Proc. Assoc. Advmt. Anim. Breed Genet. Voll2
DNA FINGERPRINTING - HOW IT WORKS AND APPLICATIONS FOR THE BEEF
INDUSTRY
D. M. Vankan’ and B. M. Burns*
’ Veterinary Blood Grouping Laboratory, University of Queensland,St. Lucia QLD 4072
* Tropical Beef Centre, P.O. Box 5545, RockhamptonMail Centre QLD 4702
SUMMARY
DNA fingerprinting is a powerful test for parentage and paternity testing of cattle. The mechanism
of genetic identification using DNA markers is outlined with a brief description of theory and
methodology and examples provided to demonstrate how it works. Theoretical expectations of the
test are discussed and results obtained to date presented.
Keywords: DNA fingerprinting, microsatellites, parentage, paternity
INTRODUCTION
Parentage testing of cattle has long been used by breed societies to maintain the reliability of their
pedigree records. This in turn helps to enhance genetic progress in breeding programs. The
accuracy of parentage tests is therefore very important to the stud cattle industry. Accurate
determination of paternity is also valuable to commercial cattle producers who wish to multiplesire mate their herds and allocate calves to their correct sires without the need to test the dams.
This enables producers to trace the fecundity of their bulls and to identify those sires and sire lines
producing high performing progeny.
Parentage and paternity testing is achieved by the detection of “markers”. Any attribute that can be
readily detected and its inheritance traced can serve as a marker. Traditionally, protein markers
found on the surfaces of red blood cells and in the blood plasma were used (blood typing). DNA
“microsatellites” provide an alternative and superior source of genetic markers (DNA
It is important that realistic expectations and appropriate uses of DNA
fingerprinting).
fingerprinting are familiar to those wishing to use this new technology.
GENETIC IDENTIFICATION USING DNA MARKERS
The Basic Theory. Parentage and paternity testing relies on two basic principles; on the detection
of genetically inherited markers that remain the same throughout the animal’s life and on the
knowledge that all animals possess two copies of every gene (or marker), one of which was
inherited from the sire and the other from the dam. If the marker shows variation, then the copy (or
variant) inherited from the dam may be different to that inherited from the sire. This provides the
foundation for parentage and paternity testing. That is, if one variant of a genetic marker is present
in a calf but absent in both alleged parents, the calf must be excluded as the offspring of that
mating. Parentage and paternity tests always work by exclusion since no test can positively identify
an animal. That is, testing can exclude a sire or dam as a possible parent or an offspring can be
excluded as being possible from a nominated mating or parent and these exclusions are absolute.
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however, if an animal or mating qualifies, this does not prove it absolutely. At best we can provide
a probability that the qualification is correct, but this will never be 100%.
The process of DNA fmgerprinting requires DNA to be extracted from a tissue sample. Blood is
the tissue of choice since it is easier and therefore cheaper to extract DNA from blood samples
compared to samples such as semen , muscle and hair follicles. All tissue samples need to be
relatively fresh in order to extract DNA of sufficient quality for testing.
An Illustration of How it Works. The results of five samples (two bulls, one cow and two calves)
tested with one DNA marker are illustrated in Figure 1. Variants of this DNA marker (labelled A,
B, C, D) appear as distinct bands. Every animal displays one or two bands. Animals with one band
have inherited the same variant from each parent (both bands being in the same position) whereas
those with two bands have inherited a different variant from each parent.
Bull 2
A
B
C
D
Figure 1. An illustration
of the results of one DNA marker tested with samples from two
bulls, one cow and two calves. Variants of the DNA marker appear as distinct bands.
Calf 1 qualifies as the offspring of the Cow and Bull 2 because it has one band in common with
each of these potential parents (band B in common with the Cow and band C in common with Bull
2). If this calf still qualifies after analysis of 11 such markers there is a greater than 99% chance
that this calf is the offspring of the Cow and Bull 2. Calf 1 does not qualify as the offspring of the
Cow and Bull I because its band C is not found in either of these potential parents. This example
highlights how the testing of only one parent (eg the bull) reduces the power of the test. If the
Cow had not been tested, Calf 1 would qualify as the offspring of both Bull 1 and Bull 2 since Calf
1 shares a band in common with both of these bulls. Although both sires have completely different
bands for this marker, and are therefore able to be distinguished from one another, this marker is
unable to allocate the calf to its correct sire because of the band contributions of the Cow. Calf 2
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Proc. Assoc. Advmt. Anim. Breed Genet. Vol12
has inherited two band B’S, one from its sire and one from its dam. Therefore, it qualifies as the
offspring of the Cow and Bull 1 but does not qualify as the offspring of the Cow and Bull 2. Single
parent analysis of Calf 2 would be able to differentiate between the two bulls.
ACCURACY AND EXPECTATIONS
Theoretical Considerations. The “accuracy” of a parentage or paternity test refers to the ability of
the test to detect an incorrect parentage or paternity (Jamieson, 1965, Rendel and Gahne, 196 1).
The overall accuracy of a parentage/paternity evaluation is determined by two factors: (1) the
number of genetic markers examined. As more markers are examined accuracy increases, but not
linearly, so a compromise must be reached between achieving a reasonable accuracy at an
affordable cost and (2) the degree of variation that exists for each genetic marker. More accuracy
is achieved if the genetic markers used show a large degree of variation. Marker variation is
reduced when animals are closely related and differs markedly between breeds of cattle. Variation,
and therefore accuracy, is reduced in those breeds of cattle with a smaller gene pool. Our studies
indicate that improved accuracy can be expected in. Bos indicus breeds as compared to Bos taurus
breeds.
A commercially viable DNA-based parentage or paternity test requires the identification of groups
of markers (three or four markers per group) that can be tested simultaneously and that show a high
degree of variability across different cattle breeds. Currently a standard set of 11 DNA markers
tested in three separate marker groups are used for DNA fingerprinting. Extra groups of markers
are available if further resolution is required. The combined results of all 11 markers produce a
DNA profile for each animal and the chances of any two animals having the same profile lies
somewhere between three in 10 million to three in 100 billion, depending on the breed.
Accuracy in Parentage and Paternity. The standard set of 11 DNA markers were evaluated in 12
breeds of cattle. When used for parentage analysis (ie the sire, dam and calf are tested) these
markers were able to detect around 99% of incorrect parentages. This figure varied from 98.5% in
Poll Herefords to 99.9% in B&mans, with the accuracy in all other breeds tested being over 99%.
When used for paternity analysis (ie only the sire and calf are tested) accuracy is reduced to 88%99%, depending on the breed (Vat&an et al. 1994).
Accuracy in Multiple-Sire Joinings. In situations where more than one bull is used and the dams
are not being tested, the paternity accuracy given above is reduced in proportion to the number of
bulls used. Table 1 indicates the theoretical level of accuracy that could be expected in a Bos
indicus herd as more bulls are used. One can expect around 90% of calves to be assigned to a
single sire if 10 bulls are used in the mating group. In a “worst-case” Bos taurus herd, by contrast,
only 28% could be expected to be assigned. These accuracy figures are further reduced if animals
in the mating group are related.
Prescreening of bulls before being placed in a mating group can assist multiple-sire analyses by
maximising the chances of being able to differentiate between bulls although this cannot be
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Table 1. The theoretical level of accuracy for correct paternity assignment expected from a
Bos indicus herd as the number of bulls increases
No. of bulls in sire group
1
5
10
15
20
Expected accuracy
99%
95%
90%
86%
82%
guaranteed. The example of Calf 1 ( Figure 1) illustrates this problem. Prescreening in this
example reveals entirely different variants of this DNA marker for Bulls 1 and 2. Since they can be
differentiated unequivocally using this marker they would be suitable candidates for being in the
same mating group. The paternity of calf 1 is nevertheless unable to be resolved without testing the
dam.
Results obtained to date are presented in Table 2. The accuracy obtained is close to that expected
assuming a paternity accuracy for Bos indicus of 99% for a single bull. If improved accuracy is
required, calves that remain unassigned after testing through the standard panel of markers can be
tested through further systems to obtain a very high level of accuracy.
One difficulty highlighted by these results is the problem of missing sires or calves that cannot be
assigned to any of the nominated sires. Although there are many explanations for these findings,
eg. cows being pregnant before being placed in a mating group, bulls and/or cows jumping fences,
bull calves, most of the herds analysed have a proportion of missing sires.
precocious
Interestingly, herd No. 15 involving 244 calves and 34 Brahman sires had no missing sires when all
bulls on the property were included in the analysis whereas the initial analysis involving only the
16 bulls in the mating group produced 5.4% of calves with missing sires.
APPLICATIONS FOR THE BEEF INDUSTRY
For the stud industry, DNA typing offers greater accuracy than blood typing although the costs of
the two procedures are similar. A gradual changeover from blood typing to DNA typing is
recommended to minimise the financial burden of having to double-test animals. Blood typing and
DNA fingerprinting are separate and quite different tests and the results are not interchangeable.
That is, the blood typing results of parents cannot be used to parentage verify a calf that has been
DNA typed and not blood typed.
For commercial breeders DNA typing offers a new and powerful test for collecting information on
their animals and for enhanced genetic improvement through the selection of high performance
progeny. The identification of those bulls with superior fecundity, or those producing progeny with
high quality carcass characteristics can assist in bull selection and the reduction of bull percentages
as well as facilitate management decisions. It is envisaged that the identification of markers for
traits of economic importance will also facilitate the selection of superior animals in the future.
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Table 2. Results obtained for multiple-sire analyses involving more than 50 calves of Santa
Gertrudis (SG) or Brahman (B) origin, showing the observed and expected percentages of
calves assigned to a single sire, the proportion of calves with missing sires (% calves 0 sires)
and the improved accuracy obtained by testing one (+l) or two (+2) more groups of markers
Herd
I.D.
Breed
Number
of Calves
Number
of Sires
Observed
% solved
Expected %
solved
10
12
28
26
6
5
7
22
10
11
19
14
6
12
34
28
91.0
90.4
88.6
75.5
77.0
94.1
95.1
93.2
80.2
90.4
89.5
82.6
86.9
94.1
88.6
71.1‘
75.5
1
SG
156
;
SG
SG
71
3
4
5
6
7
8
9
10
11
12
13
14
15
16
SG
SG
SG
SG
B
B
B
B
B
B
B
B
B
144
408
67
62
100
235
103
213
163
175
91
255
244
288
100
85.4
89.2
83.6
96.8
96.0
88.1
91.3
92.5
92.0
93.1
100
85.5
77.5
74.7
% calves
0 sires
+l
% solved
+2
%
solved
95.5
100
98.5
94.0
100
98.3
0.6
23.9
’
34.8
21.3
16.4
4.8
24
9.4
6.8
8.0
6.1
0
39.6
10.9
0
7.6
99.4
96.6
97.1
90.2
93.0
90.3
99.2
97.1
95.5
The power of DNA fingerprinting is not absolute and it is important that realistic expectations are
placed on the test. As no test works by positive identification, and it is not economically feasible to
test animals through an unlimited number of markers, the value of the test to each individual
producer will vary according to the demands placed on it.
REFERENCES
Jamieson, A. (1965) Hered. 20: 419
Rendel, J. and Gahne, B. (1961) Anim. Prod. 3: 307
Vankan, D.M., Moore, S.S., Bell, K. and Hetzel, D.J.S. (1994) Anim. Genet. 25 suppl2:41
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