PCR multiplexed microsatellite panels to expedite canine genetic disease linkage analysis

Modern dog breeds possess large numbers of genetic diseases for which there are currently few candidate genes or diagnostic tests. Linkage of a microsatellite marker to a disease phenotype is often the only available tool to aid in the development of screening tests for disease carriers. Detection of linkage to a specific disease phenotype requires screening of large numbers of markers across known affected and unaffected animals. To establish high throughput genome scanning this study placed 100 canine microsatellite markers, arranged by fragment size and fluorescent dye label, into 12 PCR multiplexed panels. The highest degree of multiplexing was 11 markers per panel while the lowest was five markers per panel; each panel was run in one gel lane on automated DNA sequencers. Selection of the markers was based upon chromosomal or linkage group locations, degree of polymorphism, PCR multiplex compatibility and ease of interpretation. The marker set has an average spacing of 22.25 centiMorgan (cM). Marker polymorphism was evaluated across 28 American Kennel Club (AKC) recognized breeds. The utility of buccal swab vs. blood samples was also validated in this study as all template DNA was derived from swabs obtained and submitted by participating dog breeders and owners. The PCR multiplexed microsatellite panels and sampling method described in this report will provide investigators with a cost effective and expedient means of pursuing linkage studies of specific canine genetic diseases.     

Power of exclusion for parentage verification and probability of match for identity in American Kennel Club breeds using 17 canine microsatellite markers

DNA analysis of microsatellite markers has become a common tool for verifying parentage in breed registries and identifying individual animals that are linked to a database or owner. Panels of markers have been developed in canines, but their utility across and within a wide range of breeds has not been reported. The American Kennel Club (AKC) authorized a study to determine the power to exclude non-parents and identify individuals using DNA genotypes of 17 microsatellite markers in two panels. Cheek swab samples were voluntarily collected at Parent Breed Club National Specialty dog shows and 9561 samples representing 108 breeds were collected, averaging 88.5 dogs per breed. The primary panel of 10 markers exceeded 99% power of exclusion for canine parentage verification of 61% of the breeds. In combination with the secondary panel of seven markers, 100% of the tested breeds exceeded 99% power of exclusion. The minimum probability match rate of the first panel was 3.6 x 10(-5) averaged across breeds, and with the addition of the second panel, the probability match rate was 3.2 x 10(-8); thus the probability of another random, unrelated dog with the same genotype is very low. The results of this analysis indicated that, on average, the primary panel meets the AKC's needs for routine parentage testing, but that a combination of 10-15 genetic markers from the two panels could yield a universal canine panel with enhanced processing efficiency, reliability and informativeness.     

PCR MULTIPLEXED MICROSATELLITE PANELS TO EXPEDITE CANINE GENETIC DISEASE LINKAGE ANALYSIS

ABSTRACT

Modern dog breeds possess large numbers of genetic diseases for which there are currently few candidate genes or diagnostic tests. Linkage of a microsatellite marker to a disease phenotype is often the only available tool to aid in the development of screening tests for disease carriers. Detection of linkage to a specific disease phenotype requires screening of large numbers of markers across known affected and unaffected animals. To establish high throughput genome scanning this study placed 100 canine microsatellite markers, arranged by fragment size and fluorescent dye label, into 12 PCR multiplexed panels. The highest degree of multiplexing was 11 markers per panel while the lowest was five markers per panel; each panel was run in one gel lane on automated DNA sequencers. Selection of the markers was based upon chromosomal or linkage group locations, degree of polymorphism, PCR multiplex compatibility and ease of interpretation. The marker set has an average spacing of 22.25?centiMorgan (cM). Marker polymorphism was evaluated across 28 American Kennel Club (AKC) recognized breeds. The utility of buccal swab vs. blood samples was also validated in this study as all template DNA was derived from swabs obtained and submitted by participating dog breeders and owners. The PCR multiplexed microsatellite panels and sampling method described in this report will provide investigators with a cost effective and expedient means of pursuing linkage studies of specific canine genetic diseases.     

Selection, acclimation, training, and preparation of dogs for the research setting

Dogs have made and will continue to make valuable contributions as animal models in biomedical research. A comprehensive approach from time of breeding through completion of in-life usage is necessary to ensure that high-quality dog models are used in studies. This approach ensures good care and minimizes the impact of interanimal variability on experimental results. Guidance related to choosing and developing high-quality laboratory dogs and managing canine research colonies is provided in this article. Ensuring that dogs are healthy, well adapted, and cooperative involves good communication between vendors, veterinarians, care staff, and researchers to develop appropriate dog husbandry programs. These programs are designed to minimize animal stress and distress from the postweaning period through the transfer and acclimation period within the research facility. Canine socialization and training programs provided by skilled personnel, together with comprehensive veterinary health programs, can further enhance animal welfare and minimize interanimal and group variability in studies.     

Fifty-four new gene-based canine microsatellite markers

Fifty-four new markers were developed to fill in gaps in the current map of canine microsatellites and to complement existing markers that may not be sufficiently informative in highly inbred canine pedigrees. Canine genes contained on the radiation hybrid map were used to obtain the sequence of the human homolog. A BLAST search versus the canine whole genome shotgun (wgs) sequence resource was used to obtain the sequence of the canine genomic contigs containing the homolog of the corresponding human gene. Canine sequences that contained microsatellites and mapped back to the correct location in the human genome were used to design primers for amplification of the microsatellites from canine genomic DNA. Heterozygosities of the markers were tested by genotyping grandparental DNAs obtained from the Nestle Purina Reference family DNA distribution center plus DNAs from unrelated Bouviers and Irish wolfhounds. Canine map positions of markers on the July 2004 freeze of the canine genome assembly were determined by in silico PCR or BLAST.     

Canine DNA subjected to whole genome amplification is suitable for a wide range of molecular applications

Molecular and genetic studies of canine disease phenotypes can be limited by the amount of DNA available for analysis. New methods have been developed to amplify the genomic DNA of a species producing large quantities of DNA from small starting amounts. Whole genome amplification (WGA) of DNA is now being used in human studies, although this technique has not been applied extensively in veterinary research. We evaluated WGA of canine DNA for suitability in a range of molecular tests. DNA from 93 canine blood extracted and 18 buccal swab samples was subjected to WGA using the GenomiPhi kit (Amersham). Genomic DNA was compared with WGA product using a range of techniques, including reference strand-mediated conformation analysis, denaturing high-performance liquid chromatography analysis, microsatellite genotyping, direct DNA sequencing, and single nucleotide polymorphism allelic discrimination. All samples amplified well, giving an average yield of 3 mug of DNA from 2.5 ng of starting material. Extremely high levels of experimental reproducibility and concordance were observed between source and WGA DNA samples for all analyses used: greater than 95% for blood extracted DNA and greater than 80% for buccal swab DNA. These studies clearly demonstrate the usefulness of WGA of canine DNA as a means of increasing DNA quantities for canine studies. This technique will have major implications for future veterinary research.     

TGB: the tobacco genetics and breeding database

The germplasm of the genus Nicotiana contains more than 5,000 accessions and plays an important role in modern biological research. Tobacco can be used as a model system to develop methodologies for plant transformation and for investigating gene function. In order to develop the study of Nicotiana, a large quantity of data on germplasm, sequences, molecular markers and genetically modified tobacco was required for in-depth and systematic collation and research. It became necessary to establish a special database for tobacco genetics and breeding. The tobacco genetics and breeding (TGB, http://yancao.sdau.edu.cn/tgb) database was developed with the aim of bringing together tobacco genetics and breeding. The database has three main features: (1) a materials database with information on 1,472 Nicotiana germplasm accessions, as well as updated genomic and expressed sequence tag (EST) data available from the public database; (2) a molecular markers database containing a total of 12,388 potential intron polymorphisms 10,551 EST-simple sequence repeat (EST-SSR) and 66,297 genomic-SSR markers; and (3) an applications database with genetic maps and some genetically modified studies in tobacco. The TGB database also makes Basic Local Alignment Search Tool and primer designing tools publicly available. As far as can be ascertained, the TGB database is the first tobacco genetics and breeding database to be created, and all this comprehensive information will aid basic research into Nicotiana and other related plants. It will serve as an excellent resource for the online tobacco research community.     

Functional markers in wheat: technical and economic aspects

Speed and cost–effective techniques for evaluation of plant materials to provide information before entering the next cycle of selection are critical for success in plant breeding. Whether or not a ‘new’ technique realizes its potential depends on technical and economic considerations. Biotechnology-based research tools such as doubled haploid technology and molecular markers have already demonstrated their value for application in plant breeding. In the area of genomics, implementation of functional markers (FMs) is currently of particular interest. The pipeline from plant materials to FM data points for any application includes maceration of plant material, DNA isolation and sample preparation. For each step of this pipeline, a number of techniques are available, and no single method is ideally suited for all applications. The challenge is to meet the needs of many different scenarios which are present in a modern breeding programme such as the use of (1) few markers for genotyping hundreds of samples (e.g., marker assisted backcrossing (MAB)), (2) few markers in thousands of samples (e.g., screening for GMO), (3) hundreds to thousands of markers for hundreds to thousands of samples (e.g., genetic characterization of breeding materials (fingerprinting)). This paper compares different techniques for each of the steps from plant material to FM data point, with the main emphasis on SNP detection platforms, assuming that multiple FMs will become available in the near future. We focus on technical and economic aspects and discuss which techniques are most suitable for each of the scenarios using wheat as a model.     

Empowering international canine inherited disorder management

The mapping of the canine genome and the study of canine breed genomic architecture has revolutionized the discovery of genetic tests for inherited disorders in dogs. As the genetics underlying complex disorders are revealed, canine breeders and their registering organisations will be required to understand genetics in a much more sophisticated way. To facilitate the management of genetic disorders in the era of new complex information, we consider how best to apply the results of new research and analytical techniques to benefit the wider canine breeding community with the aims of improving canine health and maintaining benevolent genetic diversity. If this is not done, there is a serious risk that expensive and valuable genetic research will remain unused or be misused to the detriment of breeds. In this review, we make a case for the formation of an international organisation that will exist as a central repository for breed-based genetic analysis and information sharing. This organisation ("Inter-Dog") could be modelled on a similar organisation that is monitoring genetic improvement of dairy cattle. The formation of such an organisation will require the collaboration of international kennel management organisations, researchers, and agencies offering genetic testing services.     

Recombinant DNA laboratory data manager using DBASE III+

The use of recombinant DNA technology in clinical and research laboratories involves diverse information management functions such as keeping track of patient samples, blot membranes, polymerase chain reaction products and test results. We report here the use of a PC-based database manager (DBASE III+, Ashton-Tate) for the coordinated maintenance of these functions. We have implemented a menu driven interface, developed using DBASE's programming language, which provides a data entry and maintenance system. The system is easily learned by technologists and saves time and reduces data handling errors compared to a manual method. The system can rapidly look up data and produce customized worksheets or reports correlating all available clinical and laboratory information. We will provide a copy of the program disc to interested parties.     

The Medicago Genome Initiative: a model legume database

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume MEDICAGO: truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated MEDICAGO: functional genomics program at the Noble Foundation (http://www.noble.org ), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.     

PETscan: measuring incidence of disease phenotypes to prioritize genetic studies in companion animals

Reliable incidence measurement of diseases is necessary for identification of hereditary diseases in companion animal populations. The data collection system ‘PETscan’ was developed to facilitate standardized registration of diagnoses in veterinary practice. In the development, we attempted to counter challenges known from other primary practice data systems. PETscan includes a comprehensive list of potential diagnoses and supports the veterinary professionals in the diagnostic process. Demographics, individual data and standardized diagnostic data are collected through practice management software in a central database for epidemiological analysis. A preliminary data analysis from PETscan showed specific health issues in four canine breeds. As a real‐time prospective monitoring tool, PETscan summaries can objectively assess the incidence of disorders in companion animal populations and can be used to prioritize disease–gene identification studies and evaluate the effects of breeding strategies, for example, after implementation of a new DNA test in the breeding strategy.     

Orphanet – das europäische Portal für seltene Krankheiten

Orphanet is a relational database of rare diseases and orphan drugs. This internet-based information platform was established jointly by the French Ministry of Health and the French National Institute of Health and Medical Research (INSERM) in 1997. Since 2000 Orphanet progressed as a European project. The concept was to provide all stakeholders with compiled information on rare diseases through a directory of expert services. This directory of services provides information on specialised outpatient clinics, clinical laboratories, research projects, registries, clinical trials and patient organisations from currently 38 countries. The Services are directly associated with the inventory of rare diseases; furthermore, a comprehensive encyclopaedia is available. All information is freely accessible in five languages at the website http://www.orpha.net.     

Cryopreservation of canine embryos

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.     

Genomic instability and telomere fusion of canine osteosarcoma cells

Canine osteosarcoma (OSA) is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH) using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA.     

Canine cytogenetics--from band to basepair

Humans and dogs have coexisted for thousands of years, during which time we have developed a unique bond, centered on companionship. Along the way, we have developed purebred dog breeds in a manner that has resulted unfortunately in many of them being affected by serious genetic disorders, including cancers. With serendipity and irony the unique genetic architecture of the 21st century genome of Man's best friend may ultimately provide many of the keys to unlock some of nature's most intriguing biological puzzles. Canine cytogenetics has advanced significantly over the past 10 years, spurred on largely by the surge of interest in the dog as a biomedical model for genetic disease and the availability of advanced genomics resources. As such the role of canine cytogenetics has moved rapidly from one that served initially to define the gross genomic organization of the canine genome and provide a reliable means to determine the chromosomal location of individual genes, to one that enabled the assembled sequence of the canine genome to be anchored to the karyotype. Canine cytogenetics now presents the biomedical research community with a means to assist in our search for a greater understanding of how genome architectures altered during speciation and in our search for genes associated with cancers that affect both dogs and humans. The cytogenetics 'toolbox' for the dog is now loaded. This review aims to provide a summary of some of the recent advancements in canine cytogenetics.     

Seroprevalence of canine herpesvirus in breeding kennels in the Gauteng Province of South Africa

Canine herpesvirus (CHV-1) causes neonatal deaths as well as infertility due to embryonal death, abortion and stillbirths in breeding kennels. The aim of this study was to determine the prevalence of antibodies against canine herpesvirus in the serum of dogs older than 1 year in breeding kennels in the Gauteng Province of South Africa. A serum neutralization test (SNT) and a newly developed enzyme linked immunosorbent assay (ELISA) were used to test the serum samples of 328 dogs in 38 breeding kennels. With SNT as well as ELISA, 22% of sera were positive (P>0.9). Seventeen kennels (45% of total kennels) each had at least one positive dog on SNT compared with twenty kennels (53% of total kennels) that each had at least one positive dog on ELISA (P=0.6). The prevalence of positive dogs in positive kennels was 42+/-26% (n=17 kennels) for SNT and 39+/-26% (n=20 kennels) for ELISA. Pairwise comparison of kennels showed that the prevalence of SNT positive dogs was similar to the prevalence of ELISA positive dogs (P=0.3, n=38 kennels). Seroprevalence was independent of age, gender or colony size. This study suggests that canine herpesvirus is sufficiently common in breeding dogs in the Gauteng Province of South Africa to pose a threat to neonatal survival and fertility.     

Large-scale SNP genotyping with canine buccal swab DNA

The dog is an attractive model for genetic studies of complex disease. With drafts of the canine genome complete, a large number of single-nucleotide polymorphisms (SNPs) that are potentially useful for gene-mapping studies and empirical estimations of canine diversity and linkage disequilibrium (LD) are now available. Unfortunately, most canine SNPs remain uncharacterized, and the amount and quality of DNA available from population-based samples are limited. We assessed how these real-world challenges influence automated SNP genotyping methods such as Illumina's GoldenGate assay. We examined 384 SNPs on canine chromosome 9 and successfully genotyped a minimum of 217 and a maximum of 275 SNPs using buccal swab samples for 181 dogs (86 beagles, 76 border collies, and 15 Australian shepherds). Call rates per SNP and sample averaged 97%, with reproducibility within and between analyses averaging 98%. The majority of these SNPs were polymorphic across all 3 breeds. We observed extensive LD, albeit less than reported for surveys using fewer dogs, consistent between breeds. Analyses of population substructure indicated that beagles are distinct from border collies and Australian shepherds. These results demonstrate the suitability of amplified canine buccal samples for high-throughput multiplex genotyping and confirm extensive LD in the dog.     

ICB database: the gyrB database for identification and classification of bacteria

The Identification and Classification of Bacteria (ICB) database (http:/www.mbio.co.jp/icb) contains currently available information about the DNA gyrase subunit B (gyrB) gene in bacteria. The database is designed to provide the scientific community with a reference point for using gyrB as an evolutionary and taxonomic marker. Nucleic and amino acid sequence data are currently available for over 850 strains, along with alignments at several different taxonomic levels and an exhaustive review of primer selection and background information.