Section 4 - Guidelines for DNA Technology. Version October, 2017

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1 Section 4 - Guidelines for DNA Technology Section 4 DNA Technology

2 Table of Contents Overview 1 Molecular genetics Introduction Current and potential uses of DNA technologies Parentage verification and parental assignment Traceability of beef product and authentication of beef products with certified breed origin Molecular genetic information for marker-assisted selection schemes Diseases resistance and genetic defects Technical aspects DNA collection Genetic markers commonly used Laboratory accreditation of parentage testing in cattle Accreditation of Microsatellite based paternity testing in cattle Laboratory identification Education and training of lab supervisor and operators Equipment Certification Participation and performance in ring test Microsatellite markers Marker nomenclature SNP-based parentage testing in cattle Laboratory identification Education and training of lab supervisor and operators Equipment Certification Participation and performance in ring test SNP markers Marker nomenclature Accreditation of applying the results of genotype analysis for parentage verification Organisation identification Education and training of institution Accreditation Participation and performance in ring test SNP Nomenclature SNP Markers and interpretation Recommendations Appendix 1. Rules for microsatellite-based testing parentage verification for cattle Appendix 2. Application Form for microsatellite-based parentage testing in cattle Appendix 3. ISAG recommended microsatellites Appendix 4. Rules for SNP-based testing parentage verification for cattle Appendix 5. Application form for SNP-based parentage testing in cattle Appendix 6. ISAG recommended SNP markers for parentage testing in cattle DNA Technology - Page 2 of 14.

3 10 Appendix 7. Application Form for Centres seeking ICAR Accreditation status for DNA Data Interpretation Change Summary Date of Change Nature of Change August 17 August 17 August 17 August 17 August 17 August 17 Reformated using new template. Table of contents added. Heading numbers and heading text edited for clarity and removal of redundant text. Annexes replaced by links to relevant Appendices on ICAR website. Moved the file to the new template (v2017_08_29) Links to ICAR website hidden behind here. Sept. 17 Update version to September, Sept. 17 Sept. 17 Oct Links to DNA technology websites corrected. Link to application forms on ICAR website corrected. Update version to October. Replace links for terminology. Hyperlinks have been corrected DNA Technology - Page 3 of 14.

4 1 Molecular genetics 1.1 Introduction Advances in molecular biology provide a new set of information to be incorporated into animal industry. On one hand, the use of molecular information may contribute to the enhancement of consumers trust in the ability monitor and control the animal production chain. On the other hand, molecular information will greatly contribute to achievement of genetic improvement of animal traits through the use of MAS, gene introgression, heterosis prediction, and correct pedigree control. In most cases, advantages of using molecular information comes from improving accuracy, shortening of generation interval and increasing of selection intensities. Nevertheless, there is still a need for research and development in the search for associations between genetic markers and traits of interest. In addition to that, and before using genetic information in practical selection schemes, an understanding of gene action, gene interactions and differential gene expression to avoid negative collateral effects is needed. Cooperation between animal industry (sucklers, fatteners, slaughterers and retailers) and research is required for a successful and beneficial search for genetic information, in commercial beef cattle populations. 1.2 Current and potential uses of DNA technologies Parentage verification and parental assignment Up until now, parentage verification has been one of the commercial uses of genetic markers (microsatellites type). Parentage testing is based on the exclusion of relationship when an animal has a genotype inconsistent to a putative relationship. New trends in animal production systems are tending to encourage animal production in more extensive conditions in response to environmental and production related constraints. As the cost of the analysis decreases and the number of genetic markers available increases, breed societies will be able to build up pedigree records using genetic markers to track the pedigree of calves born in a herd at a given time. This will require a prior knowledge on candidate sires and dams at a given time and a set of identified markers. The probability of assignment to a correct pair of animals will depend on the number of alleles per loci, allelic frequencies in the population, the number of parents and number of possible matting. The International Society of Animal Genetics ( has identified a panel of markers for this purpose Traceability of beef product and authentication of beef products with certified breed origin Since the BSE crisis the traceability of beef products is of great concern to consumers. Traceability is based on the availability of a verification and control system which monitors all relevant details throughout the whole beef production chain. The genetic sequence of an individual is unique and does not change. DNA remains constant from conception to consumption. Therefore, use of DNA information at a number of regions in the genome allows one to match DNA of an individual at birth to the final product. Microsatellites and SNP markers may be used for this purpose. Genetic markers for the authentication of beef products for labels of quality related to geographic location (GPI) and labels of quality related to specific breeds or their crosses are/or will be very useful. However, it implies the establishment of molecular standards for each breed. A lot of information is coming from studies of genetic diversity among breeds. Genes subject to intense selection in each population such as coat colour, horned/polled, shaped of horns etc are of interest. DNA Technology - Page 4 of 14.

5 1.2.3 Molecular genetic information for marker-assisted selection schemes Overview Quantitative traits are generally assumed to be controlled by a large number of genes. However individual genes sometimes account for a significant amount of variation of the trait. Such is the case of the double muscling gene. Since the genotype of an animal does not change during its lifetime, use of DNA information through the identification of markers linked to QTLs with effects on production traits or the identification of a gene itself will be of great interest in the near future. Nevertheless, while traits become more complex, there is a growing need of having a sufficiently large marker set to incorporate molecular information for selection decisions. Including molecular information as a selection criterion is of special interest for traits that are difficult and costly to measure or/and are measured late in life. By 2017, approximately 100,000 loci have been identified in cattle populations. Many of them are genetic markers linked to QTLs. At the present time, there is a number of QTLs associated to beef traits (see Cattle QTL DB, and Bovine Genome). Recording schemes have been collecting information for the most common production traits measured in live animals. There is an ever-increasing volume of information becoming available. Traits where genetic markers are under intense investigation are carcass and meat quality. As mentioned earlier, meat quality may be assessed from different point of views. In many cases, quality assessments of beef products are very expensive, difficult to obtain and performed late in the animal s life. Recording of quality information is commonly made on a reduced number of animals of only one sex. On the other hand, many measures taken to determine quality lower the commercial value of the final product. Genetic markers will play an important role for this type of trait. There are already a number of loci related to tenderness, marbling, lean yield etc available. Furthermore there is a growing awareness of the link between diet and health. Consumers habits are changing. Nutritional value is a concern for consumers and they demand foods with health and beneficial properties. The nutritional value of beef products may be another approach to focus on for the selection of quality traits. This is the case of traits such cholesterol content, fatty acids profiles, vitamins contents etc. Analyses to determine nutritional value or other specific traits that involve sophisticated techniques are very expensive. Thus, the molecular information available for these type of traits will be very useful and less expensive. Genetic markers will allow us to increase selection intensities since more candidates to selection will have recorded information Diseases resistance and genetic defects Another group of traits with a high potential for the use of molecular data are those linked to resistance, susceptibility to diseases. There are a number of multifactorial or complex diseases which are the result of a polygenic background and environmental components. Diseases resistance traits are among the most difficult to include in genetic improvement programs because they require good field measurement of the disease status of the animals and a systematic control of management or environmental conditions that allow for the identification of environmental influence on the health status of the animal. Infectious diseases depend very much upon environmental factors such as the degree of exposure to pathogen agent. Thus, if exposure is low, animals will show little variation. Part of the phenotypic differences for resistance may be differences in the degree of challenge. Therefore, if genes or genetic markers linked to resistance are correctly identified, resistant animals will be able to be selected on the base of their molecular information. In many diseases, identification of genes associated to resistance will require of experimental conditions to be found. DNA Technology - Page 5 of 14.

6 Genetic analysis to identify heterozygous carriers of genetic diseases caused by single recessive genes are currently in use. This is the case of BLAD (bovine leukocyte adhesion deficiency) and others. In OMIA (Online Mendelian Inheritance in Animals) there are over five hundred familial inheritance disorders and traits registered in cattle. 238 of them are single locus disorders for which 168 have the causative mutation identified 1.3 Technical aspects DNA collection Systematic collection of DNA is recommended in beef cattle population. DNA may be obtained from any nuclear cell in the body. Protocols for DNA extraction are now available for blood (white cells), semen, saliva (epithelial cells), hair follicles, muscle, skin, organs (such as liver, spleen etc.). Small amounts of tissue material are required for routine DNA analysis. However, if future used of DNA is to involve its use for many different objectives (selection for a number of traits, traceability, etc) storage costs, DNA extraction costs, DNA yield will have to be carefully examined and optimised. Common collection methods include dried blood samples kept on blotting paper and stored at room temperature, ear tag systems that deposit tissue samples in an enclosed container or hair follicles Genetic markers commonly used Microsatellites These are segments of DNA containing tandem repeats of simple motifs usually dimers or trimers. These segments are located throughout the genome and normally in non-coding regions. These regions are subject to addition or subtraction of the number of tandem repeats which make them unique at each site of the genome SNP These are a single nucleotide polymorphism located throughout the genome. The most informative SNPs are either located in coding regions, therefore different polymorphism imply a change in the structure or function of the encoded protein, or at non-coding regions that may be involved on regulatory function of the gene Data collection The centralised database may be organised in respect to the main uses to be made of the genetic information: a. Parental verification and assignment. b. Traceability of beef products. c. Breed identification or breed diversity. d. Qualitative and Quantitative trait. Tables may contain: a. Animal ID: to link to all other animals information including relatives. b. Number of genetic markers: n. c. Identification of each marker i (for i= 1, n). d. First allele for marker i. DNA Technology - Page 6 of 14.

7 e. Second allele for marker i. f. Association with other traits. g. A table of identified markers for each breed: - Standard Name of marker. - Aliases. - Complete Gene, Part of a gene, marker. - Accession number. - Allelic size. - Genotypes. For standardization purposes in respect of the nomenclature of genes or loci, a web site is available at: and markers at: 2 Laboratory accreditation of parentage testing in cattle Considering the need for high quality standards in bovine parentage testing and identity verification due to the impact incorrect parentage assignment or identity may have in the estimation of genetic indexes and in national and international genetic evaluations, and based on two years of work by the genetic analysis task force, ICAR has decided to define the minimum requirements for laboratories performing DNA parentage testing and identity verification. Guidelines for accreditation are provided for microsatellite- and SNP-based analyses in cattle. Minimum requirements for additional species and DNA tests will be defined in the future. Laboratories requesting microsatellite- and/or SNP-based accreditation will have to apply by downloading and filling out the appropriate forms (refer to sections 5 below for microsatellites; and section 8 below for SNPs) on the ICAR website. The forms must be filled out accurately and completely, providing necessary documentation as required. The application will be evaluated by a Committee of Experts appointed by ICAR that will either approve it, request additional information, or reject it. In case of rejection, the applicant may submit a new form at least one year after her/his failed application. Accreditation will be given for a two-year period, at the end of which a new application is to be completed and submitted after participation in additional ISAG comparison tests. Sections 2.1 and 2.2 respectively contain the rules and guidelines for laboratory accreditation of DNA paternity testing in cattle using microsatellites and the rules and guidelines for SNP-based parentage testing in cattle. Sections 5 and 6 respectively contain the application form for laboratory accreditation of DNA paternity testing in cattle using microsatellites and the list of recommended ISAG microsatellite markers. Sections 8 and 9 respectively contain the application form for laboratory accreditation for SNP-based testing in cattle and the list of ISAG recommended SNP markers. 2.1 Accreditation of Microsatellite based paternity testing in cattle The present Rules contain Minimum requirements for accreditation of DNA paternity testing in cattle. DNA Technology - Page 7 of 14.

8 Section 5 below contains the application form to be filled by applicants and ed to the ICAR secretariat Section 6 below contains the list of microsatellite markers recommended by ISAG and the method for calculating 1 parent and 2 parent exclusion probabilities Laboratory identification The applicant must be clearly identified by providing the following: a. Name of the laboratory and Institution if relevant, b. Institution if relevant c. Address and Country d. Contact person at the lab, as well as all information necessary for getting in touch with her/him quickly Education and training of lab supervisor and operators The minimum requirements for education and training of the laboratory supervisor and senior operator are: a. Bachelor degree, or higher, in a scientific discipline for laboratory head or supervisor, and b. At least five years of experience in molecular diagnostics for the laboratory senior operator. Experience is considered a key factor in data production and in the interpretation of results Equipment a. Equipment used to run and score microsatellite must be described. b. The year of purchase and last revision must be provided - this allows ICAR to evaluate the appropriateness of the technology being used and ensure each lab is following a proper maintenance program that should enable it to generate high quality data. c. Yearly revision is considered a minimum requirement. d. A personal opinion on the performance of the laboratory set up available is asked to foresee the need for improvements in quality standards Certification a. International ISO17025 or ISO9001 is a minimum requirement for ICAR accreditation Participation and performance in ring test a. The participation and performance in ISAG and national ring (comparison) tests must be disclosed, and certificates provided, when available. Applicants must also sign a release allowing ISAG to directly disclose their ring test results to the ICAR DNA Committee. b. The participation in at least two ISAG ring tests is a minimum requirement. c. Beginning with the ISAG ring test, lab typing performance for the official set of 12 ISAG microsatellites (see Annex II) must be disclosed (previous ISAG ring test reporting can be limited to 9 microsatellites). DNA Technology - Page 8 of 14.

9 d. The Committee of Experts will decide performance thresholds for each ring test with due consideration for the structure of the ring test and the average performance of laboratories in the ring test that year Microsatellite markers a. The names of all microsatellites typed on all animals (marker set I) and of the additional ones assayed in the case of unresolved parentage (marker set II) must be declared, as well as the number of animals typed in at least the last two years. b. The minimum requirement for international exchange is the complete set of 12 official ISAG microsatellite markers. c. To ensure sufficient experience within the lab, analysis of 500 animals per year is set as minimum requirement for certification. d. Exclusion probability (PE; 2 parents and 1 parent) of each marker and of the complete marker sets must be calculated and provided in the application. The type of population and number of animals (minimum 150) used for computations are to be described. ICAR recommends using Holstein as a reference group when possible. The ICAR Committee of Experts will evaluate that an appropriate PE is reached for accreditation, on the basis of the population analyzed Marker nomenclature a. Nomenclature of markers must be described. b. ISAG nomenclature is required for the official ISAG 12 marker set. 2.2 SNP-based parentage testing in cattle The present Rules contain Minimum requirements for accreditation of SNP-based DNA parentage testing in cattle. Sections 8 contains the application form for laboratory accreditation for SNP-based testing in cattle that has to be ed to the ICAR secretariat Section 9 contains the list of recommended SNP markers Laboratory identification The applicant must be clearly identified by providing the following: a. Name of the laboratory. b. Institution if relevant. c. Address and Country. d. Contact person at the lab, as well as all information necessary for getting in touch with her/him quickly Education and training of lab supervisor and operators The minimum requirements for education and training of the laboratory supervisor and senior operator are: a. Bachelor degree, or higher, in a scientific discipline for laboratory head or supervisor. b. At least five years experience in molecular diagnostics for the laboratory senior operator. DNA Technology - Page 9 of 14.

10 Experience is considered a key factor in data production and in the interpretation of results Equipment a. Equipment used to run and score SNPs must be described, as well as the methods used. b. The year of purchase and last revision must be provided this allows ICAR to evaluate the appropriateness of the technology being used and ensure each lab is following a proper maintenance program that should enable it to generate high quality data. c. Yearly revision is considered a minimum requirement Certification a. No certification is presently required. In the future, ISO17025 and/or ISO9001 will be a minimum requirement Participation and performance in ring test a. The participation and performance in ISAG ring (comparison) tests must be disclosed and certificates provided, when available. Applicants must also sign a release allowing ISAG to directly disclose their ring test results to the ICAR DNA Committee. b. Participation in at least one ISAG ring tests is considered a minimum requirement at this time. c. A Committee of Experts will decide performance thresholds for each ring test with due consideration for the structure of the ring test and the average performance of laboratories in the ring test that year SNP markers a. The name of all SNPs typed on all animals (marker set I) and of the additional markers assayed in the case of unresolved parentage (marker set II) must be declared, as well as the number of animals typed in at least the last two years. b. It is a minimum requirement to use at least 95 SNPs from the set recommended by ISAG (see Section 9) on all animals typed. c. To ensure sufficient experience within the lab, genotyping 500 animals per year is set as a minimum requirement for accreditation. d. Exclusion probability (PE; 2 parents and 1 parent) of the complete marker sets used must be calculated and declared. The type of population and the number of animals (minimum 150) used for computations are to be described. ICAR recommends using Holstein as a reference group when possible. The ICAR Committee of Experts will evaluate that an appropriate PE is reached for accreditation, on the basis of the population analyzed Marker nomenclature a. Nomenclature of markers must be described. b. ISAG nomenclature is required for the ISAG SNP marker set. DNA Technology - Page 10 of 14.

11 3 Accreditation of applying the results of genotype analysis for parentage verification With the advent of SNP testing, the functions of analysis and verification have the potential to be separated. Consequently, ICAR has a separate accreditation for applying the results of genotype analysis, which may be taken by laboratories, herd books and any other organisation involved in parentage verification. It is concerned with utilising the results that are delivered by the laboratories from DNA sample analysis and so is a data warehouse function. These organisations may act as service providers between laboratory accredited for SNP-based DNA parentage testing and end user. Service providers could use different laboratories for different breeds/species. Organisations requesting accreditation for applying the results of genotype analysis for SNP based parentage verification will have to apply by downloading and filling in the appropriate form (Section 10 below). This form must be filled out accurately and completely, providing necessary documentation as required, and submitted to ICAR. The form will be evaluated by a Committee of Experts appointed by ICAR that will either approve it, request further information or reject it. In the case of rejection, the applicant may submit a new form at least one year after the failed application. Accreditation will be given for a four year period, at the end of which a new application is to be completed and submitted. The present Rules contain Minimum requirements for accreditation of applying the results of genotype analysis for SNP based parentage verification. 3.1 Organisation identification The applicant must be clearly identified by providing the following: a. Name of the Organisation. b. Address and Country of the Organisation. c. Contact person at the Organisation, as well as all information necessary for getting in touch with her/him quickly ( and telephone number). 3.2 Education and training of institution The minimum requirements for education and training of the responsible person of the Organisation are: a. Bachelor degree, or higher, in a scientific discipline. b. Understanding and experience in molecular biology, data handling and interpretation of results. c. An in-depth knowledge of the principles of parentage verification using data from genotype analysis. Experience is considered a key factor in the interpretation of results from genotype analysis. 3.3 Accreditation a. Some organisations currently performing this function will be members of ISAG, or ICAR and some not members of ICAR or ISAG. There are organisations who would wish to become accredited such as herd books who won't necessarily be members of ICAR. DNA Technology - Page 11 of 14.

12 b. Organisations must provide evidence of their competence to provide parentage verification service. ISO17025 accreditation is acceptable for this. When not available, initially similar evidence of fitness will be acceptable c. If an organisation acts as a service provider for data handling and interpretation of results obtained from an ICAR accredited laboratory, such organization does not have to have ISO Participation and performance in ring test As this accreditation is concerned with data handling and interpretation of the SNPs for parentage verification, as part of the accreditation a set of animal's SNPs will be sent to the organisation, plus animals of known and unknown parentage and the question posed as to who the parents were: a. Participation in the data handling and interpretation test is mandatory. b. The test must be performed every four years. c. A panel will devise and monitor the test. 3.5 SNP Nomenclature a. The ISAG100 Primary Panel should be used for International exchange of parentage SNPs for parentage verification. Additional SNPs from the ISAG200 panel are to be used where there is an inconsistency or inconclusive parentage result. b. The name of all SNPs genotyped (ISAG200) and of the additional markers assayed in the case of unresolved parentage (other SNP sets such as 500SNP, 800SNP or larger) must be declared, as well as the number of animals typed in at least the last 2 years. c. ISAG200 - ISAG100 core panel plus additional ISAG SNPs. d. ISAG200plus - ISAG200 plus additional user defined SNPs. 3.6 SNP Markers and interpretation a. When using ISAG200, follow the guidelines here on the ISAG website. b. ISAG200plus can be used for both verification and discovery, if this is done it is a minimum requirement to use at least 100 SNPs from the set recommended by ISAG (see Seciton 9 below - ISAG100 panel) on all animals typed. c. A maximum cutoff of 1% SNP misconcordance between parent and child is allowed for parentage verification. d. If a parent is SNP excluded then 3 options can be used: - If ISAG100 or ISAG200 have been used for verification, ISAG200plus can be used for discovery. At low SNP levels genotyping errors can cause false exclusions that are just over the 1% SNP misconcordance rate - Run parentage verification using the ISAG200 panel against other possible parents suggested by the farmer or herdbook - Run parentage discovery using ISAG200plus h. For Parentage discovery at least 500 SNP (ISAG200plus or ISAG200 and 300 additional markers with MAF >0.40) are recommended. Caution is advised when DNA Technology - Page 12 of 14.

13 using smaller panels, as multiple sire and dams may be predicted. This will depend upon the population and breed composition. i. If multiple parents are predicted then with the exception of identical twins, a larger SNP set must be used to determine the most likely parent. j. To ensure sufficient experience within the organisation, it is recommended that parentage analysis of 500 animals per year is set as a minimum requirement for accreditation. k. Exclusion probability (PE; 2 parents and 1 parent) of the complete marker sets used must be calculated and declared. The type of population and the number of animals (minimum 150) used for computations are to be described. The panel will evaluate that an appropriate PE is reached for accreditation, on the basis of the population analyzed. 3.7 Recommendations a. Use the same set of SNP for parentage verification and discovery (if possible) instead of using a smaller set for initial verification and a larger set for discovery. b. A standardised format is required for data exchange from laboratory to service provider. c. It is recommended that sufficient SNPs should be used to reduce the likelihood of assigning incorrect parentage for verification. The number of SNPs will be determined by technology, in herd parentage recording and SNP availability. 4 Appendix 1. Rules for microsatellite-based testing parentage verification for cattle Please refer here on the ICAR website for the rules for microsatellite-based testing parentage verification of cattle. 5 Appendix 2. Application Form for microsatellite-based parentage testing in cattle Please refer here on the ICAR website for the Application Form for accreditation of microsatellite-based parentage testing in cattle. 6 Appendix 3. ISAG recommended microsatellites Please refer here on the ICAR website for the list of ISAG recommended microsatellites. 7 Appendix 4. Rules for SNP-based testing parentage verification for cattle Please refer here on the ICAR website for the Rules for SNP-based testing parentage verification for cattle. 8 Appendix 5. Application form for SNP-based parentage testing in cattle Please refer here on the ICAR website for the Application form for accreditation of SNPbased parentage testing in cattle. DNA Technology - Page 13 of 14.

14 9 Appendix 6. ISAG recommended SNP markers for parentage testing in cattle Please refer here on the ICAR website for the ISAG recommended SNP markers for parentage testing in cattle. 10 Appendix 7. Application Form for Centres seeking ICAR Accreditation status for DNA Data Interpretation Please refer here on the ICAR website for the Application Form for Centres seeking ICAR Accreditation status for DNA Data Interpretation. DNA Technology - Page 14 of 14.

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