Hello, in this lecture, we will discuss
genetics parentage testing in animals.
This lecture is part of module 4,
Precision Livestock Breeding. The
development of this presentation was
supported by an Erasmus+
KA2 grant within the ISAGREED
project: Innovation the content and structure
of study programmes in the field of animal
genetic and food resource management
using digitization.
At the beginning, it is necessary to
explain the basic terms that will be used
during this presentation. The first one
is parenthood - the correctness of
both parents, father and mother, is
verified. In the case of paternity,
only the father's authenticity is
verified for an individual, which is the
most important thing for humans.
If we are going to identify an
individual, We will use molecular
genetic method (called molecular
dactyloscopy) to determine whether
the DNA sample belongs to the individual
being examined. The term genetic
type or genetic profile represents the
set of all alleles (genotypes) that
is characteristic, unmistakable,
and unique to each individual.
The purpose of genetic parentage testing
is to verify the origin and
identification of an individual.
In the case of livestock bred in the
Czech Republic, the testing is governed
by Act No.
154/2000, called as
Breeders Act. For pigs, the genetic
type of boars in breeding farms is
determined. For cattle, the
origin of breeding bulls is verified,
and their genetic type is determined
before selection for breeding. For
horses, the origin of foals after
insemination is verified.
Parentage determination is of great
importance not only in animal genetics,
but also in forensic genetics and
medicine. There are mistakes in
breeding pedigrees that could cause
inaccuracy in the estimation of breeding
value. The price of animals
is often based on the genetic quality of
their parents, and it is therefore
necessary to be able to verify
parentage. Customers may
also be cheated. In the field of
foresnic genetics, paternity
disputes are commonly dealt with,
and many offenders are now convicted
based on DNA traces.
Even victims of violent crimes or
natural disasters can be
identified from their DNA.
Relationship verification is a
necessity, especially in trans-
plantation.
In the past, before the development of
molecular genetic methods, blood
groups and the protein variability were
used to verify parentage.
However,these methods were not very
reliable. Therefore, methods
based on the detection of DNA
variability (DNA fingerprinting),
initially based on the variable number of
tandem repeats (VNTR)
in minisatellites,
began to be used. Currently,
methods associated with DNA
amplification by PCR,
called STRs,are used for
microsatellites. The
advantage is that a very small amount
of biological materials is
sufficient for analysis. This
most widely used method will be the
subject of the following explanation.
The reduction of single nucleotide
polymorphism
is also increasingly used in
conjunction with modern large-scale
animal testing methods. The
basic requirements for modern methods
are high reliability of rejection and
of confirmation of parentage,
speed and simplicity of testing,
high reproducibility, and
finally, the lowest possible
cost.
What are microsatellites? They
are short repetitive sequences,
tandem repeats,that consists of
mono, di, tri or
tetranucleotide motifs. They
are highly polymorphic, i.e. that the
number of their allelic forms is very
high. We classify them as
lengths polymorphism. We can
amplify the sequence with
microsatellites by PCR
and then detect their length by
electrophoresis, which is determined by the
different number of repeats.
The designation of alleles of
individual microsatellites is
determined by the size of this sequence
in numbers of bases.
In the figure, you can see an example of
only three alleles of one microsatellite,
which differ in the number of repeats of
the GC repeat and the size of their
PCR product varies, and their
combination can give a total of
six different genotypes. The
size of the allele is determined
by gel electrophoresis.
Here is a diagram of the gel
electrophoresis for illustration.
Different set of microsatellites, or
panels, are used in different animal
species with a minimum number
of 10, which is sufficient for
highly reliable parentage
verification. In humans,
15 to 22 of these markers
are used for paternity.
Other microsatellite panels are being
developed for other animal species
and allow studying the genetic diversity
of populations in addition to
parentage tests.
In this picture, you can see the steps of
parentage verification in animals
using microsatellites. DNA
isolation is possible from any
biological material that contains
even a minimal amount of DNA.
This is followed by a multiplex PCR
reaction that specifically
multiplies the individual
microsatellites together in one
tube. Their size is then determined
by precise capillary
electrophoresis. The alleles and
genotypes for each microsatellite
are then determined. By
comparing the genetic type of parent and
offsets, parentage can be verified.
The idea of multiplex PCR
is that multiple DNA fragments are
amplified in a single reaction,
depending on the number of primers added
to the reaction. One of the primers
of a given pair must always be
labeled with a specific fluorescent
color. When designing primers,
care must be taken to ensure
that the allele size ranges
of different microsatellites in a
single color do not overlap,
otherwise they would not be able to be
differentiated from each other.
The reaction takes place in a
thermal cycler. The result
is a microtube containing
amplicons of different lengths, so
that the entire analysis can be
performed in one tube.
The mixture of PCR fragments of many
different microsatellites needs to be
distinguished by size and color.
This is done by fluorescence capillary
electrophoresis. This allows a
very precise determination of the
size of the individual fragments,
i.e. alleles, and is performed using
a genetic analyzer.
The specific size of individual alleles
is determined by comparison
with the calibration curve of size
standard. It is a mixture of DNA
fragments of known length. The size
standard is always separated with
each sample together and is
distinguished by the orange color.
The result of electrophoresis
can be monitored using raw data.
On the electrophoretogram, we can see the
peaks corresponding to the individual
microsatellites (red, blue,
green and black), and the size standard
(orange).
The software of the genetic analyzer
allows automatic evaluation -
determination of the size of
individual peaks that correspond to alleles. We thus
obtain the genotypes of individual
microsatellites of the individual, the
so-called genetic type. By
comparing with parents, we can
verify parentage or by comparing
with another sample we can identify
the individual.
And that's all for this short
presentation explaining genetics
parentage testing in animals.
Thank you for your attention.