SEQ ID NO: 21 sets out the RAGE_8Kb_5771 contig nucleic acid sequence.

SEQ ID NO: 22 sets out the RAGE_5Kb_4805 contig nucleic acid sequence.

SEQ ID NO: 23 sets out the FCGR3B_7.42Kb_4947 contig nucleic acid sequence.

Detailed description of the invention The present invention allows the identification of the nutritional requirements, disease susceptibility or behavioural characteristics of a dog by determination of its breed ancestry. Detection of the presence or absence of SNP markers in the dog allows identification of the breeds that have contributed to the dog's genome its genetic breed inheritance), allowing the genetic background of the dog to be deduced.

Dog Breeds A breed is a homogeneous group of animals within a species, which has been developed by man. Dog breeds are normally divided into seven categories, based on the uses for which the breeds were originally developed. The seven dog breed categories and examples of specific breeds that fall within each category are shown in Table 1.

Table 1 a) Hounds Breed Size Grooming Exercise Locality Life-span Afghan Hound L Con Con C B Basenji M Lt Mod T/C B Basset Bleu De Gascogne M Lt Con C B Basset Fauve De Bretagne M Mod Con T/C B Basset Griffon Vendeen (Grand) MI Mod Con C B Basset Griffon Vendeen (Petit) M Mod Con T/C B Basset Hound M Lt Con T/C B Bavarian Mountain Hound M Lt Con C B Beagle M Lt Con T/C B Bloodhound L Lt Con C A Borzoi L Mod Con C B Dachshund (Long Haired) M Mod Mod T/C B Dachshund (Miniature Long Haired) S Mod Mod T/C C Dachshund (Smooth Haired) M Lt Mod T/C B Dachshund (Miniature Smooth Haired) S Lt Mod T/C C WO 2004/113570 PCT/GB2004/002559 Dachshund (Wire Haired) M Mod Mod T/C B Dachshund (Miniature Wire Haired) S Mod Mod T/C C Deerhound L Mod Con C B Norwegian Ellkhound L Mod Con T/C B Finnish Spitz M Mod Mod C B Foxhound L Lt Con C B Grand Bleu De Gascogne L Lt Con C B Greyhound L Lt Mod C B Hamiltonstovare L Lt Con C B Ibizan Hound L Lt Con T/C B Irish Wolfhound XL Mod Con C A Norwegian Lundehund M Lt Mod T/C B Otterhound L Mod Con C B Pharaoh Hound L Lt Con C B Rhodesian Ridgeback L Lt Con T/C B Saluki L Mod Con C B Segugio Italiano L Lt Con C B Sloughi L Lt Con C B Whippet M Lt Con T/C B b) Working Dogs Breed Size Grooming Exercise Locality Life-span Alaskan Malamute L Con Con C B Beauceron L Lt Con C B Bernese Mountain Dog XL Mod Mod T/C A Bouvier Des Flandres L Con Con T/C B Boxer L Lt Con T/C B Bullmastiff L Lt Con T/C B Canadian Eskimo Dog L Mod Con C B Dobermann L Lt Con T/C B Dogue de Bordeaux L Lt Mod C B German Pinscher M Lt Mod T/C B Greenland Dog L Mod Con C B Giant Schnauzer L Con Con T/C B Great Dane XL Lt Con T/C A Hovawart L Mod Con T/C B Leonberger XL Mod Con C B Mastiff XL Lt Mod C A Neapolitan Mastiff XL Lt Mod C A Newfoundland XL Con Con C B Portuguese Water Dog L Con Mod T/C B Rottweiler L Lt Con T/C B WO 2004/113570 PCT/GB2004/002559 Russian Black Terrier L Con Con T/C B St. Bernard XL Con Mod T/C A Siberian Husky L Mod Con C B Tibetan Mastiff XL Mod Mod T/C B c) Terrier Breed Size Grooming Exercise Locality Life-span Airedale Terrier L Con Mod T/C B Australian Terrier S Mod Mod T/C B Bedlington Terrier M Mod Mod T/C B Border Terrier S Mod Mod T/C B Bull Terrier M Lt Mod T/C B Bull Terrier (Miniature) M Lt Mod T/C B Cairn Terrier S Mod Mod T/C B Cesky Terrier M Con Mod T/C B Dandie Dinmont Terrier M Mod Mod T/C B Fox Terrier (Smooth) M Lt Mod T/C B Fox Terrier (Wire) M Con Mod T/C B Glen of Imaal Terrier M Mod Mod T/C B Irish Terrier M Mod Mod T/C B Kerry Blue Terrier M Con Mod T/C B Lakeland Terrier M Con Mod T/C B Manchester Terrier M Lt Mod T/C B Norfolk Terrier S Mod Mod T/C B Norwich Terrier S Mod Mod T/C B Parson Russell Terrier M Lt Mod T/C B Scottish Terrier M Con Mod T/C B Sealyham Terrier M Con Mod T/C B Skye Terrier M Mod Mod T/C B Soft Coated Wheaten Terrier M Con Mod T/C B Staffordshire Bull Terrier M Lt Con T/C B Welsh Terrier M Con Mod T/C B West Highland White Terrier S Con Mod T/C B d) Gundogs (Sporting Group) Breed Size Grooming Exercise Locality Life-span Bracco Italiano L Lt Con C B Brittany M Lt Con C B English Setter L Mod Con T/C B German Longhaired Pointer L Mod Con C B German Shorthaired Pointer L Lt Con C B German Wirehaired Pointer L Mod Con C B WO 2004/113570 PCT/GB2004/002559 Gordon Setter L Mod Con C B Hungarian Vizsla L Lt Con C B Hungarian Wirehaired Vizsla L Mod Con C B Irish Red and White Setter L Mod Con C B Irish Setter L Mod Con T/C B Italian Spinone L Mod Con C B Kooikerhondje M Mod Mod T/C B Lagotto Romagnolo M Mod Con C B Large Munsterlander L Mod Con T/C B Nova Scotia Duck Tolling Retriever M Mod Mod T/C B Pointer L Lt Con T/C B Retriever (Chesapeake Bay) L Mod Con C B Retriever (Curly Coated) L Mod Con C B Retriever (Flat Coated) L Mod Con T/C B Retriever (Golden) L Mod Con T/C B Retriever (Labrador) L Lt Con T/C B Spaniel (American Cocker) M Con Mod T/C B Spaniel (Clumber) L Mod Mod C B Spaniel (Cocker) M Con Mod T/C B Spaniel (English Springer) M Mod Con T/C B Spaniel (Field) M Mod Con C B Spaniel (Irish Water) M Mod Con C B Spaniel (Sussex) M Mod Con C B Spaniel (Welsh Springer) M Mod Con T/C B Spanish Water Dog M Mod Mod C B Weimaraner L Lt Con T/C B e) Pastoral (Herding Group) Breed Size Grooming Exercise Locality Life-span Anatolian Shepherd Dog L Mod Con C B Australian Cattle Dog M Lt Mod C B Australian Shepherd L Mod Con C B Bearded Collie L Con Mod T/C B Belgian Shepherd Dog (Groenendael) L Mod Con T/C B Belgian Shepherd Dog (Malinois) L Mod Con T/C B Belgian Shepherd Dog (Laekenois) L Mod Con T/C B Belgian Shepherd Dog (Tervueren) L Mod Con T/C B Bergamasco L Con Mod C B Border Collie M Mod Con C B Briard L Con Con T/C B Collie (Rough) L Con Con T/C B Collie (Smooth) L Lt Con T/C B WO 2004/113570 PCT/GB2004/002559 Estrela Mountain Dog XL Mod Mod C B Finnish Lapphund M Con Mod T/C B German Shepherd Dog (Alsatian) L Mod Con T/C B Hungarian Kuvasz L Mod Mod C B Hungarian Puli M Con Mod T/C B Komondor L Con Mod C A Lancashire Heeler S Lt Mod T/C B Maremma Sheepdog L Mod Con C B Norwegian Buhund M Mod Mod T/C B Old English Sheepdog L Con Con T/C B Polish Lowland Sheepdog M Con Con T/C B Pyrenean Mountain Dog XL Con Mod T/C A Pyrenean Sheepdog M Mod Mod T/C B Samoyed L Con Con T/C B Shetland Sheepdog M Con Mod T/C B Swedish Lapphund M Con Mod T/C B Swedish Vallhund M Lt Mod T/C B Welsh Corgi (Cardigan) M Lt Mod T/C B Welsh Corgi (Pembroke) M Lt Mod T/C B f) Utility Dogs (Non-sporting) Breed Size Grooming Exercise Locality Life-span Akita L Mod Con T/C B Boston Terrier S Lt Mod T/C B Bulldog M Lt Mod T/C A Canaan Dog L Lt Mod T/C B Chow Chow L Con Mod T/C B Dalmatian L Lt Con T/C B French Bulldog S Lt Mod T/C B German Spitz (Klein) S Con Lt T/C B German Spitz (Mittel) M Con Lt T/C B Japanese Shiba Inu M Mod Mod T/C B Japanese Spitz M Con Mod T/C B Keeshond M Con Mod T/C B Lhasa Apso S Con LT T/C B Mexican Hairless M Lt Mod T/C B Miniature Schnauzer S Con Mod T/C B Poodle (Miniature) M Con Mod T/C C Poodle (Standard) L Con Con T/C C Poodle (Toy) S Con Mod T/C C Schipperke S Lt Lt T/C C Schnauzer M Con Mod T/C B WO 2004/113570 PCT/GB2004/002559 Shar Pei M Lt Mod T/C B Shih Tzu S Con Mod T/C B Tibetan Spaniel S Mod Mod T/C C Tibetan Terrier M Con Mod T/C B g) Toy Dogs Breed Size Grooming Exercise Locality Life-span Affenpinscher S Mod Lt T/C B Australian Silky Terrier S Mod Lt T/C B Bichon Frise S Con Lt T/C B Bolognese S Con Lt T/C B Cavalier King Charles Spaniel S Mod Mod T/C B Chihuahua (Long Coat) S Mod Lt T/C B Chihuahua (Smooth Coat) S Lt Lt T/C B Chinese Crested S Lt Lt T/C B Coton De Tulear S Con Lt T/C B English Toy Terrier (Black and Tan) S Lt Lt T/C B Griffon Bruxellios S Mod Lt T/C B Havanese S Con Lt T/C B Italian Greyhound S Lt Mod T/C B Japanese Chin S Mod Lt T/C B King Charles Spaniel S Mod Lt T/C B Lowchen (Little Lion Dog) S Con Lt T/C B Maltese S Con Lt T/C B Miniature Pinscher S Lt Lt T/C B Papillon S Mod Lt T/C B Pekingese S Con Lt T/C B Pomeranian S Con Lt T/C B Pug S Lt Lt T/C B Yorkshire Terrier S Con Lt T/C B

KEY

SIZE S-Small M-Medium L-Large XL-Ex Large GROOMING Lt-Little Mod-Moderate Con-Considerable EXERCISE Lt-Little Mod-Moderate Con-Considerable LOCALITY T-Town C-Country LIFESPAN A-Under 9 Yrs B-Over 9 Yrs C-Over 15 Yrs Breed-specific SNPs As used herein, a "breed-specific SNP" is a single nucleotide polymorphism 19 Jun 2O008 3 :17PM

WATERMARK

00 12 0 that can be used to distinguish between different dog breeds or to determine breed ci inheritance, either alone or in combination with other SNPs Such a breed-specific

SNP

may be unique to one breed. Alternatively, a breed-specific SNIP may be present in a _n plurality of breeds, but its presence in combination with one or more other breed-specific SNIPs can be used to determine a dog's genetic breed inheritance. In one embodiment of the invention, each SNP is present in substantially all dogs of one breed, and is absent in substantially all dogs of other breeds. The breed-specificity of a SNIP is typically assessed in a sample population of a breed that is representative of that breed. Such a sample population will typically consist only of purebred dogs. The sample population typically comprie 4 or more dogs per breed, such as at least 20, 1005 400, 1000 or 10,000 dogs of one breed, For example, the sample population tested for the SNIP may be up to 10, 200, 500, 1000, 10,000 or 1,000,000 or more dogs. The sample population may consist of from 4 to 10,000, for example 2010o 1000, or 100 to 500 dogs per breed. For example, the sample population may be from 200 to 400 dogs per breed.

Each breed-specific SNP is typically present in 70%, 80% or 90% or more of the sample population of that breed, preferably 95% or more of the sample population, more preferably 99% or more of the sample population. Each breed-specific SNP is typically absent in substantially all dogs of sample populations of other breeds. For example, a breed specific SNP may be present in 30%, 20% or 10% or less of a sample population of another breed, preferably 5% or less of the sample population, more preferably 1 or less of the sample population, In a prefered embodiment, the SNPs are present in at least of dogs in a sample population of from 400 to 10 00 dogs of a breed and/or are present in 5% or less dogs in a sample population of from 400 to 1000 dogs of any other breed. In a most preferred embodiment, the breed-specrnic SNPs will be unique to that breed, i.e. they will be present in 100% of dogs in a sample population which is representative of that breed and will be entirely absent from dogs in a sample population which is representative of any other breed.

Alternatively, the SNPs may be specific for a breed category shown in Table 1.

For example, the SNP maarkers may be specific for H-ound breeds such as the Beagle, Bloodhound, Whippet or Greyhound. The SNP markers may be specific for Working dogs, such as the Boxer, Great Dane and ST Bernard. The SN? markers may be specific for dogs in the Terrier group, such as the West Highland White Terrier and the Airedale Terrier. The SN? markers may be specific for breeds in the Utility, or Non-Sporting, COMS ID No: ARCS-195i 43 Received by IP Australia: Time (i-tm) 15:20 Date 2008-06-19

I-

19 Jun 2008 3~17PM 19Jn20831P .W0*_LrXElM 98196010 p.G 0 o group such as the Bulldog, Dalmatian and Poodle. The SNP markers may be specific for Toy dog breeds such as the Chihuahua and Shih Tzu.

;Z Inaone embodiment of the invention, the SNPs are specific to a family or subgroup of breeds within a breed category. The SNPs may be specific for (3undogs, or Sporting group dogs. This category is divided into four sub-groups: Retriever, Spaniels, HunU'Point(jRetrjeve and Setters. The SNP markers may be specific for any one or more of these four sub -groups: The SN? markers may be specific for dogs in the Pastoral, or Herding, group. This breed category includes the Collie family of breeds and Shepherd dogs. Hence the SNPs may be specific for the Collie family and/or Shepherd dogs.

In a fuirther embodiment of the invention, the breed-specific SNPs can be used to distinguish one breed of dog in a panel of dog breeds from the other breeds in the panel.

The panel may consist of from 2 to 400 breeds, for example from 2 to 200, from 5 to 100, from 5 to 30, from 5 to 20, firoml10 to 15, from 2 to 10 or from 5 to 10 breeds. The SNP markers are thus specific for one of the breeds in the panel. The SN? markers may actually be found in more than one breed, for example for 2, 3, 5, 10 or more breeds.

However, according to this particular embodiment, they will be specific for only one of the breeds in the panel. The breeds can be selected from any of the categories shown in Table 1 above. SNPs that are specific for two or more breeds within a breed category can be used to distinguish those particular breeds from other breeds in the breed category. In a most preferred embodiment, the SNP markers are present only in one breed are unique to that breed compared to all other breeds).

Each breed is not defined by a single SNP, but by the combination of SN41s present in the dog genome. Accordingly, the genetic breed inheritance of a dog may be identified from a combination of the nucleotides present at two or more SNP positions, for example at three or more, four or more, five or more, or six or more SNP positions.

Each dog breed may therefore be defined by a set of rules based on the combination of nucleotides found at each of these SN? positions. In some cases, in order to define a breed it may be necessary to provide one or more rules which specify the nucleotide found at least 7, 8, 9, 10, 11, 12, t15 or 20 or more SNP positions. Typically, the number of SNP positions used in each rule will be from 2 to 20, preferably from 2 to 12, more preferably from 2 to 6. Each dog breed may be defined by a single rule or more than one rule, for example by 2, 3, 4, 5, 10, 20 or more rules.

COMS ID No: ARC S-i 951 43 Received by IP Australia: Time 15:20 Date 2008-06-19 00 14 In order to identify the genetic breed inheritance of the dog, typically at least 2 Sdifferent SNP positions are typed, for example at least 3, 4, 5, 6, 7, 8, 9 or 10 or more ;Z positions, preferably at least 20 different SNP positions. Typically up to 10, 15, 20, 30, 50 or 100 positions will be typed, for example 10 to 50, or 10 to 25 positions. In this case, the term "typed" typically comprises determining the nucleotide present at any Cc given SNP position.

The term "genetic breed inheritance" is used herein to describe the breed ancestry of a dog, namely the one or more breeds that have contributed to the dog's genome. Therefore, in the case of a purebred dog, the term "genetic breed inheritance" will typically correspond to the breed of the dog. Accordingly, in one embodiment of the invention the nucleotide present at one or more SNP positions in the dog's genome can be used to determine the breed of the dog. In the case of a crossbred or outbred dog, the term "genetic breed inheritance" may relate to the one or more breeds that are represented in the dog's lineage. This term may further be used to describe the proportions or relative amounts of each breed that goes to make up a mongrel dog.

The method of the invention can be used to detect a genetic breed inheritance from any number of different dog breeds, such as at least 2, 3, 4, 5, 10, 20, 50, 70, 100 or 400 or more different dog breeds. In one embodiment of the invention, the nucleotide present at one or more SNP positions is used to distinguish between the following breeds: Labrador Retriever, Golden Retriever, German Shepherd, Dachshund, Shih Tzu, Yorkshire Terrier, Poodle, Rottweiler, Boxer and Cocker Spaniel.

The method of the invention may comprise: detecting the presence or absence of one or more breed-specific SNP markers indicative of a breed selected from Cocker Spaniel, Shih Tzu, Doberman and English Mastiff and determining therefrom whether one or more of said breeds has contributed to the genetic breed inheritance of the dog.

Breed Differences The present invention enables the determination of a nutritional requirement, disease susceptibility or behavioural characteristic of a dog, the method comprising determining the nucleotide present at one or more breed-specific SNP (single 00 14a nucleotide polymorphism) positions in the dog genome and thereby determining the Sbreed inheritance of the dog. A method for determining the breed inheritance of a dog according to the invention may be carried out by electronic means, for example by using C, a computer system. The presence of a breed-specific SNP in a dog indicates that it has a genetic inheritance in common with that breed, and therefore is likely to share that rr^ breed's characteristics regarding nutritional requirements, disease WO 2004/113570 PCT/GB2004/002559 susceptibility and behavioural characteristics. The absence of a particular breedspecific SNP indicates that the dog does not have any genetic inheritance from that breed. In one embodiment, a method for determining the nutritional requirements, disease susceptibility or behavioural characteristics of a dog according to the invention may be carried out by electronic means, for example by using a computer system.

Dog breeds differ from each other in (for example) size, weight, shape, digestive transit time, growth period, temperament, activity level, life span, coat type, nutritional requirements and disease susceptibility. Table 1 illustrates some of these differences. The nutritional requirement, disease susceptibility or behavioural characteristic assessed by the method of the invention may be any such nutritional, medical or behavioural need mentioned herein, such as those in Table 1 or those discussed below.

Bodyweight size in dog breeds can be grouped into 5 categories (from smallest to largest): toy, small, medium, large and giant (or extra large). Breeds also differ in the ratio of gastrointestinal weight: total bodyweight. In small breeds, the digestive tract represents 7% of their total bodyweight, whilst for giant breeds this is only 2.7%.

Digestive transit time also varies depending on the size of the dog, and can vary from hours to 4 days. The growth period of a dog varies by breed, is determined by feeding regime and feeding rate, and lasts between approximately 8 months for a small breed to up to 24 months for a giant breed. Small breeds have a much greater growth rate than large breeds. Small breed puppies typically multiply their birth weight by approximately 20 times during their first year of life. This ratio can be as great as 100 times for giant breeds. The size of a dog also affects its life expectancy.

The larger and heavier the dog, the earlier the aging process begins. Life expectancy for giant breeds is generally half that of small breeds.

Breeds differ in their dietary needs due to differences in nutrient requirement and physical form. For example, daily energy requirements to maintain body weight are higher for large, active dogs than small or inactive dogs. However, per unit of bodyweight, a small breed's energy requirements are more than twice those of large breeds. Some breeds, such as Labrador Retrievers, Basset Hounds, Beagles and Cocker Spaniels, are predisposed to obesity. Ingredient tolerance, food allergies and nutrient metabolism also differ among breeds. For example, Irish Setters often exhibit gluten intolerance. Other, well-recognised problems include vitamin A responsive WO 2004/113570 PCT/GB2004/002559 16 dermatitis in Cocker Spaniels and zinc-responsive dermatitis in Siberian Huskies and Alaskan Malamutes. Some Cocker Spaniels and Golden Retrievers have low blood taurine levels which are responsive to dietary taruine supplements.

Breeds also differ in their susceptibility to disease. For example, Dalmatians have predisposition to deafness and to the presence of uric acid crystals in the urine.

Poodles and Bichon Frise have a predisposition to periodontal disease. Bedlington Terriers and West Highland Terriers are prone to copper storage disease. German Shepherds and Beagles often experience diarrhoea caused by a gastrointestinal immune deficiency. Hip dysplasia is common in a number of breeds, particular in the Herding, Working and Sporting groups. Boxers, Doberman Pinschers and Great Danes can develop dilated cardiomyopathy. Skin and hair coat problems are frequent in breeds such as the Miniature Poodle and Chow Chow. Silky Terriers and Yorkshire Terriers are susceptible to diabetes.

Behavioural differences are also marked between dog breeds. For example, the Labrador Retriever is playful, loving to people and hardworking and is suitable for jobs such as a guide dog for the disabled, a search-and-rescue dog, and for narcotics detection. Boxers are playful and fun-loving dogs, but are also strong and defensive, so early obedience training is important. Rottweilers enjoy exercise and outdoor sports, but due to a more aggressive nature may not be suitable as a pet in households with young children. Hound breeds require a significant amount of exercise, whereas Toy dog breeds such as the Chihuahua and Shih Tzu do not need a large amount of exercise. Gundogs, or Sporting group dogs, are active dogs and require plenty of attention and regular, strenuous exercise. The temperament of these breeds makes them ideal family dogs. Knowledge of breed characteristics is therefore important when selecting a dog breed for a particular job or as a pet.

Crossbred and outbred (mongrel) dogs In one embodiment of the method of the invention, the dog that is tested may be a crossbred or outbred (mongrel) dog. A crossbred dog is the offspring of two different purebred dogs. An outbred or mongrel dog is a dog of unknown parentage, or is the result of the combination of three or more different breeds. An outbred dog may therefore represent a mixture of 3 or more breeds, for example, 4, 5 or more different breeds. The breeds that contribute to an outbred dog's genetic breed WO 2004/113570 PCT/GB2004/002559 17 inheritance may be from within the same category of breed or from different breed categories.

A mongrel will typically display a combination of physical characteristics that are not found within one particular breed, such as any characteristics mentioned herein, for example size, shape, colour, coat type, stature, gait, height or head shape.

For example, an outbred dog may have the size and shape of one breed, but have the colour or coat type of a different breed. Therefore, the method of the invention may be used to identify the genetic breed inheritance of a dog which has a mixture of characteristics typically found in different breeds. In particular, the method may be used to determine the genetic background of a dog which has the physical features of a mongrel, or is suspected of being a mongrel. The method of the invention may also be used to identify or to confirm the genetic background of a crossbred dog.

Nutritional requirements The present invention provides a means of determining a nutritional requirement of a dog, based on its genetic breed inheritance. Such a nutritional requirement is any such requirement mentioned herein, for example as discussed below. The requirement typically relates to the proportions, total amounts or types of vitamins, minerals, fat, carbohydrates, fibre, protein and water required. In one aspect, the requirement may relate to whether or not the dog requires or needs to avoid particular food components.

The protein requirement of a dog may relate to the total amount of protein or type of protein needed, as defined by the protein source or amino acid composition.

For example, the essential amino acids for dogs include lysine, arginine, histidine, isoleucine, leucine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan and valine. Essential amino acids cannot be synthesized by the dog, and so must be present in its food. The amount of each amino acid required may vary. In particular, the dog may have a requirement for an amino acid such as taurine. The dog's nutritional requirements may concern the source of protein, for example, whether the protein is derived from meat, poultry, dairy, vegetable or other protein sources. These protein sources may be defined as high or low quality protein sources. In this respect, quality is defined by digestibility and amino acid content.

For example, a high quality protein source (such as animal protein) may contain all the essential amino acids and/or have high digestibility, whereas a low quality protein WO 2004/113570 PCT/GB2004/002559 18 source (such as vegetable protein) may be missing one or more essential amino acids and/or have low digestibility.

The dog's nutritional requirement may further relate to the amount of fat needed by the dog or to a particular type of fat that is required. Fats are typically saturated, polyunsaturated or monounsaturated. A dog may require different amounts of each type of fat, or only one or more of these types of fat. For example, the nutritional requirement may be for polyunsaturated or monounsaturated fats only.

Fats also differ in their fatty acid composition. The nutritional requirement may relate to particular fatty acids, such as essential fatty acids, which cannot be made within the dog's body and so have to be provided in the diet. The essential fatty acids may be classified as omega-6 and omega-3 fatty acids, such as linoleic, linolenic and arachidonic acids, for example, gamma linolenic acid (GLA). These polyunsaturated fatty acids vary in the number of carbon atoms and the degree of unsaturation, and may be classified as short-chain and long-chain fatty acids. In one aspect of the invention, the nutritional requirement may relate to the absolute amounts of these fatty acids, or to the ratio of omega-6 to omega-3 fatty acids.

In another aspect of the invention, the dog's nutritional requirement may relate to the total amounts or proportions of vitamins or minerals needed. Vitamins may be divided into two main categories: fat soluble and water soluble. The fat soluble vitamins include vitamins A, D, E and K. The water soluble vitamins include vitamins B1, B2, B6, B 12, biotin, choline, pantothenic acid, nicotinic acid and folic acid. A dog may require particular levels of each vitamin. Minerals can be divided into two groups: macro-minerals and trace minerals. Macro-minerals include calcium, phosphorus, magnesium, sodium, potassium and chloride. Trace minerals include iron, zinc, copper, manganese, cobalt, selenium and iodine. The nutritional requirement of the dog may relate to the total amounts of each mineral or to the ratio between the minerals needed. For example, the nutritional requirement may relate to the ratio between calcium and phosphorus.

The dog's nutritional requirement may relate to the amount of carbohydrate or to the type of carbohydrate needed. Carbohydrates can be classified according to the glycemic index, which measures the ability of a food to elevate blood glucose levels.

Carbohydrates with a high glycemic index enter the bloodstream quickly, whereas those with a low glycemic index enter the bloodstream slowly and provide sustained, longer-term energy. The glycemic index of a food is typically given in relation to WO 2004/113570 PCT/GB2004/002559 19 glucose (or maltose), which has a nominal value of 100. For example, barley has a lower glycemic index than other grains such as corn, wheat or rice. Therefore, in one aspect of the invention, the dog's nutritional requirements may relate to the glycemic index of carbohydrate that is required. The nutritional requirement may further relate to the amount or proportion of fibre or the type of fibre needed. For example, fibre may be derived from different sources, such as fruit, vegetable or grains. Different types of fibre may differ in how quickly they are fermented. For example, fruit and vegetable fibres are moderately fermented whereas grain fibres are more slowly fermented.

The nutritional requirement of the dog may concern its metabolic or energy requirements. The energy requirement of the dog typically relates to its size and activity level. A large dog generally requires a greater total amount of energy than a small dog, and an active dog will normally require more energy than an inactive dog.

For example, a dog may have low activity hour per day), moderate activity (1-2 hours per day), moderate to high activity (2-3 hours per day) or high activity (>3 hours per day). The energy requirement is usually expressed as either kilocalories (kcal) or kilojoules (kJ).

The nutritional requirement may be determined on a daily, weekly basis or a monthly basis. Preferably, the dog's nutritional requirements will be determined on a daily basis, for example as a recommended daily amount (RDA) of a nutrient. The energy requirement of the dog will typically be determined on a daily basis.

The nutritional requirement of the dog may relate to food allergies or intolerance. Allergens for dogs typically fall into one of four groups: milk, eggs, soy, wheat (gluten), peanuts, shellfish, fruits, tree nuts; (ii) sesame seeds, sunflower seeds, cottonseed, poppy seed, beans, peas, lentils; (iii) tartrazine, sulphites and latex; and (iv) salicylate, amines and glutamate. The most common food allergies are to those foods in group Disease susceptibility The present invention allows for determination of a disease susceptibility of a dog, based on its genetic breed inheritance. Various dog breeds have susceptibility to different diseases and conditions. Such diseases or conditions may be cardiovascular, inflammatory, immunological, infectious, metabolic, endocrine or gastrointestinal in nature. The disease or condition may be any of the diseases or conditions mentioned WO 2004/113570 PCT/GB2004/002559 herein. For example, German Shepherd dogs commonly suffer from hip dysplasia, epilepsy, gastric torsion (bloat), perianal fistulas and exocrine pancreatic deficiency.

Labrador Retrievers are particularly susceptible to hip, elbow and retinal dysplasia, obesity and exercise-induced collapse. Golden Retrievers are also prone to hip dysplasia, and sometimes experience skin and coat problems such as pyotraumatic dermatitis (hotspots).

Dachshunds are susceptible to spinal disc injuries, diabetes, urinary stones, eye disorders, skin conditions and heart disease. Cocker Spaniels commonly suffer from hereditary eye problems (such as PRA, cataracts, glaucoma, eyelid, eyelash and retinal abnormalities), skin conditions, hemophilia, ear infections (such as otitis externa), heart disease and epilepsy. Boxers are prone to tumours, digestive problems, heart disease, corneal ulcers, skin fold infections and bloat. Rottweilers are susceptible to hip and elbow dysplasia, osteochonsrosis dessicans, panosteitis, entropieon (inverted eyelids), hypothyroidism, von Willebrand's disease and bloat.

Shih Tzu dogs commonly suffer from slipped stifle (a joint disorder) and renal dysplasia. Poodles are prone to hip dyslplasia, PRA, cataracts, epilepsy, bloat, von Willebrand's disease, skin disorders and autoimmune disorders.

Behavioural characteristics In another aspect of the invention, a behavioural characteristic of a dog may be determined. As discussed herein, different dog breeds have different activity levels and temperament. Accordingly, dogs differ in the type of environment that is suitable for them. For example, dogs have differing requirements for space, locality town or countryside), exercise, grooming and attention. Dog breeds also differ in their trainability, people fear, aggressiveness, alertness and cognitive performance.

When selecting an appropriate environment for a dog, it is important to bear in mind factors such as their size at maturity and their temperament aggressiveness).

The behavioural characteristics determined according to the present invention may be any of those in Table 1 or any other characteristics discussed herein.

Symptoms ofa problem In one aspect of the invention, a nutritional requirement, disease susceptibility or behavioural characteristic is determined of a dog that is suspected of having a nutritional, medical or behavioural problem. The dog may be displaying physical or WO 2004/113570 PCT/GB2004/002559 21 psychological symptoms that are indicative of a nutritional imbalance or deficiency, a disease or behavioural problem. A nutritional or medical problem may be indicated by changes in eye colour, gum and mouth tissue, skin condition, coat condition, energy level or muscle tone in the dog. Other symptoms of a problem may include lethargy, weight loss, bladder control loss, change in water intake, change in faeces quality, appetite loss, sudden behavioural change or alertness change.

Detection ofSNP markers The detection of SNPs according to the invention may comprise contacting a polynucleotide or protein of the animal with a specific binding agent for a breedspecific SNP and determining whether the agent binds to the polynucleotide or protein.

The method is typically it is carried out in vitro on a sample from the dog.

The sample typically comprises a body fluid and/or cells of the individual and may, for example, be obtained using a swab, such as a mouth swab. The sample may be a blood, urine, saliva, skin, cheek cell or hair root sample. The sample is typically processed before the method is carried out, for example DNA extraction may be carried out. The polynucleotide or protein in the sample may be cleaved either physically or chemically, for example using a suitable enzyme. In one embodiment the part of polynucleotide in the sample is copied or amplified, for example by cloning or using a PCR based method prior to detecting the SNP marker(s).

Tables 2 and 7 show breed-specific SNPs that can be used to type the breed inheritance of a dog. A breed-specific SNP may be a "silent" polymorphism. Such "silent" polymorphisms are those which do not result in a change in amino acid sequence. Only SNPs that change the coding sequence of the nucleic acid sequence may be detected in polypeptide sequences. The polymorphism preferably does not affect the function of the protein in any other way, for example by altering gene expression by changing promoter activity, mRNA stability, mRNA splicing or epigenetic status. Such polymorphisms may or may not be causative of a breed phenotype. Preferably, the breed-specific SNP is not causative of a nutritional requirement, disease susceptibility or behavioural characteristic of a dog, and is not in linkage disequilibrium with such a SNP. The SNP may however be specific for one or more physical characteristics of a breed, for example size, shape, colour, coat type, stature, gait, height or head shape, or other breed traits or phenotypes.

00 LL 0 O In the present invention, any one or more methods may comprise determining the Snucleotide present at one or more breed-specific SNP positions in the dog. In a preferred embodiment of the invention, the nucleotide present at least 2, 3, 5, 10, 15 or 20 or more SNP positions is detected. Preferably 10 or more breed-specific SNP positions are typed, more preferably 20 or more breed-specific positions. Any possible combination of breed-specific SNPs may be tested. In a preferred embodiment, the one or more SNP positions are any of those identified in SEQ ID NO:s 1 or 4 to 23.

The markers which are tested may be specific to a combination of different breeds. In one embodiment, the dog is tested for the presence and/or absence of one or more breed-specific SNP markers for at least 2, 3, 5 or 10 different breeds. In one embodiment the markers that are typed are specific for the following breeds: Labrador Retriever, Golden Retriever, German Shepherd, Dachshund, Shih Tzu, Yorkshire Terrier, Poodle, Rottweiler, Boxer and Cocker Spaniel. As discussed herein, in aspect of the invention the breed-specific SNPs are present in substantially all dogs of that breed, and are absent in substantially all dogs of other breeds. One or more markers specific for each breed may be typed, for example at least 2, 3, 5 or 10 markers may be tested which are specific for one breed.

Each breed-specific SNP is typically detected by directly determining the presence of the polymorphic sequence in a polynucleotide or protein of the dog. Such a polynucleotide is typically genomic DNA, mRNA or cDNA. Each SNP may be detected by any suitable method such as those mentioned below.

A specific binding agent is an agent that binds with preferential or high affinity to the polynucleotide or polypeptide having a particular nucleotide or amino acid at a SNP position but does not bind or binds with only low affinity to polynucleotides or proteins which have a different nucleotide or amino acid at the same SNP position. The specific binding agent may be a probe or primer. The probe may be a protein (such as an antibody) or an oligonucleotide. The probes or primers will typically also bind to flanking nucleotides and amino acids on one or both sides of the SNP position, for example at least 2, 5, 10, 15 or more flanking nucleotide or amino acids in total or on each side. Thus a probe or primer may be fully or partially complementary to have homology with) either all or part of the flanking 5' and/or 3' sequences shown in Tables 2 and 7. The probe may be labelled or may be capable WO 2004/113570 PCT/GB2004/002559 23 of being labelled indirectly. The binding of the probe to the polynucleotide or protein may be used to immobilise either the probe or the polynucleotide or protein.

Generally in the method, determination of the binding of the agent to the breed-specific SNP can be done by determining the binding of the agent to the polynucleotide or protein of the dog. However in one embodiment the agent is also able to bind the corresponding wild-type sequence, for example by binding the nucleotides or amino acids which flank the SNP marker position, although the manner of binding to the wild-type sequence will be detectably different to the binding of a polynucleotide or protein containing the SNP marker.

The method may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used. These probes bind to adjacent areas on the polynucleotide which contains the SNP marker, allowing after binding the two probes to be ligated together by an appropriate ligase enzyme. However the presence of single mismatch within one of the probes may disrupt binding and ligation. Thus ligated probes will only occur with a polynucleotide that contains the SNP marker, and therefore the detection of the ligated product may be used to determine the presence of the SNP marker.

In one embodiment the probe is used in a heteroduplex analysis based system.

In such a system when the probe is bound to polynucleotide sequence containing the SNP marker it forms a heteroduplex at the site where the SNP marker occurs and hence does not form a double strand structure. Such a heteroduplex structure can be detected by the use of single or double strand specific enzyme. Typically the probe is an RNA probe, the heteroduplex region is cleaved using RNAase H and the SNP marker is detected by detecting the cleavage products.

The method may be based on fluorescent chemical cleavage mismatch analysis which is described for example in PCR Methods and Applications 3, 268-71 (1994) and Proc. Natl. Acad. Sci. 85, 4397-4401 (1998).

In one embodiment a PCR primer is used that primes a PCR reaction only if it binds a polynucleotide containing the SNP marker, for example a sequence- or allelespecific PCR system, and the presence of the SNP marker may be determined by the detecting the PCR product. Preferably the region of the primer which is complementary to the SNP marker is at or near the 3' end of the primer. The presence of the SNP marker may be determined using a fluorescent dye and quenching agentbased PCR assay such as the Taqman PCR detection system.

WO 2004/113570 PCT/GB2004/002559 24 The specific binding agent may be capable of specifically binding the amino acid sequence encoded by a polymorphic sequence, preferably one of the sequences shown in Table 2. For example, the agent may be an antibody or antibody fragment.

The detection method may be based on an ELISA system.

The method may be an RFLP based system. This can be used if the presence of the SNP marker in the polynucleotide creates or destroys a restriction site that is recognised by a restriction enzyme.

The presence of the SNP marker may be determined based on the change which the presence of the SNP marker makes to the mobility of the polynucleotide or protein during gel electrophoresis. In the case of a polynucleotide single-stranded conformation SNP marker (SSCP) or denaturing gradient gel electrophoresis (DDGE) analysis may be used.

In another method of detecting the SNP marker a polynucleotide comprising the polymorphic region is sequenced across the region which contains the SNP marker to determine the presence of the SNP marker.

Polynucleotides The invention also provides a polynucleotide which comprises a breed-specific SNP. Preferably the SNP position is any one of those identified in any one of SEQ ID NO:s 1 or 4 to 23. The polynucleotide is typically at least 10, 15, 20, 30, 50, 100, 200 or 500 bases long, such as at least or up to 1kb, 10kb, 100kb, 1000 kb or more in length. The polynucleotide will typically comprise flanking nucleotides on one or both sides of or 3' to) the SNP position, for example at least 2, 5, 10, 15 or more flanking nucleotides in total or on each side. Thus such flanking sequences of the or 3' side may be fully or partially identical to or fully or partially complementary to have homology with) either all or part of the flanking 5' and/or 3' sequences identified in any one of SEQ ID NO:s 1 or 4 to 23.

The polynucleotide may differ to the sequences identified in any one of SEQ ID NO:s 1 or 4 to 23 by less than 30, 20, 10, 5, 3 or 2 substitutions and/or insertions and/or deletions in sequence, apart from at the polymorphic position. Typically, the polynucleotide will be at least 95%, preferably at least 99%, even more preferably at least 99.9% identical to the sequence comprising the SNP position as identified in any one of SEQ ID NO:s 1 or 4 to 23. Such numbers of substitutions and/or insertions and/or deletions and/or percentage homology may be taken over the entire length of WO 2004/113570 PCT/GB2004/002559 the polynucleotide or over 50, 30, 15, 10 or less flanking nucleotides in total or on each side.

The polynucleotide may be RNA or DNA, including genomic DNA, synthetic DNA or cDNA. The polynucleotide may be single or double stranded. The polynucleotide may comprise synthetic or modified nucleotides, such as methylphosphonate and phosphorothioate backbones or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.

A polynucleotide of the invention may be used as a primer, for example for PCR, or a probe. A polynucleotide or polypeptide of the invention may carry a revealing label. Suitable labels include radioisotopes such as 32P or 35S, fluorescent labels, enzyme labels or other protein labels such as biotin.

The invention also provides expression vectors that comprise polynucleotides of the invention and are capable of expressing a polypeptide of the invention. Such vectors may also comprise appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Thus the coding sequence in the vector is operably linked to such elements so that they provide for expression of the coding sequence (typically in a cell). The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

The vector may be for example plasmid, virus or phage vector. Typically the vector has an origin of replication. The vector may comprise one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh promoter. Mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium. Viral promoters such as the large T antigen promoter or adenovirus promoters may also be used.

WO 2004/113570 PCT/GB2004/002559 26 Mammalian promoters, such as P-actin promoters, may be used. Tissue-specific promoters are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR).

The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences.

This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

The polynucleotide may be a probe or primer which is capable of selectively binding to a breed-specific SNP. Preferably the probe or primer is capable of selectively binding to a SNP position as identified in any one of SEQ ID NO:s 1 or 4 to 23. The probe or primer more preferably comprises a fragment of a nucleic acid sequence of any one of SEQ ID NO:s 1 or 4 to 23 which comprises the SNP position.

The invention thus provides a probe or primer for use in a method according to the invention, which probe or primer is capable of selectively detecting the presence of a breed-specific SNP. Preferably the probe is isolated or recombinant nucleic acid.

Preferably it is at least 10, 15, 20 or 25 bases in length. It may correspond to or be antisense to the sequences set out in any one of SEQ ID NO:s 1 or 4 to 23. The probe may be immobilised on an array, such as a polynucleotide array.

The polypeptides, polynucleotides, vectors, cells or antibodies of the invention may be present in an isolated or substantially purified form. They may be mixed with WO 2004/113570 PCT/GB2004/002559 27 carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, 98% or 99%, of the proteins, polynucleotides, cells or dry mass of the preparation.

It is understood that any of the above features that relate to polynucleotides and proteins may also be a feature of the other polypeptides and proteins mentioned herein, such as the polypeptides and proteins used in the screening and therapeutic aspects of the invention. In particular such features may be any of the lengths, modifications and vectors forms mentioned above.

Homologues Homologues of polynucleotide or protein sequences are referred to herein.

Such homologues typically have at least 70% homology, preferably at least 80, 97% or 99% homology, for example over a region of at least 15, 20, 30, 100 more contiguous nucleotides or amino acids. The homology may be calculated on the basis of nucleotide or amino acid identity (sometimes referred to as "hard homology").

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the WO 2004/113570 PCT/GB2004/002559 28 cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.

The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci.

USA 89: 10915-10919) alignments of 50, expectation of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

The homologous sequence typically differs by at least 1, 2, 5, 10, 20 or more mutations (which may be substitutions, deletions or insertions ofnucleotide or amino acids). These mutations may be measured across any of the regions mentioned above in relation to calculating homology. In the case of proteins the substitutions are preferably conservative substitutions. These are defined according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: ALIPHATIC Non-polar GAP

ILV

Polar- uncharged CSTM

NQ

Polar charged D E

KR

AROMATIC HFWY WO 2004/113570 PCT/GB2004/002559 29 Antibodies The invention also provides antibodies specific for a polypeptide of the invention. The antibodies include those which are specific for proteins which have a breed-specific SNP, such as any of the SNPs mentioned herein, but which do not bind to protein sequences that do not contain the breed-specific SNP. The antibodies of the invention are for example useful in purification, isolation or screening methods involving immunoprecipitation techniques.

Antibodies may be raised against specific epitopes of the polypeptides of the invention. An antibody, or other compound, "specifically binds" to a polypeptide when it binds with preferential or high affinity to the protein for which it is specific but does substantially bind not bind or binds with only low affinity to other polypeptides. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993). Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments which bind a polypeptide of the invention. Such fragments include Fv, F(ab') and F(ab')z fragments, as well as single chain antibodies.

Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDRgrafted antibodies or humanised antibodies.

Antibodies may be used in a method for detecting polypeptides of the invention in a biological sample (such as any such sample mentioned herein), which method comprises: I providing an antibody of the invention; II incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and III determining whether antibody-antigen complex comprising said antibody is formed.

Antibodies of the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising antibody in a host animal against the whole polypeptide or a 0 0 fragment thereof, for example an antigenic epitope thereof, herein after the S"immunogen". The fragment may be any of the fragments mentioned herein (typically at least 10 or at least 15 amino acids long) and comprise a SNP marker (such as any of C the SNP markers mentioned herein).

A method for producing a polyclonal antibody comprises immunising a suitable Cr host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated j with the immunogen, blood subsequently removed from the animal and the IgG fraction Spurified.

A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat, mouse, guinea pig, chicken, sheep or horse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

Detection kit A kit suitable for use in the invention comprises means for determining the nucleotide present at one or more breed specific genomic SNP positions in a dog. In particular, such means may include a specific binding agent, probe, primer, pair or combination of primers, or antibody, including an antibody fragment, as defined herein which is capable of detecting or aiding detection of a breed-specific SNP. The primer or pair or combination of primers may be sequence specific primers which only cause PCR amplification of a polynucleotide sequence comprising a particular WO 2004/113570 PCT/GB2004/002559 31 nucleotide at the SNP position, as discussed herein. The means for determining nucleotide present at one or more breed specific SNP positions (such as the binding agent, probe, primer or antibody as discussed herein) may be provided in containers that are labelled with the breed for which the SNP is specific. The kit may further comprise buffers or aqueous solutions.

The kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments of the method mentioned above to be carried out. Such reagents or instruments may include one or more of the following: a means to detect the binding of the agent to the SNP, a detectable label such as a fluorescent label, an enzyme able to act on a polynucleotide, typically a polymerase, restriction enzyme, ligase, RNAse H or an enzyme which can attach a label to a polynucleotide, suitable buffer(s) or aqueous solutions for enzyme reagents, PCR primers which bind to regions flanking the SNP position as discussed herein, a positive and/or negative control, a gel electrophoresis apparatus, a means to isolate DNA from a sample, a means to obtain a sample from the individual, such as swab or an instrument comprising a needle, or a support comprising wells on which detection reactions can be carried out. The kit may be, or include, an array such as a polynucleotide array comprising the specific binding agent, preferably a probe, of the invention. The kit typically includes a set of instructions for using the kit.

Customised dog food In one aspect, the invention relates to a customised diet for a dog based on its nutritional needs, as determined by its breed inheritance. Such a food may be in the form of, for example, wet pet foods, semi-moist pet foods, dry pet foods and pet treats. Wet pet food generally has a moisture content above 65%. Semi-moist pet food typically has a moisture content between 20-65% and can include humectants and other ingredients to prevent microbial growth. Dry pet food, also called kibble, generally has a moisture content below 20% and its processing typically includes extruding, drying and/or baking in heat. Pet treats can be semi-moist, chewable treats; dry treats; chewable bones; baked, extruded or stamped treats; or other types of treats which are known in the art.

The ingredients of a dry pet food generally include cereal, grains, meats, poultry, fats, vitamins and minerals. The ingredients are typically mixed and put 00 jL O through an extruder/cooker. The product is then typically shaped and dried, and after Sdrying, flavours and fats may be coated or sprayed onto the dry product.

All pet food is required to provide a certain level of nutrients. For example, the Association of American Feed Control Officials (AAFCO) and the Pet Food Institute have established nutrient profiles for dog foods, based on commonly used Cc ingredients. These established profiles are called the "AAFCO dog food nutrient profiles". Under these regulations, dog foods must be formulated to contain concentrations of nutrients that meet all minimum levels and not to exceed the maximum levels as determined by AAFCO.

S 10 The AAFCO nutritional guideline provides adequate nutrition but may not provide the dog with optimal nutrition. For this reason, dog food formulations have been developed which meet the specific needs of various dog breeds or breed categories. For example, a breed specific diet for the Bedlington Terrier typically comprises a dry product containing 18% protein, 18% fat, 7% ash, 2% fibre, and a wet product containing 8% protein, 5% fat, 1% Ash and 2% fibre. The ingredients used are typically chicken, cereals and byproducts, and supplementary vitamins, minerals, and amino acids.

Accordingly, the present invention enables the preparation of customised dog food, wherein one or more nutritional requirements of the dog is determined by a method of the invention, a customised dog food formulation that corresponds to the nutritional requirements of the dog is generated, and a dog food according to the customised dog food formulation is prepared. The preparation of customised dog food may be carried out by electronic means, for example by using a computer system.

The dog food formulation may be customised according to the caloric, protein, fat, carbohydrate, fibre, vitamin or mineral requirements of the dog, as discussed herein. For example, the dog food formulation may be customised to provide the correct amounts or ratio of essential fatty acids such as omega-6 and omega-3 fatty acids. The main sources of omega-6 fatty acids are plants such as sunflower, soyabean oil, safflower and evening primrose oil, whereas omega-3 fatty acids are mainly found in linseed and marine sources, for example canola oil and salmon oil.

0 0 According to the invention, the customised dog food formulation comprises Scomponents suitable for the breed(s) which have contributed to the genetic breed inheritance of the dog, and does not comprise components that are not suitable for the breed(s) which have contributed to the genetic breed inheritance of the dog.

Accordingly, in one aspect of the invention, the customised food does not c contain ingredients which are poorly tolerated or cause allergies, are abnormally processed or stored, or contribute to diseases or conditions typically suffered by the breed(s) which have contributed to the genetic breed inheritance of the dog. In another aspect of the invention, the customised food contains ingredients which are commonly lacking in, or have nutritional or medical benefits for the breed(s) which have contributed to the genetic breed inheritance of the dog.

For example, the customised food may be formulated so that it does not contain ingredients that are poorly tolerated or cause allergies, for example glutencontaining grains such as wheat, particular protein sources such as animal proteins, milk (lactose), eggs, soy, peanuts, shellfish, fruits or tree nuts. The customised food formulation may further exclude ingredients that are abnormally processed or stored or contribute to diseases or conditions, for example copper, saturated fats and salt.

In another embodiment, the customised food may be formulated to include functional ingredients that help prevent disease or have other beneficial effects for the dog, such as: vitamins, minerals, cocoa flavanols, other plant flavanols, lycopene, curcumin, minerals, trace metals, Echineacea, phosphatidyl serine, L-arginine, ginseng, psyllium, prebiotics, probiotics, phyto-oestrogens, phyto-chemicals, soluble fibre, PUFAs, phospholipids, omega-6 and omega-3 fatty acids.

The present invention also relates to a method of providing a customised dog food, comprising providing to the dog's owner, the person responsible for feeding the dog or a vet a food which contains components suitable for the breed(s) which have contributed to the genetic breed inheritance of the dog, and which does not contain components that are not suitable for the breed(s) which have contributed to the genetic breed inheritance of the dog, wherein the breed inheritance of the dog has been identified by determining the nucleotide present at one or more SNP positions in the dog's genome.

00 O In another aspect of the invention, there is provided a method of feeding a Sdog comprising feeding a mixture of foods that have been formulated for specific breeds or breed categories, based on the dog's genetic breed inheritance. For example, an outbred dog that has breed-specific markers for two different breeds could be fed a mixture of breed-specific food formulations for those two breeds. A dog that showed the presence of breed-specific markers from one or more breeds in a particular category could be fed food that had been formulated for that breed category. It may be that the nutritional requirements of one of the breeds from which a crossbred or outbred dog is derived is dominant over the one or more other breeds represented in the dog. In that case, the customised food may be tailored to meet the requirements of the dominant breed. Alternatively, the food may be customised according to the proportion of genetic inheritance from each breed represented.

Bioinformatics The sequences of the breed-specific SNPs may be stored in an electronic format, for example in a computer database. The database may include further information about the SNP, for example the level of association of the SNP marker with the breed or the frequency of the SNP in the breed. The database may optionally comprise information relating to the nutritional requirements, disease susceptibility or behavioural characteristics of the breeds for which the SNPs are specific. The database may further comprise information regarding the food components which are suitable and the food components which are not suitable for the breeds for which the SNPs are specific.

A database as described herein may be used to determine the breed inheritance of a dog. Such a determination may be carried out by electronic means, for example by using a computer system (such as a PC). Typically, the determination will be carried out by inputting genetic data from the dog to a computer system; comparing the genetic data to a database comprising information relating to breed-specific genomic SNPs; and on the basis of this comparison, determining the nucleotide present at one or more breed-specific SNP positions, thereby identifying the breed inheritance of the dog. A method for determining the nutritional requirements, disease susceptibility or behavioural characteristics of a dog 00 0according to the invention may also be carried out by electronic means, for example

(N

Sby using a computer system (such as a PC). Typically, the method will comprise F-s inputting data of the breed-specific genomic SNPs present in the dog to a computer system; comparing this data to a database which comprises information relating to breed-specific genomic SNPs and the nutritional requirements, disease susceptibility

C

c n or behavioural characteristics of the breeds; and determining on the basis of the comparison the nutritional requirements, disease susceptibility or behavioural characteristics of the dog.

The invention also provides a computer program comprising program code means for performing all the steps of a method of the invention when said program is run on a computer. Also provided is a computer program product comprising WO 2004/113570 PCT/GB2004/002559 36 program code means stored on a computer readable medium for performing a method of the invention when said program is run on a computer. A computer program product comprising program code means on a carrier wave that, when executed on a computer system, instruct the computer system to perform a method of the invention is additionally provided.

As illustrated in Figure 1, the invention also provides an apparatus arranged to perform a method according to the invention. The apparatus typically comprises a computer system, such as a PC. In one embodiment, the computer system comprises: means 20 for receiving data of breed-specific genomic SNP markers; a module 30 for comparing the data with a database 10 comprising information relating to breedspecific genomic SNPs and optionally the nutritional requirements, disease susceptibility or behavioural characteristics of the breeds; and means 40 for determining on the basis of said comparison the breed inheritance and optionally the nutritional requirements, disease susceptibility or behavioural characteristics of the dog.

Food manufacturing In one embodiment of the invention, the manufacture of a customised dog food may be controlled electronically. Typically, the nutritional requirements of the dog may be processed electronically to generate a customised dog food formulation.

The customised dog food formulation may then be used to generate electronic manufacturing instructions to control the operation of food manufacturing apparatus.

The apparatus used to carry out these steps will typically comprise a computer system, such as a PC, which comprises means 50 for processing the nutritional requirement information to generate a customised dog food formulation; means 60 for generating electronic manufacturing instructions to control the operation of food manufacturing apparatus; and a food product manufacturing apparatus The food product manufacturing apparatus used in the present invention typically comprises one or more of the following components: container for dry pet food ingredients; container for liquids; mixer; former and/or extruder; cut-off device; cooking means oven); cooler; packaging means; and labelling means. A dry ingredient container typically has an opening at the bottom. This opening may be covered by a volume-regulating element, such as a rotary lock. The volumeregulating element may be opened and closed according to the electronic 00 O manufacturing instructions to regulate the addition of dry ingredients to the pet food.

SDry ingredients typically used in the manufacture of pet food include corn, wheat, Z meat and/or poultry meal. Liquid ingredients typically used in the manufacture of pet food include fat, tallow and water. A liquid container may contain a pump that can be controlled, for example by the electronic manufacturing instructions, to add a measured amount of liquid to the pet food.

In one embodiment, the dry ingredient container(s) and the liquid container(s) are coupled to a mixer and deliver the specified amounts of dry ingredients and liquids to the mixer. The mixer may be controlled by the electronic manufacturing I instructions. For example, the duration or speed of mixing may be controlled. The mixed ingredients are typically then delivered to a former or extruder. The former/extruder may be any former or extruder known in the art that can be used to shape the mixed ingredients into the required shape. Typically, the mixed ingredients are forced through a restricted opening under pressure to form a continuous strand.

As the strand is extruded, it may be cut into pieces (kibbles) by a cut-off device, such as a knife. The kibbles are typically cooked, for example in an oven. The cooking time and temperature may be controlled by the electronic manufacturing instructions.

The cooking time may be altered in order to produce the desired moisture content for the food. The cooked kibbles may then be transferred to a cooler, for example a chamber containing one or more fans.

The pet food manufacturing apparatus may comprise a packaging apparatus.

The packaging apparatus typically packages the pet food into a container such as a plastic or paper bag or box. The apparatus may also comprise means for labelling the pet food, typically after the food has been packaged. The label may provide information such as: ingredient list; nutritional information; date of manufacture; best before date; weight; and breed(s) or breed category or sub-group for which the food is suitable.

The invention is illustrated by the following Examples: WO 2004/113570 PCT/GB2004/002559 38 Example 1 DNA Samples Buccal cells were collected from 72 dogs of 16 different breeds by scraping the inside cheek six times with a sterile cytology brush (Rocket Medical, Cat No.

R57483), ensuring that the animal providing the sample had not consumed any food or drink for 30min prior to sample collection. The brushes were then replaced in their individual wrappers and left to dry for a minimum of 2 hours at room temperature.

DNA was extracted using standard techniques (Qiagen's QIAamp DNA Blood Mini Kit, Cat No. 51104) following the Buccal Swab Spin protocol. The DNA was then stored at -20 0

C.

PCR Amplifications The primers used were designed to amplify products ranging from 200-600bp in length. The primers were designed by eye, and were made to be approximately in length, with approximately 50% G/C, 50% A/T ratio. These were ordered from Sigma-Genosys, desalted, and at 0.025pM synthesis scale. 12.5ng of genomic dog DNA (Gibco, Cat. 69234) was added to 12.5ng of DNA from each dog and was amplified up in 25pi PCR reactions with Eurogentec HotGoldstar PCR mastermix (PK-0073-02). Reactions contained 1.5mM MgC12 and 25pmol of each primer.

Thermal cycling was performed using a Hybaid MBS 0.2S PCR machine using the following cycling conditions: an initial incubation of 95°C for 10 min, followed by cycles of 95°C for 30 sec, 60'C for 45 sec and 72 0 C for 90 sec. This was followed by a final extension step of 72°C for 5 min.

Single Nucleotide Polymorphism Identification The base sequence of the wild- type amplicon was manually inputted into the Transgenomic Wave Machine, and the PCR products run according to the manufacturers directions as described in the WAVEMAKER Software Manual, Transgenomic Inc. 1999, version 2.0 Oct 1999. Chromatograms were examined for the presence of additional peaks indicating the presence of a single nucleotide polymorphism in the sample. The PCR amplification described above was repeated on the DNA samples indicated to have SNPs present, with the following change: of the test DNA sample was added to the PCR reaction, and no other DNA was added.

WO 2004/113570 PCT/GB2004/002559 39 The PCR products were then purified using a Qiagen PCR Purification Kit (Cat No.

28104), following the method Qiaquick PCR Purification using a microcentrifuge.

DNA Sequencing Cycle sequencing was performed using 25fmol of purified PCR product with the CiEQ 2000 Dye Terminator Cycle Sequencing with Quick Start Kit (Beckman Coulter, P/N 608120). 20 h'l reactions were prepared as described in the manufacturers directions using the same primers used in the PCR step, and were subjected to 30 cycles at 95'C for 20 sec, 60'C for 20 sec and 72'C for 4 min.

Following these cycles, the samples were subjected to ethanol precipitation, and were evaporated to dryness using a vacuum pump for approximately 40 min. The samples were then resuspended in 40[dl of deionised formamide and a drop of mineral oil was placed on top. The samples were then run on a Beckman CEQ 2000 Sequencer using the LFR capillary method. SNPs were called using the CEQ2000XL DNA Analysis System Softwvare Version 4.3.9, and were confirmed using the reverse traces.

Resuits A SNP was identified in the mast cell chymase gene of the breed English Mastiff at position 5375. The SNP is shown as underlined in the sequence below (SEQ ID NO: The position 5375 is defined using standard nomenclature as can be seen in accession NCBJ Ref U89607. The following primers were used for PCR amplification: Forward Primer (5260bp-5279bp) ACT CCA OTT CAC OTO CAG C Reverse Primer (5600bp-5620bp) AGA GAT OCT GOC ACC TTG C ACTCCACTTCACCTCCAGCAAAACAGAGCATAACTTGGAAGA-ACATCTG

ACTCCACTTCACCTCCAGCAAAACAGAGCATAACTTGGAAGAAACATCTG

ATCAGAAAGATAGCCTAATATGGGAGAAGAAAAACATGACCACATAGTTC 100 AIAAAAAUTARGGG GAACTACCTG'C CTGTGGTTACCAGCCCAGCCCTTGGCTCATTGCTGGAGTTATAAAACCCA 150

CTGTGGTTACCAGCCTAGCCCTTGGCTCATTGCTGGAGTTATAAAACCCA

AGACCAGAAAATAGAAGCAGCATCTGCCCAGGGCAGCCTCACTGAGAAC-A 200

AGACCAGAAAATAGAAGCAGCATCTGCCCAGGGCAGCCTCACTGAGAAC-A

TGCATTGTCTTCCTCTCACCCTGCTGCTCCTTCTCCTATGTTCCAGAGCA 250

TGCATTGTCTTCCTCTCACCCTGCTGCTCCTTCTCCTATGTTCCAGAGCA

00 SGAAGCTGGTGAGTCTTGGGATCCTTCCCCCTGGAAACGGCAGGATCAGCA 300 0GAAGCTGGTGAGTCTTGGGATCCTTCCCCCTGGAAACGGCAGGATCAGCA CCCCAAAACCAAGTTTAGTCTGAATATAGCTGACTCATAAGCAAGGTGGC 350

CCCCAAAACCAAGTTTAGTCTGAATATAGCTGACTCATAAGCAAGGTGGC

AGGATCTCTCT

AGGATCTCTCT (SEQIDNO:1) 1 10 Table 2 SNP Breed INCBI Ref for gene ISNP and flanking sequences

SNP

Mast cell English U89607 SEQ ID NO: 1 chymase/C5375T Mastiff This SNP was found in English Mastiff dogs, and not in any of the other different breeds tested. Hence the mast cell chymase SNP identified above is unique to this breed.

Example 2 DNA Samples Dog genomic DNA was acquired from various sources. In total, 51 dogs were included in the study: 5 German Shepherds, 6 Rottweilers, 6 Dobermans, 6 Cocker Spaniels, 6 Golden Retrievers, 2 Poodles, 2 Beagles, 6 Yorkshire Terriers, 6 Shih Tzus and 6 Labradors.

Obtaining Sequence In order to obtain the canine sequence for a gene of interest it was necessary to firstly acquire the cDNA sequence of the human form. A search was carried out for the gene at www.ensembl.org, searching under the gene database. To obtain the DNA sequence for the same gene in the dog, the ncbi webpage was accessed (www.ncbi.nlm.nih.gov). A search was carried out, searching the nucleotide database for canisfamiliaris. The original human cDNA sequence was then accessed from the transcript information gained through ensembl and this sequence was copied into the cross-species megablast. Canisfamiliaris WGS (whole genome sequence) was chosen in the database field. A blast search was then carried out. From the blast results all those alignments with a score over 200 were selected.

WO 2004/113570 PCT/GB2004/002559 41 The gene sequence was then edited before primer design could take place. A blast search was carried out on the sequence using blastn (nucleotide-nucleotide blast). The results of this blast search highlighted the repeat regions within the sequence. An additional blast search using blastx (translated query vs protein database) was used to determine the position of exons in the sequence. Blast results were checked to ensure that firstly they were actually relevant to the gene of interest and also that they were in a positive reading frame. Only those results with a high identity were marked on the sequence as exons.

Primer Design Primers were manually designed along the contig at 600 bp spacing. The forward primer of each amplicon was located approximately 50 bp before the reverse primer of the previous amplicon. Primer design was concentrated around exonic regions and away from repeat regions. Primers were approximately 20 bases long with a melting temperature between 56 0 C and 64 0 C. The primers were ordered desalted at a synthesis scale of 0.025 pM (Sigma-Genosys).

PCR Amplification ul PCR reactions were carried using 25 pmol of each primer, 25 ng of commercial dog genomic DNA (Novegen, Cat. No 69234) and 12.5 l of Eurogentec HotGoldstar PCR mastermix containing a red loading dye and 1.5 mM MgCl (PK- 0073-02R). Thermal cycling was performed using a Hybrid MBS 0.2S PCR machine using the following cycling conditions: incubation at 95 0 C for 10 mins, 10 cycles of for 30 seconds, followed by 64°C (minus 1°C per cycle) for 45 seconds and 72 0 C for 90 seconds, and 28 cycles of 95°for 30 seconds, 55°C for 45 sec and 72°C for seconds. 5ul of each PCR sample and lul Gelstar nucleic acid gel stain (BioWhittaker Molecular Applications, Cat. No 50535) was run on a 2 agarose gel (Invitrogen, Cat. No 15510-027) at 100 mV to check for the presence of product.

Primers used in successful PCR reactions were plated into forward and complementary reverse plates at a concentration of 25 pmol. Samples were quantified with the Nanodrop Spectrophotometer using 1 j1l of purified DNA. Analysis was carried out using Nanodrop 2.4.7a DNA-50 software. DNA was diluted to 100 ng/pl.

WO 2004/113570 PCT/GB2004/002559 42 Sequencing All sets of DNA were amplified using each primer pair, under the same conditions as stated above. The PCR reactions were purified and a sample of purified product was sequenced. Both forward and reverse sequence traces were generated using the original primers for the reaction.

PolyPhred Analysis In order to identify SNPs (single nucleotide polymorphisms) the PolyPhred computer programme was employed. PolyPhred automatically detects the presence of heterozygous single nucleotide substitutions by comparing the pattern of the fluorescence dye incorporation between traces (Nickerson et al. Nucleic Acids Research 25 (14):2745-2751, 1997). PolyPhred is not used alone but in conjuction with Phred automated base calling, Phrap sequence assembly and Consed sequence assembly editing. The output from PolyPhred was then reformatted for the genetic algorithm software (G-max).

Gmax The objective of the next stage was to derive a way of determining breed status using a pattern of SNPs found via sequencing. The Gmax software (http://www.thegmax.com) uses a genetic algorithm to extract such patterns from large data sets. The pattern is extracted in the form of a rule. Each rule is expressed as a Boolean formula, where is "AND", is and is "NOT".

Gmax was used to screen thousands of SNPs to find a combination of a smaller number that define the breed well. For example, using 5 SNPs from a possible 1000, there will be 1017 possible combinations to search through. Randomly picking rules to fit the data would not work very well. However, a fitness test can determine how well a random rule performs at separating the data and comparing it to how close to a solution it is. If small changes are made to the rule and retest a second score for a new rule is generated. A continuation of this process will evolve the rule. This way of working is called 'hill climbing'. The problem with hill climbing is that for complex fitness tests there are local maximums. If a local maximum is reached then the overall solution will not be found. A genetic algorithm solves this problem by keeping a large population of rules and applying a form of Darwinian evolution.

WO 2004/113570 PCT/GB2004/002559 43 Results Rules were generated for 4 breeds, namely Cocker Spaniel, Shih Tzu, Doberman and Golden Retriever. Tables 3 to 6 show the rules for each breed in the form of a Boolean formula. Table 7 shows the sequences surrounding each SNP, and which bases may be present at each polymorphic position (options). The full name of each gene is abbreviated as follows: FCGR2A: FC gamma receptor RIIa; FCGR3B: FC gamma receptor RIIIb; RAGE: receptor for advanced glycation end product; and PAIl: plasminogen activator inhibitor 1.

WO 2004/113570 WO 204/13570PCTIGB2004/002559 Table 3 Spaniel A B _C Boolean formula SNP Allele SNP Allele, SNP Allele A B RAGE 8Kb 6000 AA RAGE 8Kb 6002 AA (A I(B C) RAGE 8Kb 6002 AA RAGE 8Kb 5959 TT FCGR3B 7.42Kb 5238 CC (Al B) RAGE 8Kb 6002 AA PAIIlO1KB 2979 TT (Aj B) RAGE 8Kb 6002 AA PAIIlO1KB 3317 GG (A B) RAGE 8Kb 6002 AA RAGE _5KB 43301AA Table 4 Shili Tzu A B __C Boolean formula SNp Allele SNP Allele SNP Allele FCGR3B_7.42Kb FCGR3B_7.42Kb (A RAGE 8Kb 6006 CC 5264 AG 5137 IT FCGR3B 7.42Kb FCGR3B_7.42Kb (A)I(B 5002 AG IRAGE 8Kb 6006 CC 5264 AG FCGRJB_-7.42Kb FCGR3B_7.42Kb (B 5167 TT RAGE 8Kb 6006 CC 5264 A FCGR3B_7.42Kb (A C) RAGE 8Kb 5820 AA PAll 10KB 2979 TT 5137 Tr Table Doberman A B C Boolean formula SNp Allele SNP Allele SNP Allele FCGR2A_9.86Kb I(B C)I D) I E 8708 GG RAGE 8Kb 5847 GG RAGE 5KB 4329 AA ID IE ISPl~ll Table 6 Golden Retriever A B C Boolean formula SNP Allele SNP JAllele SNP Allele A IC)) I FCGR3B_7.42Kb 5239 CC RAGE 8Kb 5771 CC RAGE 8Kb 6182 C D I E F SNP JAllele ISNP _Allele ISNP JAllele RGE5K 405GG FCGR3B_-7.421K'b [FT 4805GG 4947 CC RAGE 8Kb 6006 ITT_ WO 2004/113570 WO 204/13570PCT/GB2004/002559 Table 7 Sequences around SNIPs SNP Name Before Options After RAGE_8Kb GGTGGTTGGiGCAAAGTGCGCGCG A G GAGCTTGGCGAGGTAGCAAATG 6000 CGCAGCAGGCGGCGGGAAGGGG CTATGGTCGCAGGGCTCAAAATG CGGGCCAGGCCGAGAGTGCTCG GCTCCGGCCTCCTTCGGTGACGT GCTTTCTCTGGCCCCACCCCCTCC AGAGGGCAGAACTGGGGCTCGC GCCGCGGTCTTGTCGCCGTGGTG CCCTCCCTTTCAGTGAAGAAGGG ACTGCTCTACGATTGGCGGGCT.T AGCAGAACTGGCATCAGCTCGAC TGGGGTTCAAAGGCATCAGCGCG GTGTGGGTGGTGCGAGCATCGAC GGCTCATCCGGGCCCTCCGACTG GTGCGGCGCTGGTGTTAACCCAT CGGTCTCTCCGGGCTGATTGGCT CTCCCTCGOCTGCGGGACGCAGC AGTTTCTTGCAGCCCTGATTGGC CCGCTCCTCCTTAGGTGACTTGC CGAGATCGGAAAAGACGGCCG AGAACAGATCTGAAGGCCCAC RAGE_8Kb TGGTTCGGCAAAGTGCGCGCGCG A TG GCTTGGCGAGGTAGCAAATGCTA 6002 CAGCAGGCGGCGGGAAGGGGCG TGGTGGCAGGGCTCAAAATGGCT GGCCAGGCCGAGAGTGCTCGGCT CCGGCCTCCTTCGGTGACGTAGA 7FECTCTGGCCCCACCCCCTCCGC GGGCAGAACTGGGGCTCGCCCCT CGCGGTCTTGTCGCCGTGGTGAC CCCTTTCAGTGAAGAAGGGAGCA TGCTCTACGATTGGCGGGCTTTG GAACTGGCATCAGCTCGACGTGT GGGTTCAAAGGCATCAGCGCCGC GGGTGGTGCGAGCATCGACCTGC CTCATCGGGGCCTCCGACTGCG GGCGCTGGTGTTAACCCATCTCC GTCTCTCCGGGCTGATTGGCTAG CTCGGCTGCGGGACGCAGCCCGC 'TTTCTTGCAGCCCTGATTGGCCG TCCTCCTTAGGTGACTTGCAGAA AGATCGGAAAAGACGGCCGGG CAGATCTCAAGGCCCACAC RAGE_8Kb GAGCTCCGAAGCTCTGGATTGGC C T TCTTGCAGCCCTGATTGGCCGAG 5959 TGOAACTCGACGGGAGATGGTG ATCGGAAAAGACGGCCGGGAGC GTTCGGCAAAGTGCGCGCGCGCA TTGGCGAGGTAGCAAATGCTATG GCAGGCGGCGGGAAGGGGCGGG GTCGCAGGGCTCAAAATGGCTCC CCAGGCCGAGAGTGCTCGGCTTT GGCCTCCTTCGGTGACOTAGAGG CTCTGGCCCCACCCCCTCCGCCG GCAGAACTGGGGCTCGCCCCTCC CGGTCTTGTCGCCGTGGTGACTG CTTTCAGTGAAGAAGGGAGCAG CTCTACGATTGGCGGGCTTTGGG AACTGGCATCAGCTCGACGTGTG GTTCAAAGGCATCAGCGCCGCCT GGTGGTGCGAGCATCGACCTGCG CATCCGGGCCCTCCOACTGCOGT GCGCTGGTGTTAACCCATCTCCC CTCTCCGGOCTGATTGGCTAGT TCGGCTGCGGGACGCAGCCCG FCGR3B_7. TCCTCCGAGGGCCCCATTTCTC C T GAGTTCTGGAGGAAAACAAGAG 42Kb_5238 ACCCCTGCCCCTCGCCTGGGCTT ACCTCTTCAGGGAATGCCCTTCT CTCTTAACAGAAGGTGTGAATCT CCTTGGGTCTCATCCCCAGGGTT CATGGTCAAATTCATGCTAAGAA GTGATATCTTCTTGTTCTTGGCAC GTGCGTCATAGACCTCAACACAC TGAGTGGATCCAAACCCGCCAGG ATGCTTTTTGAATTCTTGAAAAA TAGGGAAACTTATTTTGAGACTG TAAGCTGGTGGAGACAACACTGC ATCAGATTTATCCATGTCACTAG AGGGAGGGACCCTTCCTCAGGGT ATGCTTGCCTGTAGTCTCCTGTG CTGAAGATTTAAATGAATACAGG GACCCGAGGGTGCTCATCCACCT CAGGTAGGTCCAGGTGGATGGA CAGCTTCTCTTTCCGGCTTTTTTC GCCGGGTCTGGGCCAGGCAGG CTCCCCTTCCTGCCCCAT TCCCCTCAAAGACACACATCCAA C T AGCACTCITCTCTGTGCCCATGAT 2979 TCTGAGGACTGAAGGGAACCATC ___GGCTCAGACCAACAAGTTCAACT WO 2004/113570 WO 204113570PCTiGB2004/002559 TAAAAGGATTAAAAAATATCTAG ACAGTAAGTTCAAGACTTCTTGC CCCCTTTACCCCCCAGCTCACCT AAAAGTCCCACAACTACTGCACC GGCTTTCTCATAGGCATGATTGG CCATCCCCTCGGGTATTCCCCTCT CAACTTAGTGGGCAGAGGGGCC CAGGGAGGAGAAACCTTTGAAT GTGGACCAGCTGACGCGTCTGAT GTAGCCCAGCTTTGCCAGAGCCT GCTGGTAAATGCCCTCTACTTCA CCCCAGGCAGGAGCTACTGGGAT ACGGCCAGTGGAAGACGCCCTTC GACAGGAGCAGCAGAAAGTAGC CGGAGTGAGGCACCCACCACCGC TTCATCTCATGCAAGCCAAAGCT CTCTTCCACAAATCTGACGG GACATCCAGAAAGTCCCTCC RAGE_8Kb TCGGCAAAGTGCGGGCGCGCAG C T GGCGAGGTAGCAAATGCTATGGT 6006 CAGGCGGCGGGAA6GGGCGGGC COCAGGGCTCAAAATGGCTCCGG CAGGCCGAGAGTGCTCGGCTTTC CCTCCTTCGGTGACGTAGAGGGC TCTGGCCCCACCCCCTCCGCCGC AGAACTGGGGCTCGCCCCTCCCT GGTCTTGTCGCCGTGGTGACTGG TTCAGTGAAGAAGGGAGCAGAA TCTACGATTGGCGGGGCTTTGGGG CTGGCATCAGCTCGACGTGTGGG TTCAAAGGCATCAGCGCCGCCTC TGGTGCGAGCATCGACCTGCGGC ATCCGGGCCCTCCGACTGCGGTC GCTGGTGTTAACCCATCTCCCTC TCTCCGGGCTGATTGGCTAGTTTr GGCTGCGGGACGGAGCCCGCTCC CTTGCAGCCCTGATTGGCCGAGA TCCTTAGGTGACTTGCAGAACAG TCGGAAAAGACGGCCGGGAGC ATCTCAAGGCCCACACCTTT FCGR3B_7. CCTGCCCCTCGCCTGGGCTTCTCT A G CTTCAGGGAATGCCCTTCTCCTT 42Kb5264 TAACAGAAGGTGTGAATCTCATG GGGTCTCATCCCCAGGGTTGTGA GTCAAATTCATGCTAAGAAGTGC TATCTTCTTGTTCTTGGCACTGAG GTCATAGACCTCAACACACATGC TGGATCCAAACCCGGCAGGTAGG TTTTTGAATTCTTGAAAAATAAG GAAACTTATTTTGAGACTGATCA CTGGTGGAGACAACACTGCAGG GATTTATCCATGTCACTAGATGC GAGGGACCCTTCCTCAGGGTCTG TTGCCTGTAGTCTCCTGTGGACC AAGATTTAAATGAATACAGGGA CGAGGGTGCTCATCCACCTCAGC GGTAGGTCCAGGTGGATGGAGC TTCTCTTTCCGGCTTTTTTCCTCC CGGGTCTGGGCCAGGCAGGAGA CCTTCCTGCCCCATCCTGGGGCT GTTCTGGAGGAAAACAAGAGAC CACTTGTCAGAATTCAG

C

FCGR3B_7. TGATGGTGGGTTGAAGCTAAAGA T G GACAACACTGCAGGGAGGGACC 42Kb_5137 GCTCCTGTCCTCTCCTGCCCGCTG CTTCCTCAGGGTCTGAAGAT7FTA TCCTTCCCTCTGCGCCTCCTTCTC AATGAATACAGGCAGGTAGGTC TGCCTTCCCTTCAACCAATTAGT CAGGTGGATGGAGCCGGGTCTG c3ACTTCCTCCCTCCCAGGGCCCC GGCCAGGCAGGAGAGTTCTGGA ATTTCTCACCCCTGCCCCTCGCCT GGAAAACAAGAGACCTCTTCAG GGGCTTCTCTTAACAGAAGGTGT GGAATGCCCTTCTCCTTGGGTCT GAATCTCATGGTCAAATTCATGC CATCCCCAGGGTTGTGATATCTT TAAGAAGTGCGTCATAGACCTCA CTTGTTCTTGGCACTGAGTGGAT ACACACATGCTTTTTGAATTCTT CCAAACCCGCCAGGTAGGGAAA GAAAAATAAGCTGGTGG CTTATTTTGAGACTGATCAGATT

TA

FCGR3B_7. ACTTCCTGGCCACTGCACTTCCA A G CATTTCTCACCCCTGCCCCTCCCC 42Kb_5002 CCTTTTCCAATAAGGCACCCCGG TGGGCTTCTCTTAACAGAAGGTG AGCCAGGGCTACAGGCTCACAG TGAATCTCATGGTCAAATTCATG ACCAGCCCAGGCCAGTGGGTCTC CTAAGAAGTGCGTCATAGACCTC CGAGGGGCTGAGCTCACCTGGCT AACACACATGCTTTTTGAATTCT AGTGTCAGTGCTCAGCCCTGGTG TGAAAAATAAGCTGGTGGAGAC ATGGTGGGTI'GAAGCTAAAGAG AACACTGCAGGGAGGGACCCTTC CTCCTGTCCTCTCCTGCCCGCTGT CTCAGGGTCTGAAGATTTAAATG WO 2004/113570 WO 204113570PCTiGB2004/002559

GCTTCCGTCTGCGGCTCCTTGTCT

GCCTTCCCTTCAACCAATTAGTG

ACTTCCTCCCTCCCAGGGCC

AATACAGGGAGGTAGGTCCAGG

TGGATGGAGCCGGGTCTGGGCCA

GGCAGGAGAGTTCTGGAGGA

FCGR3B_7. TCCTCTCCTGCCCGCTGTCCTTCC C T GGGTCTGAAGATTTAAATGAATA 42Kb_5167 CTCTGCGCCTCCTTCTCTGCCTTC CAGGCAGGTAGGTCCAGGTGGA CCTTCAACCAATTAGTGACTTCC TGGAGCCGGGTCTGGGCCAGGC TCCCTCCCAGGGCCCCATTTCTC AGGAGAGTTCTGGAGGAAAACA ACCCCTGCCCCTCGCCTGGGCYI7 AGAGACCTCTTCAGGGAATGCCC CTCTTAACAGAAGGTGTGAATCT TTGTCCTTGGGTCTCATCCCCAG CATGGTCAAATTCATGCTAAGAA GGTTGTGATATCTTCTTGTTCTTG GTGCGTCATAGACCTCAACACAC GCACTGAGTGGATCCAAACCCGC ATGCTTTTTGAATTCTTGAAAAAk CAGGTAGGGAAACTTATTTTGAG TAAGCTGGTGGAGACAACACTGC ACTGATCAGATTTATCCATGTCA AGGGAGGGAGCCTTCCTC CTAGATGCTTGCCTGTAGTCT RAGE_8Kb TCCGAGTAGCTGCCAGTCAGGGC A T TCTCTGGCCCCACCCCCTCCGCC 5820 CAAGGGCCAGAAGCAATTGGTC GCGGTCTTGTCGCCGTGGTGACT CGGGACCACACAGGCCTCGCCTC GCTCTACGATTGGCGGGCTTTGG CTCCGAGCCCTTTCTTTGCTTCAC GGTTCAAAGGCATCAGCGCCGCC TTCCGCTTTCCGAGAACGTCCGG TCATCCGGGCCCTCCGACTGCGG ATTCCTATTGGACTTTGGAGCGT TCTCTCCGGGCTGATTGGCTAGT AGAGCTCCGAAGCTCTGGATTGG TTCTTGCAGCCCTGATTGGCCGA CTGGAACTCGACGGGAGATGGT GATCGGAAAAGACGGCCGGGAG GGTTCGGCAAAGTGCGCGCGCGC CTTGGCGAGGTAGCAAATGCTAT AGCAGGCGGCGGGAAGGGGCGG GGTCGCAGGGCTCAAAATGGCTC GCCAGGCCGAGAGTGCTCGGCT CGGCCTCCTTCGGTGACGTA FCGR2A_9. TCCTTTCTCTTTCCCCTCCTCTCA A G GGGCCTTGTTCTCTGAACAGAAA 86Kb_8708 GAGAAGCAGAGGATAGGCAGCC TAGGAAGAGATTGATTGATTGAT ATGGTGCACAGGTGCTTTAACCC TGCACCTCGGTGAAGTACATGCT TTCTGGTTCTGAGAGGGTGAGAC GCTGCGCACTCCTTACTCAACAC ATCACAGATATTGTCCCAGAAAA TAGGAATCTCCCACCTCCCAGGC TAACTCACCCCCTCTCTCAGTA TCCCAGGGAGGGGATGGGGGTG AAATCAAGAGCCCAAACATTTTT CAGTTCTCCCTGGGGCACTGACC CGTCTTAGCTGCACGCGAAATCC CCAGGGCTCCTTAGACTAGACCT CATCATCTGCCTAGATTATCTCC CCAGCCTTTCTTTCTTTTTCTTTC ATGTGTTGATAAATCCTCCACTT TGAGGCCACAGAGACCCCTCTGT TGCATGACTCTGAGGGCTTC ACTTTGGTGCCAAGACAGG RAGE_8Kb GGCCAGAAGCAATTGGTCCGGG C TG TCTTGTCGCCGTGGTGACTGCTC 5847 ACCACACAGGCCTCGCCTCCTCC TACGATTGGCGGGCTTTGGGGTT GAGCCCTTTCTTTGCTTCACTTCC CAAAGGCATCAGCGCCGCCTCAT CCTTTCCGAGAACGTCCGGATTC CCGGGCCCTCCGACTGCGGTCTC CTATTGGACTTTGGAGCGTAGAG TCCGGGCTGATTGGCTAGTTTCT CTCCGAAGCTCTGGATTGGCTGG TGCAGCCCTGATTGGCCGAGATC AACTCGACGGGAGATGGTGGTTC GGAAAAGACGGCCGGGAGCTTG GGCAAAGTGCGCGCGCGCAGCA GCGAGGTAGCAAATGCTATGGTC GGCGGCGGGAAGGGGCGGGCCA GCAGGGCTCAAAATGGCTCCGGC GGCGGAGAGTGCTCGGCTTTCTC CTCCTTCGGTGACGTAGAGGOCA TGGCCCCACCCCCTCCGCCGCG GAACTGGGGCTCGCCCCTCC CTTGGGCAGGGCTGGATTCAGTA A T AAAAGCCAGGTGTGGGGGAAAG 4329 ATTTTGAGGAAGCGCCACCTTCC TCAAATGACCAGTGTCCCATCCT _______CCTGTGAGTGACACATCTTTAAG TGGCCAGAACCCTACCATCTGAG WO 2004/113570 WO 204113570PCTiGB2004/002559 TCTTCTTTTTAACCTATTTGCAGA TCCCTCAAACATCCTCAGGATTT TTGGAGAGGGAAGAACAGGGAG TATAAGACTGTCATAGTGGGGAA GGGGTTATTGCCAAATATGTTAA CCTCTCCTGTCAAAGACCAGGCA ATGTGGGTTGGOGTGCTTGTGTA GGAGTGGAGGGGAGCAGGTTAG TGTATCTCCCTCAATTTCCCCAG ATGGGTGATGGGTGGAGGGTGG AAACGAGGCATTCTTTThI7TCTC GAGGCACGGGCCGGGGGCAGTT AGTCTAAAAtCAAGAGGGTGGG CTCTCCTCACTTGTAAACTTGTA GGGAGAGAGGAGGCATGTCAT GTTTCACAGAAAAGGAAAAAAA

A

ACGGGCCGGGGGCAGTTCTCTCC T G CAAGGCCAGGCCAGGCCTGAAC 4766 TCACTTGTAAACTTGTAGTTTCA CCTGTTGCCCAGCAACCTTACGT CAGAAAAGGAAAAAAAAAATGC AAGCAACATGGGGCTCCCATCGT AGTTTTAAATAAAGAAATTTGTT CCACCAGGCAAGCCCTCAGTGGA TT'ITCCCTGGGTTTAGTTGAGCAT CTGATGGAATGGGTTAGGGGTCC TATTTTCAAAAACATGATAAACC TGAATACTAAGAAACCTTAGGAG CCAGAATAAAATTCTTfCATAAA GTCCACTCCCAACCCCCACGGAC ACCCCAAACGGTGTTTTCCCTTC ATGGCTGTGCCCAGACTGGCACT CAGCTACCCACTCCCAACCTTAC GCCTAAGGGTGGGGTGATCATTG CCTCACCACCCAGGAGCACCCAT TTTCTCCTAGTACCTGAAGGACT GGTTCACCCTCAACCCTCCC CTTGTCTAAGAAGCATGAAT RAGE_8Kb GCGGTCTCTCCGGGCTGATTGGC C T CTCCCTCGGCTGCGGGACGCAGC 6182 TAGTT[CTTGCAGCCCTGATTGG CCGCTCCTCCTTAGGTGACTTGC CCGAGATCGGAAAAGACGGCCG AGAACAGATCTCAAGGCCCACA GGAGCTTGGCGAGGTAOCAAAT CCTTTCTAACGTTGACACAGGAT GCTATGGTCGCAGGGCTCAAAAT GACAGAGTTGACCCCGGCCCCGT GGCTCCGGCCTCCTTCGGTGACG TTTAAACCTGAAAAGCGAACTAG TAGAGGGCAGAACTGGGGCTCG CTCCACCCCTTCGTGAGTAGGTG CCCCTCCCTTTCAGTGAAGAAGG CCGAGGGGGCAAGGCCCGCCCT GAGCAGAACTGGCATCAGCTCG CCTGAGCGACCCGCGGCGGAAT ACGTGTGGGTGGTGCGAGCATCG GGGGTTAGGCCCGCCCCTTCCGT ACCTGCGGCGCTGGTGTTAACCC CCTGTAGTGTGTCCCGCAGAAG

A

FCGR3B_7. CCCTCCCAGGGCCCCATTTCTCA C T AGTTCTGGAGGAAAACAAGAGA 42Kb_5239 CCCCTGCCCCTCGCCTGGGCTTC CCTCTTCAGGGAATGCCCTTCTC TCTTAACAGAAGGTGTGAATCTC C'FrGGGTCTCATCCCCAGGGTTG ATGGTCAAATTCATGCTAAGAAG TGATATCTTCTTGTTCTTGGCACT TGCGTCATAGACCTCAACACACA GAGTGGATCGAAACCCGCCAGGT TGCTTTTTGAATTCTTGAAAAAT AGGGAAACTTATTTTGAGACTGA AAGCTGGTGGAGACAACACTGC TCAGATTTATCCATGTCACTAGA AGGGAGGGACCCFJ7CCTCAGGGT TGCTTGCCTGTAGTCTCCTGTGG CTGAAGATTTAAATGAATACAGG ACGGGAGGGTGCTCATCCACCTC CAGGTAGGTCCAGGTGGATGGA AGC'YICTCTTTCCGGCTTTTTTCC GCCGGGTCTGGGCCAGGCAGGA TCCCCTTCCTGCCCCATC RAGE_8Kb 5771

CATGCGACAGAATTGGTGTCCGT

TGGACCTGGTCGGGGAGCTTGAT

TCGTCCGAGTAGCTGCCAGTCAG

GGCCAAGGGCCAGAAGCAATTG

GTCCGGGACCACACAGGCCTCGC

CTCCTCCGAGCCCTTTCTTTGCTT

CACTTCCCCTTTCCGAGAACGTC

CGGATTCCTATTGGAGTTTGGAG

CGTAGAGCTCCGAAGCTCTGGAT

GCGCAGCAGGCGGCGGGAAGGG

GCGGGCCAGGCCGAGAGTGCTC

GGCTTTCTCTGGCCCCACCCCCT

CCGCCGCGGTCTTGTCGCCGTGG

TGACTGCTCTAGGATTGGCGGGC

TTTGGGGTTCAAAGGCATCAGCG

CCGCCTCATCCGGGCCCTCCGAC

TGCGGTCTCTCCGGGCTGATTGG

GTAGTTTCTTGCAGCCCTGATTG

I

WO 2004/113570 WO 204/13570PCT/GB2004/002559

TGGCTGGAACTCGACGGGAGAT

GGTGGTTCGGCAAAGTGCGCG

GCCGAGATGGGAAAAGACGGCC

GGGAGCTTGGCGAGGTAGCAAA

AGTTTCACAGAAAAGGAAAAAA T G YYACCTAAGCAACATGGGCICTCC 4805 AAAATGCAGTTTTAAATAAAGAA CATCGTCCACCAGGCAAGCCCTC ATTTCTTTYCCCTGGGTTTAGT AGTGGACTGATGGAATGGGTTAG TGAGCATTATTTTCAAAAACATG GGGTCCTGAATACTAAGAAACCT ATAAACCCCAGAATAAAATTCTT TAGGAGGTCCACTCCCAACCCCC TCATAAAACCCCAAACGGTGTTT ACGGACATGGCTGTGCCCAGACT TCCCTTCGAGCTACCCACTCCCA GGCACTGCCTAAGGGTGGGGTG ACCTTACCCTCACCACCCAGGAG ATCATTGTTTCTCGTAGTACCTGA CACCCATGGTTCACCCTCAACCC AGGACTCTTGTCTAAGAAGCATG TCCCCCAAGGCCAGGCCAGGCCT AATTCCTAGCATTCCCCGTGGCC GAACCCTGTTGCCCAGCAAC GGATAGGACAGGATGGAAA FCGR3B_7. GCCGTGTGTTGOGGGGATGCGGC C T CCTCCTTCTCTOCCTTCCCTTCAA 42Kb_4947 TAGGGAGAGTAGAACAGGGTAG CCAATTAGTGACTTCCTCCCTCC CAATCTTAAGACTTCCTGGCCAC CAGGGCCCCATTTCTCACCCCTG TGCACTTCCACCTTTTCCAATAA CCCCTCGCCTGGGCTTCTCTTAA GGCACCCCGGAGCCAGGGCTAC CAGAAGGTGTGAATCTCATGGTC AGGCTCACAGACCAGCCCAGGC AAATTCATGCTAAGAAGTGCGTC CAGTGGGTCTCCGAGGGGCTGAG ATAGACCTCAAGACACATGCTTT CTCACCTGGCTACTGTCACTGCT TTGAATTCTTGAAAAATAAGCTG CAGCCCTGGTGATGGTGGGTTGA GTGGAGACAACACTGCAGGGAG AOCTAAAGAGCTCCTGTCCTCTC GGACCCTTCCTCAGGGTCTGAAG CTGCCCGGTGTCCTTCCGTCTGC ATTTAAATGAATACAGGCA

Claims

1. A method of determining the genetic breed inheritance of a dog, the method comprising: detecting the presence or absence of one or more breed- specific SNP markers in the dog; and c identifying therefrom the genetic breed inheritance of the dog.

2. The method according to claim 1, wherein the one or more breed-specific SNP 10 markers are used to distinguish one breed of dog in a panel of dog breeds from the other breeds in the panel.

3. The method according to claim 2, wherein the panel consists of from 5 to breeds.

4. The method according to any one of the preceding claims, wherein the one or more breed-specific SNP markers are specific for any of the breed categories selected from Hounds, Working dogs, Terrier, Gundogs, Pastoral, Utility dogs or Toy dogs.

5. The method according to claim 4, wherein the one or more breed-specific SNP markers are capable of distinguishing between Labrador Retriever, Golden Retriever, German Shepherd, Dachshund, Shih Tzu, Yorkshire Terrier, Poodle, Rottweiler, Boxer and Cocker Spaniel.

6. The method according to any one of claims 1 to 4, comprising: detecting the presence or absence of one or more breed-specific SNP markers indicative of the Cocker Spaniel breed; and determining therefrom whether the Cocker Spaniel breed has contributed to the genetic breed inheritance of the dog.

7. The method according to any one of claims 1 to 4, comprising: detecting the presence or absence of one or more breed-specific SNP markers indicative of the Shih Tzu breed; and 00 determining therefrom whether the Shih Tzu breed has contributed to 0 Sthe genetic breed inheritance of the dog. n 8. The method according to any one of claims 1 to 4, comprising: N 5 detecting the presence or absence of one or more breed-specific SNP markers indicative of the Doberman breed; and c determining therefrom whether the Doberman breed has contributed to the genetic breed inheritance of the dog. S 10 9. The method according to any one of claims 1 to 4, comprising: S(a) detecting the presence or absence of one or more breed-specific SNP markers indicative of the English Mastiff breed; and determining therefrom whether the English Mastiff breed has contributed to the genetic breed inheritance of the dog. The method according to any one of the preceding claims, wherein the one or more breed-specific SNP markers are present in 70% or more of a sample population of that breed and/or present in 30% or less of a sample population of another breed.

11. The method according to claim 10, wherein the sample population comprises four or more dogs per breed.

12. The method according to any one of the preceding claims, wherein the one or more breed-specific SNP markers are present in at least 95% of dogs in a sample population of from 400 to 1000 dogs of a breed and/or are present in 5% or less of dogs in a sample population of from 400 to 1000 dogs of any other breed.

13. The method according to any one of claims 10 to 12, wherein each breed- specific SNP marker is present in 99% or more of a sample population of that breed and/or present in 1% or less of a sample population of another breed.

14. The method according to any one of the preceding claims, wherein the positions of at least 10 or more breed-specific SNP markers are typed in step 00 15. The method according to any one of the preceding claims, wherein the dog is a 0 mongrel. n 16. The method according to any one of the preceding claims, wherein detecting N 5 the presence or absence of the breed-specific SNP markers is carried out in vitro on a sample from the dog and optionally the sample is blood, urine, saliva, skin, cheek cell Cc or hair root.

17. The method according to any one of the preceding claims, wherein the presence or absence of the breed-specific SNP markers in the dog is detected by contacting a polynucleotide or protein from the dog with a specific binding agent and determining whether the agent binds to the polynucleotide or protein.

18. Use of one or more breed-specific SNP markers for identifying the genetic breed inheritance of a dog.

19. The use according to claim 18, wherein the SNP markers are as defined in any one of claims 2 to 13.

20. The use according to claim 18 or 19, wherein the positions of at least 10 or more breed-specific SNP markers are typed.

21. The use according to any one of claims 18 to 20, wherein the dog is a mongrel.

22. The use according to any one of claims 18 to 21, wherein detecting the presence or absence of the breed-specific SNP markers is carried out by a method according to claim 16 or 17.

23. A method for identifying the genetic breed inheritance of a dog, the method comprising: inputting genetic data from the dog to a computer system; (ii) comparing the data to a computer database, which database comprises information relating to breed-specific genomic SNPs; and (iii) determining on the basis of the comparison the presence or 00 absence of one or more breed-specific SNP markers, thereby identifying the breed inheritance of the dog. S24. The method according to claim 23, wherein the breed-specific SNP markers S 5 are as defined in any one of claims 2 to 13. c 25. The method according to claim 23 or 24, wherein the presence or absence of at least 10 or more breed-specific SNP markers are determined in step (iii).

26. The method according to any one of claims 23 to 25, wherein the dog is a 0 mongrel.

27. A computer program comprising program code means that, when executed on a computer system, instruct the computer system to perform all the steps of any one of claims 23 to 26.

28. A computer system arranged to perform a method according to any one of claims 23 to 26 comprising: means for receiving genetic data from the dog; (ii) a module for comparing the data with a database comprising information relating to breed-specific genomic SNPs; and (iii) means for determining on the basis of said comparison the breed inheritance of the dog.

29. A method of preparing customised food for a dog, comprising: determining the genetic breed inheritance of the dog by a method according to any one of claims 1 to 17 or 23 to 26; generating a customised dog food formulation comprising components suitable for the breed(s) which contributed to the genetic breed inheritance of the dog, and which does not comprise components that are not suitable for the breed(s) which contributed to the genetic breed inheritance of the dog; and preparing a dog food according to the customised dog food formulation. 54 00 30. A method according to claim 29, wherein the food does not comprise O Singredients which are poorly tolerated, cause allergies, are abnormally processed or Sstored, or contribute to diseases or conditions typically suffered by the breed(s) which have contributed to the genetic breed inheritance of the dog or wherein the food S 5 contains ingredients which have nutritional or medical benefits for the breed(s) which have contributed to the genetic breed inheritance of the dog.

31. A method according to claim 29 or 30, wherein the food contains: cocoa flavanols, other plant flavanols, lycopene, curcumin, minerals, trace metals, Echineacea, phosphatidyl serine, L-arginine, ginseng, psyllium, prebiotics, probiotics, phyto-oestrogens, phyto-chemicals, soluble fibre, PUFAs or phospholipids; and/or does not contain or has low levels of: gluten-containing grains such as wheat, animal proteins, milk, eggs, soy, peanuts, shellfish, fruits, tree nuts, copper, saturated fats or salt.

32. A method according to any one of claims 29 to 31 further comprising providing the food to the dog's owner, the person responsible for feeding the dog or a vet.

33. A method of providing a customised dog food, the method comprising providing to the dog's owner, the person responsible for feeding the dog or a vet a food which contains components suitable for the breed(s) which have contributed to the genetic breed inheritance of the dog, and which does not contain components that are not suitable for the breed(s) which have contributed to the genetic breed inheritance of the dog, wherein the breed inheritance of the dog has been identified by detecting the presence or absence of one or more breed-specific SNP markers in the dog genome.

34. A method according to claim 23, further comprising: (iv) electronically processing the genetic breed inheritance information to generate a customised dog food formulation; generating electronic manufacturing instructions to control the operation of food manufacturing apparatus in accordance with the customised dog food formulation; and 19 Jun 2008 3:17PM WATERMARK 98196010 00 o (vi) manufacturing the customised dog food according to the electronic manufacturing instructions. ;Z A computer system according to claim 28, further comprising: (iv) means for processing the genetic breed inheritance information to generate a customised dog food formulation; M means for generating electronic manufacturing instructions to control Nthe operation of food manufacturing apparatus in accordance with the customised dog food formulation; and (vi) a food product manufacturing apparatus.

36. Use of a computer system as defined in claim 35 to make a customised dog food product.

37. A method according to claim 1 substantially as hereinbefore described in any one of the Examples. COMS ID No: ARCS-195143 Received by IP Australia: Time 15:20 Date 2008-06-19