For decades cattle registries worldwide have relied on genetic testing to protect the integrity of pedigree records. Bloodtyping tests have provided a highly affective means of verifying parentage and solving breeding problem cases. The last few years have seen the emergence of a DNA typing method that has all the requirements to be the new generation of genetic marker testing. This method, known as microsatellite-DNA testing, has been adopted by the American-International Charolais Association (AICA) to provide validation of pedigree records.AICA has undertaken a major reorganization of its parentage verification program. Beginning March 1996, service will be provided by the Veterinary Genetics Laboratory (VGL), University of California at Davis. Animal identification and parentage analyses will be based on DNA typing tests. Hair roots will be used as the source of DNA for testing. A new kit was developed by AICA for collection and shipment of hair samples to VGL. Bar codes are being used to identify individual samples. Electronic transfer of computer files between AICA and VGL has been implemented to reduce paperwork and personnel time, to increase accuracy of sample logging and to expedite communication and turnaround time for results.Breeders will want to become familiar with the new procedures. AICA is striving to provide all necessary information to the membership to make a smooth transition. This article is intended to explain some of the procedures being used and to familiarize breeders with the process of DNA testing.
Sampling Procedures: For purposes of routine testing of cattle, DNA can be easily obtained from white blood cells, hair roots or semen. Indeed, one of the appealing aspects of DNA testing is that it is not dependent on blood samples to arrive in the laboratory in good condition for testing. Hair roots have several characteristics to make them the sample of choice: hair roots can be easily obtained by pulling hair from the tail switch; owners can collect samples themselves; the procedure is non-invasive, thus reducing the risk of injury or discomfort to animals and to the people collecting samples. Also, hair samples can be shipped to the laboratory in a envelope by First Class mail. They do not require special shipping procedures or refrigeration. Overall, the use of hair roots for DNA testing will lead to substantial savings to breeders.For this procedure, two bunches of about 20-30 hairs per bunch are to be pulled from the tail switch of each animal. A hair root, or follicle, is visible to the naked eye as a small bulb at the end of the hair shaft. The new sample kits contain instructions for the correct placement of hairs in the kit, with hair roots at the right side of the form. Adhesive tape is put over hair shafts, but not over the roots, to secure hair in place. About five to eight roots will be used for testing. The remaining roots will be placed in storage for future use.
DNA Typing Tests: The DNA tests used for animal identification and parentage testing detect genetic variation of a class of DNA markers known as microsatellite-DNA, which are also known as short tandem repeats, or STRs for short. STRs consist of a series of end-to-end repeats of 1-6 base pairs (the building blocks of DNA). The vast majority of cattle STRs found to date are 2 base pair repeats (dinucleotide repeats). Genetic variation occurs in the number of repeats within each system. The following table (Table 1) shows common DNA variants for three cattle STRs. Within each marker system, variants differ from each other by 2 or a multiple of 2. The numbers represent the size of each variant in base pairs.Variants are transmitted from parents to offspring, and follow a simple codominant mode of inheritance. Each variant is detectable by testing procedures. By contrast, 9 out of 11 cattle blood grouping systems have recessive types that are obscured whenever they occur with dominant variants.For the routine testing of Charolais cattle, DNA testing is based on a panel of 12 independent STR systems that include the three shown above. A back up panel of 12 additional markers is also available for difficult cases that require further testing.
Efficacy of DNA Tests: A compelling reason for switching to a new procedure is to increase efficacy of genetic testing. That means increasing the power of tests to detect pedigree errors and to solve parentage problems. The theoretical efficacy of the DNA test panel, measured as the probability to detect incorrect parentage, is estimated to be about 99.99% when sire, dam and offspring are compared. For single parent comparisons, sire only or dam only, the efficacy is about 95%. These figures mean that DNA tests are expected to detect virtually all pedigree errors and exclude the incorrect parent. The actual efficacy of DNA tests is slightly lower than theoretical values because these do not take into account breeding schemes that frequently involve closely related animals. In contrast, blood grouping tests alone have efficacy rates that are at best about 90% for sire and dam comparisons and about 75% for single parent comparisons.The higher efficacy of DNA tests will result in more accurate pedigree records and contribute significantly to the validity of studbook records.
DNA Testing Procedures: Microsatellite-DNA typing is made possible by a patented method known as Polymerase Chain Reaction (PCR). PCR mimics DNA replication that occurs naturally within cells, but at a much faster pace. It is designed to make millions of copies of very specific segments of DNA in a three hour processing time. From the small amount of DNA extracted from hair roots, copies are made for each of the 12 systems of the test panel. During PCR, DNA products are marked with fluorescent tags. After PCR, DNA samples are put through a process known as gel electrophoresis to separate DNA variants in each system. Light from a laser beam causes tagged DNA molecules to fluoresce (to emit light in specific wavelengths). Detectors capture the fluorescence of DNA molecules and transmit the information to a computer for processing and imaging analysis. Computer software uses internal size standards run with each sample to classify DNA variants according to size. Analysts familiar with the genetic makeup of each marker system verify computer results for correctness.Once DNA types for an animal are determined, the information is stored in a computer database and can be retrieved at any time for genetic analyses.
Parentage Verification: Parentage verification based on DNA types follows the same genetic rules as those used with bloodtypes. Each parent contributes half of its genetic material (one chromosome of each pair) to the offspring. Thus, all DNA variants found in an offspring must also be present in one or both parents. The presence of DNA variants in an offspring that are not accounted for by either parent is basis for parentage exclusion. The majority of pedigree errors in cattle are due to incorrect sire assignments.When multiple bulls are involved in a parentage case, they are often related as sire-son, sibs and other close relatives. It is important in such cases that the markers used for testing not be genetically linked (close together on the same chromosome) because the greater likelihood of sharing DNA types among relatives reduces the effectiveness of tests to solve a paternity question. The standard panel of 12 DNA systems used for Charolais testing are independent of one another.The DNA types of the calf are genetically compatible with the types of the given sire and dam and thus qualifies as an offspring from this mating. The calf shares one DNA type with each of the given parents in all systems.In the example above, the calf has DNA types (highlighted) that are not found in either the given dam or sire. Because it shares DNA types with the dam, it can be concluded that the dam qualifies. The calf has DNA types in systems BM1818 and BM1824 that are not present in the given sire and is thus excluded as an offspring of that bull. As a rule, parentage exclusions using STR markers are based on at least two independent systems to discount the possibility that a mutational event, although rare, could account for single system incompatibilities.
Additional Tests Available
Chromosome Screening: The genetic material of cattle is organized in 30 pairs of chromosomes. Each pair consists of homologous chromosomes, meaning that members of a pair contain the same array of genes. Increasingly, cattle registries require screening of breeding stock for chromosomal abnormalities known as Robertsonian translocations, the joining of two non-homologous chromosomes. Translocations lead to abnormal segregation of chromosomes during production of ova and sperm and can cause reduced fertility and increased return to service. Translocations in cattle frequently involve chromosomes 1 and 29 (1/29 translocation) and chromosomes 14 and 20 or 21 (14/20 or 14/21 translocation). A study of Charolais cattle in France reports that 3.8% of animals are carriers of the 1/29 translocation. The frequency in American Charolais is not known. Translocations can be detected by chromosome screening procedures. AICA members who wish to have breeding stock checked for translocations should contact AICA for instructions on how to submit samples for chromosome screening.
Freemartin Diagnosis: About 92% of females co-twins of male calves are infertile ("freemartin" heifers). Although the cause of infertility is not fully understood, it is associated with the exchange of cells between twin fetuses via fused blood vessels of fetal placental membranes (placental anastomosis). Freemartins have both female and male cells in their blood. A DNA test that detects the presence of male cells in female twins is available to AICA members. The test requires a blood sample from the female twin. Calves can be tested at any age after birth. Breeders should contact AICA for instructions on how to submit samples for freemartin diagnosis.By adopting a DNA-based parentage testing program, AICA has taken a leading role among cattle registries. The success of this program will serve as a model to other registries that are pursuing the introduction of DNA tests for pedigree validation. AICA's program heralds a new age of genetic testing for cattle and holds many exciting opportunities for the betterment of the breed. |
