© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL DNA Testing to Determine Breed GENETIC HEALTH ANALYSIS™ 3000 Dr. Cynthia Cole DVM, PhD, DACVCP Director R&D Mars Veterinary

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL There is more to a “Breed” than meets the eye. It is in the DNA! How do you determine what breed a dog is?

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL How do we get different breeds? Within one species of domestic animals, a breed is a specific group generally having a homogenous appearance (phenotype), homogeneous behavior, and/or other characteristics that distinguish them from other organisms of the same species and that were arrived at through selective breeding. Canis lupus familiaris or Canis familiaris

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL Breed Determination- How it Works Each dog in a breed is more similar genetically to other members of that breed than to dogs in other breeds.

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL Genetically similar = more of their DNA in common

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL DNA Think of DNA as a series of letters that make up words and sentences  genes WENSDFLEENTHIVMESTIFMMKLW MEBRCSWEACKMAECNETLSEMSEIL NMSTAILENVMESEIVMESTTHEQUIC KFOXJUMPEDOVERTHESLEEPYDOG WNEMSDKVESTMEASEACVEIEMTE AOWAMLMOIMEWLTNISVESMLTEA IDFMLAEABIEMSELKAAGESAOKLLJH GNLEIUGNESEUVCNESLKEJNTNSEK ASEUCVENNKSEJKTLENSUEKABBET RENSUEKVNESUTKMEWLASIEWEJO BRDRDSMRMESLE

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL DNA Think of DNA as a series of letters that make up words and sentences  genes WENSDFLEENTHIVMESTIFMMKLW MEBRCSWEACKMAECNETLSEMSEIL NMSTAILENVMESEIVMESTTHEQUIC KFOXJUMPEDOVERTHESLEEPYDOG WNEMSDKVESTMEASEACVEIEMTE AOWAMLMOIMEWLTNISVESMLTEA IDFMLAEABIEMSELKAAGESAOKLLJH GNLEIUGNESEUVCNESLKEJNTNSEK ASEUCVENNKSEJKTLENSUEKABBET RENSUEKVNESUTKMEWLASIEWEJO BRDRDSMRMESLE

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL DNA  RNA  Protein cellbio.utmb.edu/ cellbio/ribosome.htm

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL DNA mutations create differences in sequences resulting in different “alleles”

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL One Gene- Two Alleles  One gene controls whether you get a black or a chocolate Labrador Retriever.  B- (Black color) is dominant over b (brown color) B_ bb

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL By selecting for a common phenotype; we selected for a common set of genes and other markers. 11 Parker, H. Mammalian Genomics 2012

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL Insight Into Breed Differences Key Learnings:  More than 99% of genetic material is common to all breeds  Breeds could be differentiated using Single Nucleotide Polymorphisms (SNP) patterns  Not necessarily associated with a gene. Dog Genome Project completed in 2005 Site of Variation Identified DNA sequence for all 39 chromosome pairs in dogs

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL Understanding SNPs  SNPs can be found in dog DNA ~ every 1000 base pairs along the chromosomes  Different breeds have very different SNP patterns –Some SNPs will be found in almost all breeds- not very useful for breed ID. –Some SNPs will be found in only a few breeds- very useful in breed identification AAAT TTAATTATAA TGGGTG TTTGGGTG ATTTATTTAATT AT GG AAAG GGAG SNP A SNP B SNP D SNP C SNPs are considered among the best markers for DNA analysis because of their extremely low relative mutation rate.

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL Family Tree predicted that best fits data: Tested dog The algorithm scans all SNP markers used in the test and looks for matches to breed signatures in our database of 250 breeds, types and varieties (gives marker by marker certainty score for each breed match): Scores are totalled across all markers and best breed matches given significance score for each tree: Each breed detected has a numerical confidence score and lowest confidence breeds are called out in the report It then scans the DNA data fitted to each of the 11 different family trees and predicts which tree is best fit for best explaining the DNA data SNP marker data Algorithm analyses data Algorithm – How it works Confidential - Mars Veterinary

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL These pups are F1 crosses from what two purebreds? Why do we need a DNA test to tell what breed?

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL 16

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL Why do we care what the breeds are in a dog? Size or weight predications –Puppies and young dogs –Older dogs- the difficult discussion he is not just big boned Behavior –High energy breeds versus laid back –Territorial or guarding tendencies? –Herding tendencies? Health related issues –Breed disease risks not genetically defined Shar-Pei Fever Golden Retriever- increased cancer risk? 17

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL Results of Testing

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL Limitations of the Test Can only detect breeds in the database –US purebred in some cases have a very different genetic pattern than the same purebred in Europe. –Large validation project underway for initial launch of product in France, Germany, Netherlands, and UK. Certain behaviors are associated with certain breeds –Behaviors in cross breds??? 19

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL ROYAL CANIN® Genetic Health Analysis ™ 3000 New Version

© COPYRIGHT MARS INCORPORATED | CONFIDENTIAL Questions? RoyalCaninGHA.com Thank You For Your Time!