DNA typing and its applications in verifying parentage

Every living being has the privilege of being unique, different from others of the same species. He is the expression of the information coded by his genome : the long  DNA molecule contained in the nucleus of each of his cells. Identification of an individual, and its direct application of parentage verification, is based on this principle.

DNA is composed of several chemical compounds, particularly nitrogenous bases. There are four different bases (adenine A, Cytosine C, Guanine G, and Tymine T), which when assembled with the other components form nucleotides.

On the long DNA molecule, which supports heredity, there are areas which code proteins (the genes) and other areas which do not. Microsatellites, which are repeated sequences of 2,3 or 4 nucleotides, are situated in the non-coding areas. It is estimated that there are between 50 000 to 100 000 microsatellites in a genome. The most frequently used in horses are repetitions of 2 nucleotides.

For a given microsatellite:

• There are different alleles defined by the number of repetitions, this varies from one individual to another (ex : 6 repetitions define allele A, 8 define allele B, … (figure 2);
• This number is stable during the animal’s life and is transmitted to his descendance.

For any individual, a microsatellite has two alleles (variants) : one from the paternal side, and one from the maternal side. When the two alleles are identical, the individual is qualified as being homozygous ; when they are different, he is heterozygous.

The set of alleles in a microsatellite forms genetic polymorphism. The higher the number of alleles, the more the microsatellite will be effective in identifying an animal and distinguishing it from another animal. Typing the set of microsatellites studied will define the individual’s genotype.

The microsatellite study methods are replicable, can be automated, and are extremely precise, therefore reliable and efficient. The molecular biology techniques implemented enable to carry out the steps needed for the analysis.

First step : extracting DNA

Any cell with a nucleus contains nuclear DNA. This is notably the case in white blood cells, the only blood cells in horses whch contain a nucleus. DNA can also be extracted from many other cells (hair bulbs, sperm, mouth cells….).

Taking a blood sample is the most frequently used technique of obtaiing DNA as it is a good compromise between :

• ease of obtaning the sample,
• reliability of the sample,
• automation of the results.

This DNA can be kept for several years and used for other studies.

Second step : amplifying the microsatellites in the DNA

Polymerase chain reaction or PCR is a molecular biology technique which, in vitro, amplifies up to one billion times a fragment of DNA.

This technique is similar to photocopying many times over a fragment of DNA containing the microsatellites to be analysed.

The size of the amplified product varies depending on the number of repeated sequences of nucleotides. Each possible size corrresponds to one allele of the microsatellite.

3rd step : separating the different alleles by electrophoresis

The amplified products are seperated according to their size. All the data is processed by a computer equipped with an analysis software program.

Final step : Result of the genotype analysis

The first equine microsatellites were discovered in 1992 by a Swedish team and pathed the way for research conducted by numerous different international teams. The latter also dicoverd that equine microsatellites were also found in the genome of Equus species (donkeys, zebras, Przewalski horses).

This collaboration conducted by an international research company, ISAG (international society for animal genetics), enabled to establish an international DNA typing sample group composed of 12 microsatellites. This sample group is used today by over 60 laboratories worldwide. It is monitored every two years by an international comparison test, and enables new labiratories to join the group.

The laboratories have the latitude to add microsatellites to the range. Thus, horse identification in France has been carried out since 2000, thanks to 14 microsatellites.

Each allele, initially interpreted by size, is then turned into letters following an international naming system. When identifying a horse by DNA typing, a genotype is presented following the diagram presented in figure 3 :

• In this example it can be noted that for the first microsatellite analysed called VHL20, the horse analysed is heterozygous L N, since the L allele came from one of the two parents, and the N allele came from the other.
• For the microsatellite noted HMS7, the animal is homzygous since the N allele came from both Sire and Dam.

The use of genotypes is defined by the decree dated 24th April 2009, relating to equine identification and parentage verifications through DNA typing. The regulations can be specified further by different stud-book regulations.

Principle

Once the genotypes have been established, parentage verification consists in comparing the genotype of the offspring to the genotypes of his supposed sire and dam.
For each microsatellite, the offspring should have inherited an allele from the sire, and one from the dam. When this is not the case, parentage is declared as being incompatible.

On the other hand, if no incompatability is detected on any of the 14 microsatellites, then parentage is declared compatible.

The parentage verification presented in the example in figure 4 shows an incompatibility with the sire for 4 microsatellites : the alleles P (HTG4), N(HTG10), M (HMS3) and J (HMS1) present in the offspring cannot have been transmitted from the sire, and therefore enable to declare the foal incompatible with the sire.

Guarantee of the individual’s identity

For one microsatellite, there is a different number of alleles (variants) for each breed, as well as a frequency with which each of the alleles will appear. This will allow to determine the  probability of having two identical animals.

This means calculating the probability of testing two random individuals in a given population (breed), who would have the same genotype. It is quite easy to see that the probability of randomly finding two identical individuals in the quoted populations is almost nil (chart 2). For thoroughbreds for example, a probability of 4,6.10-10 means that there can be no two identical individuals out of 250 million horses, which is far more than the world population.

Chart 2 : Exclusion and identity probabilities according to microsatellites in two breeds of hotblood horses.

A blood sample (5ml tube containing an anticoagulant solution of potassium EDTA) is required and is enough for each horse to be analysed. This sample should be taken by a certified person qualified for equine identification (IFCE agent, certified vet….).

Laboratory equipment should be requested before any intervention.

Each sample should be correctly identified, accompanied with the detailed description form, or the laboratory slip mentioning the animal’s detailed identity and his SIRE number, and sent to the laboratory.