The Benefit of a National Genomic Testing Scheme.

Although a significant cost, genotyping an entire population offers many benefits, many of which can reduce the workload and effort in decision-making on farm. As well as providing more accurate predictions of the genetic merit of individuals (and by extension their expected performance), national genotyping strategies enable complete traceability from the cradle to the grave as well as parentage discovery. The information available per animal aids more informed breeding and management decisions, including mating advice, and determining the optimal role and eventual fate of each animal.

A study on methods for preimplantation genetic testing (PGT) on in vivo- and in vitro-produced equine embryos, with emphasis on embryonic sex determination.

Two methods for preimplantation genetic testing (PGT) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocoele fluid aspiration (BFA). While TCB is widely applied for both in vivo- and in vitro-produced embryos, BFA has been mostly utilized for in vivo-produced embryos. Alternative methods for PGT, including analysis of cell-free DNA (CFD) in the medium where in vitro-produced embryos are cultured, have been reported in humans but not for equine embryos. In Experiment 1, in vivo- (n = 10) and in vitro-produced (n = 13) equine embryos were subjected to BFA, cultured for 24 h, then subjected to TCB, and cultured for additional 24 h. No detrimental effect on embryonic diameter or re-expansion rates was observed for either embryo group (P > 0.05). In Experiment 2, the concordance (i.e., agreement on detecting the same embryonic sex using two techniques) among BFA, TCB, and the whole embryo (Whole) was studied by detecting the sex-determining region Y (SRY) or testis-specific y-encoded protein 1 (TSPY) (Y-chromosome), and androgen receptor (AR; X-chromosome) genes using PCR. Overall, a higher concordance for detecting embryonic sex was observed among techniques for in vivo-produced embryos (67-100 %; n = 14 embryos) than for in vitro-produced embryos (31-92 %; n = 13 embryos). The concordance between sample types increased when utilizing TSPY (77-100 %) instead of SRY (31-100 %) as target gene. In Experiment 3, CFD analysis was performed on in vitro-produced embryos to determine embryonic sex via PCR (SRY [Y-chromosome] and amelogenin - AMEL [X- and Y-chromosomes]). Overall, CFD was detected in all medium samples, and the concordance between CFD sample and the whole embryo was 60 % when utilizing SRY and AMEL genes. In conclusion, equine embryos can be subjected to two biopsy procedures (24 h apart) without apparent detrimental effects on embryonic size. For in vivo-, but not for in vitro-produced equine embryos, BFA can be considered a potential alternative to TCB for PGT. Finally, CFD can be further explored as a non-invasive method for PGT in in vitro produced equine embryos.

Development of PCR-based genetic test for detection of TTF1 mutation causing abortion in Holstein Friesian cattle.

A stop-gain mutation (rs715966442; BTA11: 1,02,463,944 nucleotide position) in transcription termination factor, RNA polymerase I (TTF1) gene causes abortion in Holstein Friesian (HF) cattle. A PCR-restriction fragment length polymorphism (PCR-RFLP)-based genetic test has been developed and validated to screen the TTF1 mutation locus in HF cattle. The mutation locus was screened in 80 HF and HF crossbreds using the protocol, which revealed two animals as carriers of the mutant TTF1 allele. The test employed is cost-effective, rapid and precise and can be utilized as an effective tool for the screening of TTF1 mutation carriers in HF cattle population.

Congenital heart defects in Arabian horses and the prospects of genetic testing: A review.

Congenital heart defects (CHDs) can have profound and potentially life-threatening consequences on horses' health and performance capability. While CHDs are rare in the general horse population, the Arabian breed is disproportionately overrepresented and thus is widely suspected to be genetically predisposed. This review discusses the most common CHDs in Arabian horses, including ventricular septal defect (VSD), tetralogy of Fallot (TOF), patent duct arteriosus (PDA), tricuspid valve atresia (TVA) and atrial septal defect (ASD). This review also explores how future research into the genetic factors that likely underpin many CHDs can revolutionise the way these disorders are managed in Arabian horses.

Incorporating Genetic Testing into a Breeding Program.

Genetic tests are powerful tools that enable (1) a focus on genetic diversity as mating outcomes can be predicted and thus optimized to minimize or even avoid exclusion and (2) working toward breeding goals by improving a phenotype.

Evaluation of the value of genetic testing for cystinuria in the Danish population of English bulldogs.

Cystinuria is a genetic disease that can lead to cystine urolith formation. The English bulldog is the dog breed most frequently affected. In this breed, three missense mutations have been suggested to be associated with cystinuria: c.568A>G and c.2086A>G in SLC3A1 and c.649G>A in SLC7A9. In this study, the occurrence of these three mutations in the Danish population of English bulldogs was investigated. Seventy-one English bulldogs were genotyped using TaqMan assays. The dogs' owners were given questionnaires concerning the medical histories of their dogs. Allele frequencies of 0.40, 0.40, and 0.52 were found for the mutant alleles in the three loci: c.568A>G, c.2086A>G, and c.649G>A, respectively. For both mutations in SLC3A1, a statistically significant association was found between cystinuria and homozygosity for the G allele among male, English bulldogs. For the mutation in SLC7A9, there was no statistically significant association between homozygosity for the mutant allele and cystinuria. Due to high allele frequencies, limited genetic diversity, continued uncertainty about the genetic background of cystinuria, and more severe health problems in the breed, selection based on genetic testing for the mutations in SLC3A1 cannot be recommended in the Danish population of English bulldogs. However, results of the genetic test may be used as a guide to recommend prophylactic treatment.

Genetics of inherited skin disorders in dogs.

Canine genodermatoses represent a broad spectrum of diseases with diverse phenotypes. Modern genetic technology including whole genome sequencing has expedited the identification of novel genes and greatly simplified the establishment of genetic diagnoses in such heterogeneous disorders. The precise genetic diagnosis of a heritable skin disorder is essential for the appropriate counselling of owners regarding the course of the disease, prognosis and implications for breeding. Understanding the underlying pathophysiology is a prerequisite to developing specific, targeted or individualized therapeutic approaches. This review aims to create a comprehensive overview of canine genodermatoses and their respective genetic aetiology known to date. Raising awareness of genodermatoses in dogs is important and this review may help clinicians to apply modern genetics in daily clinical practice, so that a precise diagnoses can be established in suspected genodermatoses.

A retrospective case series of clinical signs in 28 Beagles with Lafora disease.

BACKGROUND: Clinical signs and their progression in Beagles with Lafora disease are poorly described. OBJECTIVES: To describe clinical signs in Beagles with Lafora disease. ANIMALS: Twenty-eight Beagles with Lafora disease confirmed by genetic testing or histopathology. METHODS: Retrospective multicenter case series. Data regarding signalment, clinical signs, diagnostic tests and treatment were retrieved from hospital data files. A questionnaire was sent to owners asking about neurological deficits, changes in cognitive functions, behavioral changes, response to treatment and survival time. RESULTS: Onset of clinical signs was 8.3 years (mean; range, 6.3-13.3). All dogs had myoclonic episodes as an initial clinical sign with tonic-clonic seizures in n = 11/28 (39%) and n = 12/28 (43%) later developing tonic-clonic seizures. Deficits of coordination (n = 21/25; 84%), impaired vision (n = 15/26; 58%), and impaired hearing (n = 13/26; 50%) developed later. Mental decline was observed as loss of house training (urination; n = 8/25; 32%), difficulties performing learned tasks (n = 9/25; 36%), and difficulties learning new tasks (n = 7/23; 30%). Common behavioral changes were: increased photosensitivity (n = 20/26; 77%), staring into space (n = 16/25; 64%), reduced stress resistance (n = 15/26; 58%), increased noise sensitivity (n = 14/26; 54%), and separation anxiety (n = 11/25; 44%). Twenty-one dogs were alive (median age 11.9 years; range, 9.8-18.6), and 7 dogs were dead (mean age 12.1 years; SD: 1.3; range, 10.5-12.6) at time of writing. CONCLUSIONS AND CLINICAL IMPORTANCE: Lafora disease in Beagles causes significant behavioral changes, and mental decline as well as neurological deficits in addition to myoclonic episodes and generalized tonic-clonic seizures. Nevertheless, a relatively normal life span can be expected.

The PMEL gene and merle (dapple) in the dachshund: cryptic, hidden, and mosaic variants demonstrate the need for genetic testing prior to breeding.

One of the most unique coat color patterns in the domestic dog is merle (also known as dapple in the dachshund breed), characterized by patches of normal pigmentation surrounded by diluted eumelanin pigment. In dogs, this striking variegated pattern is caused by an insertion of a SINE element into the PMEL gene. Differences in the length of the SINE insertion [due to a variable-length poly(A)-tail] has been associated with variation in the merle coat color and patterning. We previously performed a systematic evaluation of merle in 175 Australian shepherds and related breeds and correlated the length of the merle insertion variants with four broad phenotypic clusters designated as "cryptic", "atypical", "classic", and "harlequin" merle. In this study, we evaluated the SINE insertions in 140 dachshunds and identified the same major merle phenotypic clusters with only slight variation between breeds. Specifically, we identified numerous cases of true "hidden" merle in dachshunds with light/red (pheomelanin) coats with little to no black/brown pigment (eumelanin) and thus minimal or no observable merle phenotype. In addition, we identified somatic and gonadal mosaicism, with one dog having a large insertion in the harlequin size range of M281 that had no merle phenotype and unintentionally produced a double merle puppy with anophthalmia. The frequent identification of cryptic, hidden, and mosaic merle variants, which can be undetectable by phenotypic inspection, should be of particular concern to breeders and illustrates the critical need for genetic testing for merle prior to breeding to avoid producing dogs with serious health problems.

L2HGDH Missense Variant in a Cat with L-2-Hydroxyglutaric Aciduria.

A 7-month-old, spayed female, domestic longhair cat with L-2-hydroxyglutaric aciduria (L-2-HGA) was investigated. The aim of this study was to investigate the clinical signs, metabolic changes and underlying genetic defect. The owner of the cat reported a 4-month history of multiple paroxysmal seizure-like episodes, characterized by running around the house, often in circles, with abnormal behavior, bumping into obstacles, salivating and often urinating. The episodes were followed by a period of disorientation and inappetence. Neurological examination revealed an absent bilateral menace response. Routine blood work revealed mild microcytic anemia but biochemistry, ammonia, lactate and pre- and post-prandial bile acids were unremarkable. MRI of the brain identified multifocal, bilaterally symmetrical and T2-weighted hyperintensities within the prosencephalon, mesencephalon and metencephalon, primarily affecting the grey matter. Urinary organic acids identified highly increased levels of L-2-hydroxyglutaric acid. The cat was treated with the anticonvulsants levetiracetam and phenobarbitone and has been seizure-free for 16 months. We sequenced the genome of the affected cat and compared the data to 48 control genomes. L2HGDH, coding for L-2-hydroxyglutarate dehydrogenase, was investigated as the top functional candidate gene. This search revealed a single private protein-changing variant in the affected cat. The identified homozygous variant, XM_023255678.1:c.1301A>G, is predicted to result in an amino acid change in the L2HGDH protein, XP_023111446.1:p.His434Arg. The available clinical and biochemical data together with current knowledge about L2HGDH variants and their functional impact in humans and dogs allow us to classify the p.His434Arg variant as a causative variant for the observed neurological signs in this cat.

Web resource on available DNA variant tests for hereditary diseases and genetic predispositions in dogs and cats: An Update.

Vast progress has been made in the clinical diagnosis and molecular basis of hereditary diseases and genetic predisposition in companion animals. The purpose of this report is to provide an update on the availability of DNA testing for hereditary diseases and genetic predispositions in dogs and cats utilizing the WSAVA-PennGen DNA Testing Database web resource (URL: http://research.vet.upenn.edu/WSAVA-LabSearch ). Information on hereditary diseases, DNA tests, genetic testing laboratories and afflicted breeds added to the web-based WSAVA-PennGen DNA Testing Database was gathered. Following verification through original research and clinical studies, searching various databases on hereditary diseases in dogs and cats, and contacting laboratories offering DNA tests, the data were compared to the resource reported on in 2013. The number of molecularly defined Mendelian inherited diseases and variants in companion animals listed in the WSAVA-PennGen DNA Testing Database in 2020 drastically increased by 112% and 141%, respectively. The number of DNA variant tests offered by each laboratory has also doubled for dogs and cats. While the overall number of laboratories has only slightly increased from 43 to 47, the number of larger corporate laboratories increased, while academic laboratories have declined. In addition, there are now several laboratories that are offering breed-specific or all-breed panel tests rather than single-DNA tests for dogs and cats. This unique regularly updated searchable web-based database allows veterinary clinicians, breeders and pet owners to readily find available DNA tests, laboratories performing these DNA tests worldwide, and canine and feline breeds afflicted and also serves as a valuable resource for comparative geneticists.

Genetic and demographic history define a conservation strategy for earth's most endangered pinniped, the Mediterranean monk seal Monachus monachus.

The Mediterranean monk seal (Monachus monachus) is a flagship species for marine conservation, but important aspects of its life history remain unknown. Concerns over imminent extinction motivated a nuclear DNA study of the species in its largest continuous subpopulation in the eastern Mediterranean Sea. Despite recent evidence of partial subpopulation recovery, we demonstrate that there is no reason for complacency, as the species still shares several traits that are characteristic of a critically endangered species: Mediterranean monk seals in the eastern Mediterranean survive in three isolated and genetically depauperate population clusters, with small effective population sizes and high levels of inbreeding. Our results indicated male philopatry over short distances, which is unexpected for a polygynous mammal. Such a pattern may be explained by the species' unique breeding behavior, in which males defend aquatic territories near breeding sites, while females are often forced to search for new pupping areas. Immediate action is necessary to reverse the downward spiral of population decline, inbreeding accumulation and loss of genetic diversity. We propose concrete conservation measures for the Mediterranean monk seal focusing on reducing anthropogenic threats, increasing the population size and genetic diversity, and thus improving the long-term prospects of survival.

PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers.

BACKGROUND: The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs. RESULT: First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. CONCLUSION: In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

Evaluation of the efficiency of TaqMan duplex real-time PCR assay for non-invasive pre-natal assessment of foetal sex in equine.

Accurate diagnosis of foetal sex in pregnant mare is helpful for many breeders, both for private or commercial purposes. In this study, in order to pre-natal foetal sexing in equine, we used TaqMan duplex real-time PCR to detect the specific regions of SRY and TSPY genes on extracted cell-free foetal DNA from maternal blood. Peripheral blood samples from 50 pregnant Arabian mares with singleton foetuses were collected. Cell-free foetal DNA was extracted from maternal plasma, and duplex real-time PCR assays were performed with TaqMan probes and primers. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as control of DNA extraction procedure. From the 50 sampled mares, 28 cases had female and 22 mares had male foetuses. The final results for 46 samples were conclusive, and from them, 43 cases were predicted correctly. Sensitivity, specificity and accuracy of the test were 90.48%, 96% and 93.48%, respectively. In conclusion, a TaqMan duplex real-time PCR was set up to pre-natal detection of foetal sex in equine. The method was fast and decreased the false-positive and false-negative results. The technique can be used as a routine procedure in farms by collecting only a blood sample.

Commercial genetic testing for type 2 polysaccharide storage myopathy and myofibrillar myopathy does not correspond to a histopathological diagnosis.

BACKGROUND: Commercial genetic tests for type 2 polysaccharide storage myopathy (PSSM2) and myofibrillar myopathy (MFM) have not been validated by peer-review, and formal regulation of veterinary genetic testing is lacking. OBJECTIVES: To compare genotype and allele frequencies of commercial test variants (P variants) in MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323), FLNC (P3b; rs1142918816) and MYOZ3 (P4; rs1142544043) between Warmblood (WB) and Arabian (AR) horses diagnosed with PSSM2/MFM by muscle histopathology, and phenotyped breed-matched controls. To quantify variant frequency in public repositories of ancient and modern horse breeds. STUDY DESIGN: Cross sectional using archived clinical material and publicly available data. METHODS: We studied 54 control-WB, 68 PSSM2/MFM-WB, 30 control-AR, 30 PSSM2/MFM-AR and 205 public genotypes. Variants were genotyped by pyrosequencing archived DNA. Genotype and allele frequency, and number of variant alleles or loci were compared within breed between controls, PSSM2/MFM combined and MFM or PSSM2 horses considered separately using additive/genotypic and dominant models (Fisher's exact tests). Variant frequencies in modern, early domestic and Przewalski horses were determined from a public data repository. RESULTS: There was no significant association between any P locus and a histopathological diagnosis of PSSM2/MFM, and no difference between control and myopathic horses in total loci with alternative alleles, or total alternate alleles when PSSM2/MFM was considered combined or separately as PSSM2 or MFM. For all tests, sensitivity was <0.33. Allele frequencies in WB (controls/cases) were: 8%/15% (P2), 5%/6% (P3a/b) and 9%/13% (P4); in AR, frequencies were: 12%/17% (P2), 2%/2% (P3a/b) and 7%/12% (P4). All P variants were present in early domestic (400- to 5500-year-old) horses and P2 present in the Przewalski. CONCLUSIONS: Because of the lack of significant association between a histopathological diagnosis of PSSM2 or MFM and the commercial genetic test variants P2, P3 and P4 in WB and AR, we cannot recommend the use of these variant genotypes for selection and breeding, prepurchase examination or diagnosis of a myopathy.

Genetic analysis of resilience indicators based on milk yield records in different lactations and at different lactation stages.

Resilience is the ability of cows to cope with disturbances, such as pathogens or heat waves. To breed for improved resilience, it is important to know whether resilience genetically changes throughout life. Therefore, the aim was to perform a genetic analysis on 2 resilience indicators based on data from 3 periods of the first lactation (d 11-110, 111-210, and 211-340) and the first 3 full lactations, and to estimate genetic correlations with health traits. The resilience indicators were the natural log-transformed variance (LnVar) and lag-1 autocorrelation (rauto) of daily deviations in milk yield from an expected lactation curve. Low LnVar and rauto indicate low variability in daily milk yield and quick recovery, and were expected to indicate good resilience. Data of 200,084 first, 155,784 second, and 89,990 third lactations were used. Heritabilities were similar based on different lactation periods (0.12-0.15 for LnVar, 0.05-0.06 for rauto). However, the heritabilities of the resilience indicators based on full first lactation were higher than those based on lactation periods (0.20 for LnVar, 0.08 for rauto), due to lower residual variances. Heritabilities decreased from 0.20 in full lactation 1 to 0.19 in full lactation 3 for LnVar and from 0.08 to 0.06 for rauto. For LnVar, as well as for rauto, the strongest genetic correlation between lactation periods was between period 2 and 3 (0.97 for LnVar, 0.96 for rauto) and the weakest between period 1 and 3 (0.81 for LnVar, 0.65 for rauto). Similarly, for both traits the genetic correlation between full lactations was strongest between lactations 2 and 3 (0.99 for LnVar, 0.95 for rauto) and weakest between lactations 1 and 3 (0.91 for LnVar, 0.71 for rauto). For LnVar, genetic correlations with resilience-related traits, such as udder health, ketosis, and longevity, adjusted for correlations with milk yield, were almost always favorable (-0.59 to 0.02). In most cases these genetic correlations were stronger based on full lactations than on lactation periods. Genetic correlations were similar across full lactations, but the correlation with udder health increased substantially from -0.31 in lactation 1 to -0.51 in lactation 3. For rauto, genetic correlations with resilience-related traits were always favorable in lactation period 1 and in most full lactations, but not in the other lactation periods. However, correlations were weak (-0.27 to 0.15). Therefore, as a resilience indicator for breeding, LnVar is preferred over rauto. A multitrait index based on estimated breeding values for LnVar in lactations 1, 2, and 3 is recommended to improve resilience throughout the lifetime of a cow.

Genetic analysis of pathogen-specific intramammary infections in dairy cows.

Mastitis is one of the most common diseases in dairy cattle, causing severe economic losses to dairy farmers. Mastitis usually occurs due to intramammary infection (IMI) caused by a variety of pathogenic bacteria. Although good progress has been made in understanding genetics of pathogen-specific clinical mastitis, studies involving genetic analysis of pathogen-specific IMI are scarce. The overall objective of this study was, therefore, to assess genetic variation of overall and pathogen-specific IMI in nonclinical primiparous and multiparous cows using bacterial culture. Data and milk samples were collected over a 2-yr interval as part of the Canadian Bovine Mastitis Research Network. The final data set contained records of 46,900 quarter milk samples from 3,382 clinically healthy primiparous and multiparous Holstein cows from 84 dairy herds. For the genetic analysis, we considered the following 7 traits: overall IMI, non-aureus staphylococci (NAS) IMI, contagious pathogen IMI, environmental pathogen IMI, major pathogen IMI, minor pathogen IMI and somatic cell score (SCS). Data were analyzed at the quarter level using a threshold-probit model via Gibbs sampling in BLUPF90. Prevalence of IMI traits at the quarter level in multiparous cow from 0 to 400 DIM ranged from 6.8 to 45.5%. Posterior mean of quarter heritability estimates (on the underlying scale, posterior SD in brackets) of overall IMI and pathogen-specific IMI traits ranged from 0.017 to 0.073 (±0.009 to 0.030). Weak to strong genetic correlations [ranging from 0.18 to 0.97 (±0.01 to 0.29)] among pathogen-specific IMI traits and with overall IMI indicated that not all of these traits were genetically similar. Weak to moderate Spearman rank correlations between estimated breeding values for overall IMI and pathogen-specific IMI traits (from 0.31 to 0.87) indicated possible substantial reranking of sires. The percentage of daughters with IMI caused by various pathogen groups ranged from 13 to 80% and from 38 to 94% for the best (10% decile) and worst sires (90% decile) according to their IMI trait-specific estimated breeding values, respectively. Pathogen-specific IMI traits and overall IMI had weak to moderate positive genetic correlations [ranging from 0.11 to 0.81 (±0.11 to 0.22)] with SCS. Therefore, selection for lower SCS will improve resistance to IMI. However, based on the observed weak to moderate rank correlations (0.04 to 0.47) between pathogen-specific IMI traits and SCS, selection for lower SCC will not improve resistance to IMI from every pathogen-specific IMI group in the same manner. Therefore, despite low heritability estimates, there was sizeable genetic variation for pathogen-specific IMI traits, indicating that long-term direct genetic selection for pathogen-specific IMI can improve pathogen-specific IMI resistance.

Expanding the genetic toolbox.

Knowledge of canine genetics has advanced rapidly in recent years. Researchers say even deeper understanding is ahead, and that it could support a shift to more preventative veterinary care. But the involvement of a wide group of people will be vital in progressing towards that future.

Genetic analysis of the endangered Cleveland Bay horse: A century of breeding characterised by pedigree and microsatellite data.

The Cleveland Bay horse is one of the oldest equines in the United Kingdom, with pedigree data going back almost 300 years. The studbook is essentially closed and because of this, there are concerns about loss of genetic variation across generations. The breed is one of five equine breeds listed as "critical" (<300 registered adult breeding females) by the UK Rare Breeds Survival Trust in their annual Watchlist. Due to their critically endangered status, the current breadth of their genetic diversity is of concern, and assessment of this can lead to improved breed management strategies. Herein, both genealogical and molecular methods are combined in order to assess founder representation, lineage, and allelic diversity. Data from 15 microsatellite loci from a reference population of 402 individuals determined a loss of 91% and 48% of stallion and dam lines, respectively. Only 3 ancestors determine 50% of the genome in the living population, with 70% of maternal lineage being derived from 3 founder females, and all paternal lineages traced back to a single founder stallion. Methods and theory are described in detail in order to demonstrate the scope of this analysis for wider conservation strategies. We quantitatively demonstrate the critical nature of the genetic resources within the breed and offer a perspective on implementing this data in considered breed management strategies.