1
00:00:00,801 --> 00:00:03,205
Hello, in this lecture, we will discuss

2
00:00:03,205 --> 00:00:05,409
genetics parentage testing in animals.

3
00:00:06,010 --> 00:00:08,094
This lecture is part of module 4,

4
00:00:08,574 --> 00:00:10,818
Precision Livestock Breeding. The

5
00:00:10,818 --> 00:00:12,982
development of this presentation was

6
00:00:12,982 --> 00:00:15,466
supported by an Erasmus+

7
00:00:15,867 --> 00:00:18,832
KA2 grant within the ISAGREED

8
00:00:18,832 --> 00:00:21,596
project: Innovation the content and structure

9
00:00:21,596 --> 00:00:24,200
of study programmes in the field of animal

10
00:00:24,200 --> 00:00:26,444
genetic and food resource management

11
00:00:26,765 --> 00:00:28,848
using digitization.

12
00:00:32,294 --> 00:00:34,217
At the beginning, it is necessary to

13
00:00:34,297 --> 00:00:36,862
explain the basic terms that will be used

14
00:00:36,862 --> 00:00:39,827
during this presentation. The first one

15
00:00:39,827 --> 00:00:42,231
is parenthood - the correctness of

16
00:00:42,231 --> 00:00:44,995
both parents, father and mother, is

17
00:00:44,995 --> 00:00:47,600
verified. In the case of paternity,

18
00:00:48,000 --> 00:00:49,924
only the father's authenticity is

19
00:00:49,924 --> 00:00:52,768
verified for an individual, which is the

20
00:00:52,808 --> 00:00:54,852
most important thing for humans.

21
00:00:55,613 --> 00:00:57,616
If we are going to identify an

22
00:00:57,616 --> 00:01:00,020
individual, We will use molecular

23
00:01:00,020 --> 00:01:02,344
genetic method (called molecular

24
00:01:02,344 --> 00:01:05,309
dactyloscopy) to determine whether

25
00:01:05,470 --> 00:01:08,194
the DNA sample belongs to the individual

26
00:01:08,194 --> 00:01:10,999
being examined. The term genetic

27
00:01:10,999 --> 00:01:13,884
type or genetic profile represents the

28
00:01:13,884 --> 00:01:16,127
set of all alleles (genotypes) that

29
00:01:16,969 --> 00:01:19,653
is characteristic, unmistakable,

30
00:01:19,974 --> 00:01:21,977
and unique to each individual.

31
00:01:24,421 --> 00:01:26,945
The purpose of genetic parentage testing

32
00:01:26,986 --> 00:01:28,989
is to verify the origin and

33
00:01:28,989 --> 00:01:31,072
identification of an individual.

34
00:01:31,834 --> 00:01:33,797
In the case of livestock bred in the

35
00:01:33,837 --> 00:01:36,562
Czech Republic, the testing is governed

36
00:01:36,642 --> 00:01:37,964
by Act No.

37
00:01:38,244 --> 00:01:41,209
154/2000, called as

38
00:01:41,209 --> 00:01:44,174
Breeders Act. For pigs, the genetic

39
00:01:44,174 --> 00:01:46,578
type of boars in breeding farms is

40
00:01:46,578 --> 00:01:49,022
determined. For cattle, the

41
00:01:50,104 --> 00:01:52,348
origin of breeding bulls is verified,

42
00:01:52,989 --> 00:01:54,912
and their genetic type is determined

43
00:01:54,912 --> 00:01:57,557
before selection for breeding. For

44
00:01:57,557 --> 00:02:00,201
horses, the origin of foals after

45
00:02:00,241 --> 00:02:01,964
insemination is verified.

46
00:02:04,689 --> 00:02:07,173
Parentage determination is of great

47
00:02:07,173 --> 00:02:10,138
importance not only in animal genetics,

48
00:02:10,578 --> 00:02:12,942
but also in forensic genetics and

49
00:02:12,942 --> 00:02:15,787
medicine. There are mistakes in

50
00:02:15,827 --> 00:02:18,071
breeding pedigrees that could cause

51
00:02:18,071 --> 00:02:20,956
inaccuracy in the estimation of breeding

52
00:02:20,956 --> 00:02:23,560
value. The price of animals

53
00:02:24,001 --> 00:02:26,966
is often based on the genetic quality of

54
00:02:26,966 --> 00:02:29,690
their parents, and it is therefore

55
00:02:29,690 --> 00:02:32,014
necessary to be able to verify

56
00:02:32,175 --> 00:02:34,859
parentage. Customers may

57
00:02:35,059 --> 00:02:37,704
also be cheated. In the field of

58
00:02:37,704 --> 00:02:40,068
foresnic genetics, paternity

59
00:02:40,068 --> 00:02:42,592
disputes are commonly dealt with,

60
00:02:43,313 --> 00:02:45,797
and many offenders are now convicted

61
00:02:45,797 --> 00:02:47,560
based on DNA traces.

62
00:02:48,602 --> 00:02:51,246
Even victims of violent crimes or

63
00:02:51,246 --> 00:02:53,650
natural disasters can be

64
00:02:54,051 --> 00:02:56,535
identified from their DNA.

65
00:02:57,417 --> 00:02:59,941
Relationship verification is a

66
00:02:59,941 --> 00:03:02,666
necessity, especially in trans-

67
00:03:02,666 --> 00:03:03,267
plantation.

68
00:03:05,751 --> 00:03:08,475
In the past, before the development of

69
00:03:08,475 --> 00:03:10,959
molecular genetic methods, blood

70
00:03:10,959 --> 00:03:13,684
groups and the protein variability were

71
00:03:13,684 --> 00:03:15,447
used to verify parentage.

72
00:03:16,328 --> 00:03:18,893
However,these methods were not very

73
00:03:18,893 --> 00:03:21,617
reliable. Therefore, methods

74
00:03:21,617 --> 00:03:23,701
based on the detection of DNA

75
00:03:23,701 --> 00:03:26,425
variability (DNA fingerprinting),

76
00:03:27,026 --> 00:03:29,510
initially based on the variable number of

77
00:03:29,510 --> 00:03:32,435
tandem repeats (VNTR)

78
00:03:33,237 --> 00:03:35,080
in minisatellites,

79
00:03:35,721 --> 00:03:38,445
began to be used. Currently,

80
00:03:38,766 --> 00:03:41,010
methods associated with DNA

81
00:03:41,010 --> 00:03:43,093
amplification by PCR,

82
00:03:43,654 --> 00:03:46,539
called STRs,are used for

83
00:03:46,539 --> 00:03:48,983
microsatellites. The

84
00:03:48,983 --> 00:03:51,828
advantage is that a very small amount

85
00:03:51,908 --> 00:03:54,152
of biological materials is

86
00:03:54,152 --> 00:03:56,876
sufficient for analysis. This

87
00:03:56,876 --> 00:03:59,561
most widely used method will be the

88
00:03:59,561 --> 00:04:02,085
subject of the following explanation.

89
00:04:02,886 --> 00:04:05,050
The reduction of single nucleotide

90
00:04:05,130 --> 00:04:05,851
polymorphism

91
00:04:09,377 --> 00:04:11,741
is also increasingly used in

92
00:04:11,741 --> 00:04:14,105
conjunction with modern large-scale

93
00:04:14,185 --> 00:04:16,629
animal testing methods. The

94
00:04:16,669 --> 00:04:19,554
basic requirements for modern methods

95
00:04:20,115 --> 00:04:22,960
are high reliability of rejection and

96
00:04:23,040 --> 00:04:24,883
of confirmation of parentage,

97
00:04:25,564 --> 00:04:27,808
speed and simplicity of testing,

98
00:04:28,529 --> 00:04:30,893
high reproducibility, and

99
00:04:30,893 --> 00:04:33,457
finally, the lowest possible

100
00:04:33,498 --> 00:04:33,898
cost.

101
00:04:37,745 --> 00:04:40,710
What are microsatellites? They

102
00:04:40,790 --> 00:04:42,953
are short repetitive sequences,

103
00:04:43,394 --> 00:04:45,918
tandem repeats,that consists of

104
00:04:45,918 --> 00:04:48,443
mono, di, tri or

105
00:04:48,643 --> 00:04:51,528
tetranucleotide motifs. They

106
00:04:51,608 --> 00:04:54,412
are highly polymorphic, i.e. that the

107
00:04:54,412 --> 00:04:56,977
number of their allelic forms is very

108
00:04:56,977 --> 00:04:59,541
high. We classify them as

109
00:04:59,541 --> 00:05:02,506
lengths polymorphism. We can

110
00:05:02,506 --> 00:05:04,790
amplify the sequence with

111
00:05:04,910 --> 00:05:07,234
microsatellites by PCR

112
00:05:07,635 --> 00:05:09,438
and then detect their length by

113
00:05:09,518 --> 00:05:12,483
electrophoresis, which is determined by the

114
00:05:12,523 --> 00:05:14,606
different number of repeats.

115
00:05:15,568 --> 00:05:17,731
The designation of alleles of

116
00:05:17,972 --> 00:05:20,536
individual microsatellites is

117
00:05:20,536 --> 00:05:23,501
determined by the size of this sequence

118
00:05:23,942 --> 00:05:25,424
in numbers of bases.

119
00:05:28,870 --> 00:05:31,675
In the figure, you can see an example of

120
00:05:31,875 --> 00:05:34,439
only three alleles of one microsatellite,

121
00:05:35,000 --> 00:05:37,845
which differ in the number of repeats of

122
00:05:37,845 --> 00:05:40,650
the GC repeat and the size of their

123
00:05:40,650 --> 00:05:43,495
PCR product varies, and their

124
00:05:43,535 --> 00:05:46,339
combination can give a total of

125
00:05:46,339 --> 00:05:49,264
six different genotypes. The

126
00:05:49,304 --> 00:05:51,949
size of the allele is determined

127
00:05:52,109 --> 00:05:54,192
by gel electrophoresis.

128
00:05:54,673 --> 00:05:56,597
Here is a diagram of the gel

129
00:05:56,597 --> 00:05:59,161
electrophoresis for illustration.

130
00:06:06,423 --> 00:06:08,827
Different set of microsatellites, or

131
00:06:08,827 --> 00:06:11,431
panels, are used in different animal

132
00:06:11,431 --> 00:06:14,396
species with a minimum number

133
00:06:14,396 --> 00:06:17,111
of 10, which is sufficient for

134
00:06:17,111 --> 00:06:19,154
highly reliable parentage

135
00:06:19,194 --> 00:06:21,759
verification. In humans,

136
00:06:22,159 --> 00:06:24,724
15 to 22 of these markers

137
00:06:25,044 --> 00:06:26,887
are used for paternity.

138
00:06:29,291 --> 00:06:32,016
Other microsatellite panels are being

139
00:06:32,096 --> 00:06:34,740
developed for other animal species

140
00:06:35,141 --> 00:06:37,946
and allow studying the genetic diversity

141
00:06:37,946 --> 00:06:40,670
of populations in addition to

142
00:06:40,670 --> 00:06:41,872
parentage tests.

143
00:06:45,318 --> 00:06:48,123
In this picture, you can see the steps of

144
00:06:48,123 --> 00:06:50,767
parentage verification in animals

145
00:06:51,088 --> 00:06:53,812
using microsatellites. DNA

146
00:06:53,812 --> 00:06:56,136
isolation is possible from any

147
00:06:56,136 --> 00:06:58,861
biological material that contains

148
00:06:58,941 --> 00:07:01,585
even a minimal amount of DNA.

149
00:07:02,467 --> 00:07:04,871
This is followed by a multiplex PCR

150
00:07:04,871 --> 00:07:07,034
reaction that specifically

151
00:07:07,034 --> 00:07:09,118
multiplies the individual

152
00:07:09,118 --> 00:07:11,762
microsatellites together in one

153
00:07:11,762 --> 00:07:14,567
tube. Their size is then determined

154
00:07:14,567 --> 00:07:16,851
by precise capillary

155
00:07:16,851 --> 00:07:19,295
electrophoresis. The alleles and

156
00:07:19,455 --> 00:07:22,180
genotypes for each microsatellite

157
00:07:22,660 --> 00:07:25,225
are then determined. By

158
00:07:25,225 --> 00:07:28,150
comparing the genetic type of parent and

159
00:07:28,190 --> 00:07:30,754
offsets, parentage can be verified.

160
00:07:34,280 --> 00:07:36,924
The idea of multiplex PCR

161
00:07:37,165 --> 00:07:40,050
is that multiple DNA fragments are

162
00:07:40,170 --> 00:07:42,293
amplified in a single reaction,

163
00:07:42,694 --> 00:07:45,178
depending on the number of primers added

164
00:07:45,258 --> 00:07:48,143
to the reaction. One of the primers

165
00:07:48,183 --> 00:07:50,948
of a given pair must always be

166
00:07:50,948 --> 00:07:53,672
labeled with a specific fluorescent

167
00:07:53,672 --> 00:07:56,477
color. When designing primers,

168
00:07:56,717 --> 00:07:59,121
care must be taken to ensure

169
00:07:59,442 --> 00:08:02,086
that the allele size ranges

170
00:08:02,247 --> 00:08:05,051
of different microsatellites in a

171
00:08:05,051 --> 00:08:07,135
single color do not overlap,

172
00:08:07,776 --> 00:08:10,100
otherwise they would not be able to be

173
00:08:10,100 --> 00:08:12,744
differentiated from each other.

174
00:08:13,706 --> 00:08:16,551
The reaction takes place in a

175
00:08:16,551 --> 00:08:19,235
thermal cycler. The result

176
00:08:19,315 --> 00:08:21,158
is a microtube containing

177
00:08:21,639 --> 00:08:24,604
amplicons of different lengths, so

178
00:08:24,604 --> 00:08:27,088
that the entire analysis can be

179
00:08:27,088 --> 00:08:29,052
performed in one tube.

180
00:08:31,816 --> 00:08:34,781
The mixture of PCR fragments of many

181
00:08:34,781 --> 00:08:37,666
different microsatellites needs to be

182
00:08:37,666 --> 00:08:40,150
distinguished by size and color.

183
00:08:40,951 --> 00:08:43,436
This is done by fluorescence capillary

184
00:08:43,516 --> 00:08:46,200
electrophoresis. This allows a

185
00:08:46,320 --> 00:08:49,125
very precise determination of the

186
00:08:49,125 --> 00:08:51,369
size of the individual fragments,

187
00:08:51,930 --> 00:08:54,895
i.e. alleles, and is performed using

188
00:08:54,895 --> 00:08:56,337
a genetic analyzer.

189
00:08:59,222 --> 00:09:02,147
The specific size of individual alleles

190
00:09:02,147 --> 00:09:04,912
is determined by comparison

191
00:09:05,072 --> 00:09:07,917
with the calibration curve of size

192
00:09:07,917 --> 00:09:10,761
standard. It is a mixture of DNA

193
00:09:10,761 --> 00:09:13,686
fragments of known length. The size

194
00:09:13,686 --> 00:09:16,411
standard is always separated with

195
00:09:16,531 --> 00:09:18,935
each sample together and is

196
00:09:18,935 --> 00:09:21,259
distinguished by the orange color.

197
00:09:24,064 --> 00:09:26,387
The result of electrophoresis

198
00:09:26,788 --> 00:09:29,352
can be monitored using raw data.

199
00:09:29,993 --> 00:09:32,518
On the electrophoretogram, we can see the

200
00:09:32,558 --> 00:09:35,362
peaks corresponding to the individual

201
00:09:35,362 --> 00:09:37,847
microsatellites (red, blue,

202
00:09:38,167 --> 00:09:40,852
green and black), and the size standard

203
00:09:41,212 --> 00:09:41,773
(orange).

204
00:09:44,177 --> 00:09:46,822
The software of the genetic analyzer

205
00:09:47,062 --> 00:09:49,306
allows automatic evaluation -

206
00:09:49,706 --> 00:09:51,950
determination of the size of

207
00:09:53,152 --> 00:09:55,877
individual peaks that correspond to alleles. We thus

208
00:09:55,877 --> 00:09:58,201
obtain the genotypes of individual

209
00:09:58,201 --> 00:10:00,925
microsatellites of the individual, the

210
00:10:00,925 --> 00:10:03,490
so-called genetic type. By

211
00:10:03,490 --> 00:10:05,974
comparing with parents, we can

212
00:10:05,974 --> 00:10:08,899
verify parentage or by comparing

213
00:10:08,899 --> 00:10:11,743
with another sample we can identify

214
00:10:11,823 --> 00:10:12,945
the individual.

215
00:10:15,429 --> 00:10:17,793
And that's all for this short

216
00:10:17,793 --> 00:10:19,997
presentation explaining genetics

217
00:10:19,997 --> 00:10:22,321
parentage testing in animals.

218
00:10:23,042 --> 00:10:24,645
Thank you for your attention.