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Parentage Identification in the Bovine Using “Deoxyribonucleic Acid Fingerprints”

Authors:

Abstract

Parentage determination in cattle by means of DNA fingerprints is examined at a theoretical level and its implementation illustrated by way of a practical example.
Parentage Identification in the Bovine Using
“Deoxyribonucleic Acid Fingerprints”’
Y.
KASHI,
E.
LIPKIN, A. DARVASI, A. NAVE,
Y. ORUENBAUM,
J.
S.
BECKMANNF
and
M.
SOLLER
Department
of
Genetics
The
Hebrew
University
of
Jerusalem
Jerusalem
91904,
Israel
ABSTRACT
Parentage determination in cattle by
means of
DNA
fingerprints is examined
at a theoretical level and its implementa-
tion
illustrated by way of a practical ex-
ample.
(Key
words:
DNA
fingerprints,
minisatellites, parentage identification)
INTRODUCTION
Families of hypervariable loci in higher or-
ganisms are uncovered
by
a variety of
mini-
or
microsatellite core sequences. Each such core
sequence is present at numerous loci in the
genome
in
the form of tandem repeats
with
varying repeat numbers between different loci
in the same individual and the same locus
in
different individuals. Consequently, on hybridi-
zation of genomic
DNA
digested with an ap
propriate enzyme to
a
probe containing the
mini-
or microsatellite core sequence, an indiv-
idual-specific
DNA
band pattern termed a
“DNA
fingerprint” is obtained. These
DNA
fingerprints were first uncovered in man
(3,
4,
5,
8)
and somewhat later in domestic animals
(2,
7,
11).
Jeffreys
et
al.
(4)
were the first to demon-
strate the power of
DNA
fingerprints in parent-
age identification
in
man.
Soller
and Beckmann
(10)
considered theoretical aspects of the use
of
diallelic restriction fragment length polymor-
phism markers for parentage identification in
domestic animals.
In
the present paper, we
Received January 11,
1990.
Accepted June
6,
1990.
kontribution
from
the
Agricultural
Research
OrganiZa-
tion,
‘Izle
Volcani Center, Bet
Dagaa
Israel,
Number
2862-E,
1989
series.
*Depuknent
of
Plant
Genetics
and
Breeding,
Agricd-
tural
Research
Organization.
The
Vokani
Center
POB
6,
Bet
Dagan
50250,
Israel.
derive some theoretical aspects of parentage
identification by means
of
DNA
fingerprints
in
domestic animals, and present, for illustrative
purposes and as an introduction to the tech-
niques involved, a practical case of parentage
identification by
DNA
fingerprints
in
the
bo-
vine.
THEORY
Parentage identification in segregating popu-
lations generally takes place by means
of
the
“exclusion principle”
(10).
That is, presence at
some genetic locus in the offspring of an allele
not found in either of the putative parents effec-
tively excludes the particular parental pair from
biological parenthood.
In
DNA
fingerprints, each band represents
a
particular allele, but alleles of different loci can
have indistinguishable fragment
lengths.
Never-
theless, because of the codominance of
DNA
fragment bands,
all
bands appearing in the
DNA
fingerprint of an offspring must derive
from one or
the
other
of
its biological parents.
Consequently, presence of a band in the
DNA
fingerprint
of
an offspring that is absent in both
of the putative parents excludes at least one of
these
as
biological parents. Thus, by
scrutinii-
ing
DNA
fingerprints
of
all putative parental
pairs
in
turn,
all but the true parental
pair
can
be
excluded. The remaining parental pair will
be
identified
as
the
“true”
parents, by default.
The effectiveness of
DNA
fingerprints for
parentage identification derives
ftom
the fact
that over an entire population, each minisatel-
lite locus exhibits a wide range of alleles,
dif-
fering in their fragment lengths.
As
a result,
over the population
as
a whole, numerous
bands, differing in fragment length, can
be
identified, but only a few of these bands will
be
present in any one individual. There is thus
only a small probability that two randomly
chosen individuals will share all, or even
a
large proportion of the bands in their respective
1990
J
Dairy
Sci 73:3306-3311
3306
IDENnmCATION
USING
DEOXYRJBONUCLEIC
FINGERPRINTS
3307
DNA
fingerprints. Consequently,
there
is a high
probability that at least one of the bands
trans-
mitted
to
an
offspring by the true parent(s) of
an individual
will
not be found in either mem-
ber of a false putative "parental pair", leading
to exclusion.
The general situation considered in livestock
parentage identification
is
one in which it is
desired to identify the particular parents
of
a
given individual from among a group of puta-
tive male and female parents, among which the
true male and female parents of the individual
are found. Within
this
general situation,
three
specific cases can be considered.
Case
1
In
the first case one true
parent
is
known,
and the other parent must be identified
from
among a group
of
putative parents.
In
this
case,
presence of a band in the offspring, which
is
absent
from
both
known
and putative parent,
will necessarily exclude the putative parent.
The probability of exclusion of any single puta-
tive parent for
this
case (to be calculated later)
will
be
denoted,
P,
and
will
equal
the
probabil-
ity that the true parent contributes a band to the
offspring that is not found in the given putative
parent or in the
known
parent.
If
there are a
number,
k,
of putative parents, the probability
that
all
but the true parent will be excluded
by
a
single fingerprint will
be
The probability
that
all
but the true parent
will
be excluded b t
independent fingerprints will
be
1
-
(1
-
Pk-r)?
Case
2
In
the second case, neither parent
is
known,
but
it
is
known
which male mated with which
female, and it is necessary to identify the mat-
ing pair that produced a given offspring from
among a group of putative
known
mating pairs.
In
this
case, presence of a band in the offspring
that
is
absent
in
both parents of a putative
mating pair
will
exclude that mating pair.
Bs
cause
two
true parents
are
involved in each
mating pair, the probability of exclusion for a
single putative mating pair will equal the prob-
ability that either or both of the true parents
contributed a band
to
the offspring that
is
not
found among the combined bands of the puta-
tive mating
fair.
This
probability will equal
1
-
(1
-
P)
.
If
there
are
k
putative mating
pairs, the probability that
all
but the true mating
pair will
be
excluded by a single fingerprint
will
be
[l
-
(1
-
P)2]k-'.
while the probability
of exclusion
by
t independent fingerprints will
be
1
-
{l
-
[l
-
(1
-
P)2]k--l}t.
Case
3
In
the
third
case, neither parents nor mating
pairs are
known,
and it
is
necessary to identify
the specific male and female parents that
produced a given offspring, from among a
group of putative male and female parents.
In
this
case, males and females can
be
paired
in all
possible putative parental pairs. Again, pres-
ence
of
a band in the offspring that
is
absent in
both putative parents,
will
exclude that parental
pair, with probability
1
-
(1
-
P)2.
Given
M
putative male parents and
F
putative female
parents, the total number of putative parental
pairs
will
be
MF,
and the probability that
all
but the true pair will
be
excluded by a single
fingerprint
will
be
[I
-
(1
-
P)~I=',
white the
probability of exclusion by
t
independent
fm-
gerprints will be
1
-
(1
-
[I
-
(1
-
P)2]1wL*)t.
The probability of exclusion,
P,
for a
known
parent and a single putative parent
will
equal
the
probability that the true parent has bands
that are not present in the putative parent
or
in
the
known
parent and that at least one of these
bands is transmitted to the offspring.
It
is con-
venient to calculate
this
as
P
=
1
-
P',
where
P'
is the probability that the true parent transmits
to the offspring only bands present in both the
horn
and the putative parent.
The
P'
will
be
calculated
as
follows. Let m
be
the total number of bands uncovered by the
particular
mini-
or microsatellite probe over the
entire population and
n
be
the average number
of bands found
in
an
individual.
For
simplicity,
although the number of bands per individual
will
have a hypergeometric
(12)
distribution
(since band sampling is without replacement),
in
all
that follows
this
will
be
treated as a
constant, with value
n,
and the number of bands
transmitted
by
parent to offspring will also be
treated
as
a constant with value
$2.
Let
c be the number of bands that
are
com-
mon to both the
known
parent and the putative
parent. The probability of a given value for c
will
be given by
the
hypergeometric distribu-
tion: H(c;mp,n). The total number of different
bands
found
in
either or both the
known
parent
Journal
of
Dairy
Science
Vol.
73,
No.
11,
1990
3308
KASHI
and the putative parent will then equal
T
=
2n
-
c.
Given
T
bands in the putative parental
pair
consisting
of
known
parent and putative parent,
the probability that the true parent will carry at
least x
of
these bands will be given by the
hypergeometric distribution: H(x;m,n,T).
Given that the true parent carries x bands
found in
the
putative parental pair, the probabil-
ity that all
n/2
bands transmitted by the true
parent will come from the x bands common to
the putative parental pair,
will
equal
0
for x<(n/
2),
while for
x2(n/2)
it
will be given
by
the
hypergeometric distribution: H(n/2;n,nDs).
Hence, the overall probability that all bands
transmitted by the true parent to the offspring
correspond to bands found in the
known
parent
and in the putative parent
will
be
APPLICATION
On
September 2,
1989,
three
calves were
born
to
two
dams
at Kibbutz Ginegar. Female
calf,
01,
stood next to
dam
A,
which had been
inseminated by
sire
1.
Female calf,
03,
stood
next to
dam
B,
which had
been
inseminated by
sire
2.
Male calf,
02,
was situated between
dam
A
and
dam
B,
and could not
be
unequivocally
assigned to either.
Because
female calves that
are twin to a male are generally sterile
(free
martins), we were requested
by
E.
Lipkin, a
graduate student
in
our
department and man-
ager
of
the
dairy
herd
at
Kibbutz Ginegar,
to
identify the female twin
of
the male using
DNA
fingerprint procedures in use in
our
laboratory
(6.
7).
Blood
samples were available from
dams,
calves, and
sire
2, but could not
be
obtained for
sire
1.
DNA
extraction, labeling, and
hybridiza-
tion procedures were
as
previously described
(6).
Three
minisatellite
probes
were utilized
the Jeffkeys-33.6 probe
(3,
courtesy of
A.
Jeffreys;
a
new minisatellite found in
a
2.7-kb
bovine
DNA
fragment
(KTN2.7),
which
was part
of
a
cluster of
mini-
and microsatel-
lites
described
elsewhere
(6);
and a 700-bp
ET
AL.
fragment, part of the same cluster, containing a
poly
(TG)
microsatellite sequence.
The lack of
DNA
from
sire
1
complicated
the analysis because the exclusion principle
could not
be
applied to family
1
(sire
1
and
dam
A).
Hence, identification in
this
example
was based solely on the exclusion principle as
applied to calf
02
and family 2
(sire
2 and
dam
B
(Le., case 2
of
the theoretical analysis with a
single putative family).
RESULTS
Theory
Table
1
shows the probability of exclusion
(P)
for a single fingerprint for the case of one
parent
known
and one putative parent. Proba-
bility of exclusion increases with
the
total num-
ber of bands observed over the entire popula-
tion (m) and is relatively independent of the
mean number of bands observed in a single
individual (n),
so
long as n/m
is
4.
For values
of n/m greater than
this,
probability
of
exclu-
sion drops markedly.
A
value of
P
=
.9
seems a
useful
overall average.
In
this case, likelihood
of identifying the true parent among
10
putative
parents, say, with one parent
known,
would
be
.39,
for a single fingerprint; and
.62,
.77,
.86,
and
.91
for
2,
3,
4,
and
5
independent finger-
prints, respectively.
For the same value of
P
=
.9,
the probability
of excluding a putative parental pair, when
mating
pairs
are
known,
would be
.99
for a
single fingerprint, whereas probability of iden-
tifying the true parental
pair,
among
10
putative
parental pairs, say, would be
.91
for a single
fingerprint, and
.99
for two independent finger-
TABLE
1.
The
probability
of
exclusion
(P)
for
a
single
fingerprint,
for
the
case
of
one
parent
known
and
one
putative
parent
as
a
function
of
total
number
of
bands
observed
over
thc
enlire
population
(m)
and
mean
number
of
bands
observed
in
a
single
individual
(n).
m
n
10
20
30
40
4
.61
.88
.94
.97
6
.31
.88
.%
.98
8
. . .
.86
.%
.99
10
. .
.
.79
.%
.99
12
.
.
.
.65
.95
.99
Journal
of
Dairy
Science
Vol.
73,
No.
11.
1990
IDENTIFICATION
USING DEOXYRIBONUCLEIC
IW4-S
3309
23
9.4
6.7
4.4
$B
0201A
S2
0
0,
B
0201
A
Figure.
1.
The
DNAfingeqrintpnttemsof
the
six
animals
examined.
The
DNAfromthese
auimals
was
digested
with
either
Hi@
(panel
A)
OT
AluI
(panels
B
and
C)
and
hybridized
to
eik
KTN2.7
or
Jeffrey
33.6
minisate
probes
(panels
A
and
B,
and
panel
C,
respectively).
S2,
A,
B,
01.02,
and
03
represent
sin
2.
dam
A,
dam
B,
and calves
01,02,
and
03.
respectively. Nnmbm
on
the
left
represent
the
sizes
in
kilobases
of
the
k
HindIl
DNA sizemarker fragments.
The
asterisk
denotes
bands
found
in
the
(problematical)
calf
02,
which
me
not
found
in
sire
2
or
dam
B
black
square
denotes
a
band
found
in
calf
02
that
is
not
found
in
dam
A
or
calf
01.
prints. Similarly, probability of identifying the
true parental pair among, say,
20
females and
five males without any prior infonnation as to
mating pairs, would
be
.37
for a single finger-
print and
.60,
.75,
.84,
and
.90
for
2,
3,
4,
or
5
independent fingerprints, respectively.
Appllcatlon
Figure
1
shows the
DNA
fingerprint patterns
obtained upon hybridization
of
Southern blots
of
Hinfl
or
AIuI
digested genomic DNA
of
dams,
offspring,
and
sire
2
to the Jeffreys-33.6
and
KTN2.7
minisatellite
probes.
It
should
be
noted that the blots presented are
of
only
rnedi-
ocre
quality.
These blots were originally
pre
pared “on the
m”,
as
a service and not for
purposes
of
publication.
Higher
quality blots
would show sharper, straighter,
and
darker
bands. Nevertheless, the blots were adequate
for their intended purpose. Bands
are
somewhat
clearer on the original autoradiographs than
on
the photographs. With more attention
to
tech-
nique, it is possible to
obtain
routinely blots
of
higher quality and discriminating power.
Both
probes
yielded informative DNA
fin-
gerprint patterns. Consideration
of
these pat-
terns
showed that
a
number
of
bands found
in
calf
02.
were not found in sire
2,
dam
B, or
calf
03.
These
are
shown by an asterisk in
figure
1.
This
indicates that
calf
02
is not a
biological offspring of family
2,
and hence, not
a sib to calf
03.
Conversely,
all
bands found
in
calf
02
(with one exception, shown by black
square
in
Figure
1C)
had
a counterpart
in
either
dam
A
or
calf
01.
The exceptional band may
derive from the
unknown
sire
1.
These results
indicate that calf
02
is a biological offspring of
Journal
of
Dairy
Science
Vol.
73,
No.
11,
1990
3310
KASHI
ET
AL.
family
1
and a sib to calf
01.
Thus, calf
01
is
likely
to
be a freemartin.
With the
KTN2.7
probe, a single discrimina-
tive band was found both with
Hi@
(Figure
1A)
and AluI (Figure
1B).
With the Jeffreys-
33.6
probe, two discriminative bands were
found with AluI
(FGgufe
lc),
and
one dis-
criminative band was found with
Hi4
(data
not shown).
Hybridization
of
the blots to the
700-bp
microsatellite fragment
also
yielded in-
formative
DNA
fingerprints with two dis-
criminative bands for the AluI digest and one
discriminative band for
the
Hi@
digest (data
not shown).
DISCUSSION
Theory
The theoretical analyses suggest that
DNA
fingerprints
can
be a highly effective means of
parentage identification in domestic
animals.
More exact evaluation with respect to
dairy
cattle will require defining the total number of
different bands observed over a population with
various probes and enzymes, and the average
frequency of each band in the population.
Nevertheless, over a
broad
range
of
possibili-
ties, the results indicate that one or
two
finger-
prints per family
should
generally
suffice
for
progeny identification when exclusion is aimed
at a putative parental pair (known mating pairs,
case
2,
of the theoretical analysis).
A
larger, but
still
plausible, number of fingerprints
will
be
required
when one parent is
horn,
and the
other parent must be identified from among a
group of putative parents (case 1) or when
nothing is known other than the group of males
and females among which the mating produc-
ing the offspring was consummated (case
3).
It
should
be
noted that additional fingerprints can
be obtained by using different probes on the
same blots
so
that the marginal cost of addi-
tional fingerprints
is
low.
Illustrative
Experlmental Example
In
the illustrative experimental example,
six
different
DNA
fingerprints
(three
different
mini-
or
microsatellite
probes
x
two enzymes)
were each independently able to provide une
quivocal parentage assignments in which exclu-
sion was
aimed
at a single known mating pair
(case
2).
This
supports the view that
DNA
Journal
of
Dairy
Science
VoL
73,
No.
11,
1990
fingerprints can be an effective means of par-
entage identification in
dairy
cattle and con-
firms
the theoretical analysis indicating the
great power of
DNA
fingerprints for
this
case.
The mean number of discriminative bands was
1.33/fingerprint.
On
this
basis,
by
Poisson dis-
tribution, the probabilities
are
.26,
.35. .23,
and
.10
that
0,
1,
2,
or
3
discriminative bands will
be found for any particular fingerprint.
This
does not
Mer
greatly from the observed distri-
bution
of
discriminative bands between finger-
prints.
Comparlson
with
Other
Methods
In
man and domestic animals, parentage
identification has been routinely carried out
by
means of blood
type
and biochemical polymor-
phisms. These methods have proven themselves
in
practice
and
are
effective and rapid.
Never-
theless, they
do
have a number of technical
limitations, requiring fairly large samples
of
fresh
blood, specialized laboratories, and
in-
volving numerous independent marker determi-
nations, while obtaining much
of
their power
by scoring allelic status at the highly poly-
morphic major histocompatibility complex 10-
cus.
In
ccmtrast,
DNA
fingerprints simulta-
neously examine a number
of
highly
polymorphic loci,
can
be
carried out on small,
not necessarily fresh, samples and can be car-
ried out with equal effectiveness
on
any tissue.
A
further
attractive
aspect
of
DNA
finger-
prints is that they do not
require
a dedicated
laboratory. The procedure involved is relatively
simple and
can
be
carried
out readily on
an
ad
hoc basis, by a suitably trained technician in
any laboratory minimally
equipped
for work
with recombinant
DNA.
It
does not require
preparation
of
numerous special reagents.
In
the
future,
nonradioactive labeling
of
DNA
probes
will
be possible
(13),
which
will
allow the use
of
commercially available reagents for finger-
print determinations. Thus,
DNA
fingerprints
may provide a
useful
alternative to blood poly-
morphisms in some circumstances.
In
particu-
lar, the
ability
to increase the discriminatory
power
of
DNA
fingerprints,
by
adding addi-
tional probes and enzymes, gives
this
approach
adequate power to deal with the complex case
3
situation.
This
would appear to be beyond the
capability of classical blood
type
and biochemi-
cal polymorphisms.
IDENTIFICATION
USING
DEOXYRIBONUCLEIC
FI"TS
3311
In
our
laboratory, fingerprinting of
30
in-
dividuals with
two
enzymes and one probe
(two
hybridizations) would
require
1
wk of work by
a single technician. We estimate
labor
costs
of
a technician and costs
of
expendable materials
and supplies involved
in
DNA fingerprinting
as
being roughly
equal
and
totaling
about
$lOOO/
wk.
Thus,
our
costs would come out
to
about
$30
per
fingerprinted individual, not including
costs of blood sample collection.
This
should
be
a reasonable estimate for
costs
in
a labom-
tory that occasionally carries out DNA finger-
prints
on
an
ad
hoc
basis. A dedicated labora-
tory could probably carry out
the
same
operations at
half
the cost.
The present study
was
prompted
by
a
re-
quest to identify a possible freemartin. Howev-
er,
the availability
of
this
powerful methodol-
ogy
may
find other useful applications
in
cattle
breeding, including parentage identification
in
beef
herds where a number of bulls
run
with
the females and control of misidentification in
dairy
cattle progeny testing programs
(1,
9).
ACKNOWLEDGMENTS
We thank R.
Arbel
for providing the
blood
samples examined
in
this
study.
This
research
was
supported by the Israel Beef Cattle Council
and
by
the
US-Israel Binational Agricultural
Research and Development Fund
(BARD).
Y.
Kashi
is
the recipient
of
a fellowship from the
Levi
Eshkol
Fund.
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Dairy Science Vol. 73, No. 11, 1990
... The use of molecular marker information to increase accuracy and reduce generation intervals has been studied in recent decades, and implemented in a limited fashion in some breeding programs. Marker-assisted selection (MAS) was applied in dairy cattle for the pre-selection of animals, and to select young bulls for entry into progeny testing programs (Kashi et al., 1990a(Kashi et al., , 1990bMackinnon and Georges, 1998). MAS simultaneously uses phenotypic information and data about molecular markers in LD with QTLs, and was adopted to increase annual genetic gain for traits of economic importance in several animal species (Dekkers, 2004). ...
... Before the advancement in high-throughput SNP data, blood groups (Stormont, 1967) and mini-and s (Kashi et al., 1990a(Kashi et al., , 1990b were the basic means of inferring parentage. Even though micro-satellites are still used, with the recent availability of SNP markers and with large numbers of sires (Harris and Johnson, 2010;Fritz et al., 2013; VanRaden et al., 2013b) and dams (Spelman et al., 2013;VanRaden et al., 2013b) genotyped in the USA, Canada, Australia, New Zealand, Ireland, and France among others, parentage and pedigree errors are increasingly identified using SNP genotypes. ...
... Parentage assignment is aimed at excluding individuals ("exclusion principle") from the list of potential parents. This means that a large number of potential sires and dams are examined and only one or a few individuals are retained based on their marker data by using simple segregation rules (Kashi et al., 1990a(Kashi et al., , 1990bHayes, 2011). In addition to marker genotypes, additional accuracy can be achieved if information such as birth dates and mating records are considered. ...
... In the past, parentage testing in cattle has been carried out through the blood group and the protein polymorphism analysis, but because of some drawbacks, these tests have been replaced with new ones that are based on detection of certain "genetic markers". There are different approaches based on DNA polymorphism for paternity testing including RFLPs (Kashi et al., 1990), multilocus, minisatellite and oligosynthetic probes (Trommelen et al., 1993), and PCR based amplification of minisatellites and microsatellites (Schnabel et al., 2000). However, RFLPs generally suffer from low heterozygosities and low PIC, while the DNA fingerprints are difficult to interpret owing to complex nature of banding pattern revealed. ...
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Full-text available
The study was undertaken with an objective to develop and validate a panel containing maximum number of microsatellite markers that can be amplified in a single PCR reaction for precise parentage verification in Indian HF cattle population. The study was based on a total of 210 HF cattle (100 calf, 100 dam and 10 sires). Genomic DNA was extracted from blood and semen samples. A panel of 12 microsatellite markers (BM1824, BM2113, INRA023, SPS115, TGLA122, TGLA126, TGLA227, ETH10, ETH225, BM1818, ETH3, TGLA53) was amplified in a single multiplex reaction and analyzed by capillary electrophoresis on an automated DNA sequencer. The observed heterozygosity (Ho) of 12 markers ranged from 0.607 (TGLA53) to 0.904 (BM2113) while the expected heterozygosity (He) ranged from 0.581 (ETH225) to 0.873 (INRA23). Eleven out of 12 microsatellite loci revealed relatively high polymorphic information content (>0.6). The results suggest that multiplex microsatellite panel can be effectively used to verify the parentage as well as to assign the putative sire to daughters under progeny testing.
... The main aim of this study was to research the determination of standards of population genetics and various loci which can be used in sheep parentage testing and researching the use of various loci for animal identification under proper experimental conditions. This method, known as DNA fingerprting, which was obtained from the use of a probe with crosshybridisation, cutting genomic DNA with the proper enzymes and obtaining individual-specific DNA band samples, including mini or microsatellite sequences in many polymorphic segments, was first used in humans (2)(3)(4) then in plants (5,6) and finally in domestic animals (7)(8)(9). The DNA fingerprint is unique to each individual; half of this fingerprint is maternal and the other half is paternal. ...
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Article
In this study, the parental relationship among one sire, three dams and four calves raised in Chiayi farm was identified by RAPD fingerprinting and further attempting was made to identify the twin from these four calves. Three dam (A, B and C) raised together in the same lot had given one male and three female calves (1, 2 and 3) at night of the same date and a vague relationship between the dam and progeny was occurred. Dam A had been inseminated with artificial insemination but no more frozen semen was available by the time that this experiment was done. Dam B and C had been mated same bull. The results showed that genomic DNA of dam A, male calf and female calf 1 could be amplified to produce 1300 bp band with OPE-01 primer, 500 bp band with OPAA-11 primer, and 2200 bp band with OPE-09 primer, respectively. On the other hand, genomic DNA of dam B and C and female calf 2 and 3 could be amplified to produce 800 bp band with OPE-01 primer. However, genomic DNA of dam B and female calf 2 could be amplified to produced 1600 bp band with OPE-09 primer. It might be suggested, therefore, that male calf and female calf 1 were twin from dam A. Female calf 2 and 3 could be a biological offspring of dam B and C, respectively.
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Identification of DNA polymorphism is the main objective to launch markers assisted selection for genetic improvement of livestock species. Parentage verification is necessary to make any selection programme successful specially under field conditions. Many microsatellite sequences have been found highly polymorphic and ascribed for parentage verification in buffaloes. Microsatellite ETH 131, an anonymous site used in cattle for the same purpose, has been tested in Murrah buffaloes to determine the similarities between sire progenies' and Dam progenies' DNA patterns. Four sire families each comprising two progenies alongwith sire and dams were genotyped using ETH 131 primer sequence. Band pattern matrix (0/1) was analysed with the help of NTSYS-PC analysis to determine the similarity index of parents and progenies. Higher similarities between the progenies and dams's DNA patterns were determined i. e. 80 to 90% in comparison to a little lower similarity percentage of 74 to 79% with respective sires. ETH 131 may be used for parentage verification in Murrah buffaloes. It is promising for parentage verification in buffaloes and may be tested on larger number of animals towards achieving improved herd performance.
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For the present study, blood samples from 35 Murrah dam-progeny pairs and semen samples of their sires were collected. All six primers successfully amplified the genomic DNA from all the animals. The mean observed heterozygosity (Ho) within the population was found to be 0.5438 and it ranged from 0.375 (CSSM43) to 0.775 (CSSM61). Average expected Levene's and Nei's heterozygosity (Hep) were 0.6209 ±0.0894 and 0.6170 ±0.0889, ranging from 0.5155 to 0.7575 and 0.5123 to 0.7528, for loci CSSM43 and CSSM61, respectively. Both observed and expected heterozygosity was above 0.5, which shows that there is sufficient variability in the population and reflects presence of large number of polymorphic loci in the breed. The mean PIC value was 0.5661, ranging from 0.476 to 0.718, for the loci CSSM43 and CSSM61, respectively. In the present study, the probability of exclusion (PE) ranged from 0.0994 to 0.5535 for the loci CSSM43 and CSSM61, respectively. The combined PE (PEc) for all 6 loci was 0.845. Based on PEc, it was shown that DNA analysis in the examined herd of Murrah buffaloes allows incorrect parentage to be excluded with 84.5% probability.
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Screening of a bovine genomic library with the human minisatellite 33.6 probe uncovered a family of clones that, when used to probe Southern blots of bovine genomic DNA digested with the restriction enzyme HaeIII or MboI, revealed sexually dimorphic, but otherwise virtually monomorphic, patterns among the larger DNA fragments to which they hybridized. Characterization of one of these clones revealed that it contains different minisatellite sequences. The sexual dimorphism hybridization pattern observed with this clone was found to be due to multiple copies of two tandemly interspersed repeats: the simple sequence (TG)n and a previously undescribed 29-bp sequence. Both repeats appear to share many genomic loci including autosomal loci. In contrast, Southern analysis of AluI- or HinfI-digested bovine DNA with the (TG)n repeat used as a probe yielded substantial polymorphism. These results show that (i) different minisatellites can be found in a cluster, (ii) both simple and more complex repeated sequences other than the simple quaternary (GATA)n repeat can be sexually dimorphic, and (iii) simple repeats can reveal substantial polymorphism.
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The 23rd Colworth Medal Lecture, delivered on 25 September 1986 at Trinity College, Dublin. This is a licensed copy kindly supplied by the Biochemical Society.
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Four probes known to allow DNA fingerprinting in the human (M13, Jeffreys' core sequence, the human alpha globin hypervariable region [HVR], and a mouse probe related to the Drosophila Per gene) were checked for their ability to reveal "genetic bar codes" in cattle, horses, pigs, dogs, chickens, and a European cyprinid fish, the barbel (Barbus barbus L.). Individual-specific patterns were obtained in cattle using M13, Jeffreys' core sequence, and the alpha globin HVR, in horses, dogs, and pigs using M13, Jeffreys' core sequence, and the Per probe, and in chicken and fish using the four different probes. Although we observed a considerable heterogeneity in the extent of interindividual variation, depending on the particular probe-species combination, the fingerprints are polymorphic enough to be used efficiently in animal identification, paternity testing, and as a source of genetic markers for linkage analysis. These markers should substantially accelerate the mapping of genes affecting economically important traits.
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The human genome contains many dispersed tandem-repetitive 'minisatellite' regions detected via a shared 10-15-base pair 'core' sequence similar to the generalized recombination signal (chi) of Escherichia coli. Many minisatellites are highly polymorphic due to allelic variation in repeat copy number in the minisatellite. A probe based on a tandem-repeat of the core sequence can detect many highly variable loci simultaneously and can provide an individual-specific DNA 'fingerprint' of general use in human genetic analysis.
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The human genome contains a set of minisatellites, each of which consists of tandem repeats of a DNA segment containing the 'core' sequence, a putative recombination signal in human DNA. Multiallelic variation in the number of tandem repeats occurs at many of these minisatellite loci. Hybridization probes consisting of tandem repeats of the core sequence detect many hypervariable minisatellites simultaneously in human DNA, to produce a DNA fingerprint that is completely individual-specific and shows somatic and germline stability. These DNA fingerprints are derived from a large number of highly informative dispersed autosomal loci and are suitable for linkage analysis in man, and for individual identification in, for example, forensic science and paternity testing. They can also be used to resolve immigration disputes arising from lack of proof of family relationships. To illustrate the potential for positive or inclusive identification, we now describe the DNA fingerprint analysis of an immigration case, the resolution of which would have been very difficult and laborious using currently available single-locus genetic markers.