Microsatellite and SNP analysis for parentage verification
using bovine nasal samples with Performagene™
E.J. Maclean, J.O. Niles, C.M. James and R.M. Iwasiow
DNA Genotek Inc., Ottawa, Canada
Introduction

Results

Parentage analysis of livestock animals is becoming increasingly utilized in
the agriculture industry to improve the accuracy of herd books and to selectbreed crosses that increase production traits and herd efficiency. To date,the international standard of parentage analysis is done using microsattelites(ISAG Conference, 2008), however, parentage can also be determined using
SNP genotyping (Heaton et al, 2002). Although both the microsattelite and SNP
genotyping method have been shown to be effective for parentage analysis,
the benefit of analyzing parentage using SNP genotyping is that additional
SNP’s for production traits can be analyzed in parallel. Obtaining both parentage
and trait information through a single sample and test offers producers more
information and decreases the cost of genetic based breeding decisions.

Table 1: Quantification results for 59 nasal samples collected for the Illumina
Bovine3K analysis and the Illumina BovineSNP50 BeadChip analysisAs the use of parentage analysis transitions from microsattelite testing to morecost effective and informative SNP genotyping, the total use of genomics isincreasing. With growing adoption of genetic testing, the demand for efficientand reliable solutions for DNA sample collection and processing is increasing.
Performagene is an all-in-one solution for the collection, stabilization andextraction of DNA from nasal samples. This method enables more efficientparentage analysis by streamlining sample collection, transport and processing.
Using the Performagene DNA collection kit developed by DNA Genotek, Inc.,
we demonstrate the reliable collection and processing of high yield, highquality genomic DNA from cattle nasal samples. Nasal samples were collectedfrom a total of 349 purebred Holstein, Jersey, Limousin and Charolais cattle
during routine parentage analysis. Microsattelite analysis was conductedon the nasal samples using the ABI PRISM® 3100 Genetic Analyzer. The results
demonstrate the suitability and reliability of Performagene nasal samples foruse in parentage analysis in the existing ISAG standard. SNP genotyping wasperformed using the Illumina BovineSNP50TM BeadChip and Bovine3KTM assay
shows the reliability of Performagene nasal samples for use in a combinedparentage and production trait analysis.

Sample ID
1234
5
678
910111213
1415
1617
18
1920
21

Sample ID
222324
25
26
27
28
2930
31
32
33
34
35
36
37
38
39
40
41
42

DNA yield per 1 mL ofsample (µg)
71.64
15.03
7.29
6.79
4.01
11.61
2.75
2.71
3.05
8.73
2.29
21.18
10.12
24.75
11.90
2.70
4.83
4.51
16.85
3.58
3.40

Sample ID
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
AverageMedianMaxMinDNA yield per 1 mL ofsample (µg)
10.58
1.24
9.71
22.02
3.27
37.59
19.97
19.72
16.57
22.24
10.79
14.33
5.96
38.22
9.94
1.68
12.92
30.79
17.49
155.401.24

Figure 2: Representative gel of 16 nasal samples. 50 ng of total DNA was loaded
into a 0.8% agarose gel and run at 80 volts for 45 minutes

23.1 kb

Table 2: Summary of call rate results from SNP Genotyping and Microsattelite analysis
Assay

Processing method (nasal)

Average call rate (nasal)

Illumina Bovine3K

Full purification

98.19%

Illumina Bovine SNP50™ BeadChip

Full purification

99.63%

Microsatellite Analysis ABI PRISM Genetic Analyzer

Quick to PCR

97.15%

Material and methods
Figure 1: Nasal sample collection using Performagene

DNA yield per 1 mL ofsample (µg)
28.67
97.61
87.59
101.72
17.97
39.24
58.99
31.56
59.40
30.82
144.82
69.53
47.52
45.39
17.49
83.44
37.63
155.40
35.63
46.81
83.14

1

5 sec
1 No calls were not included in the calculation of call rates (6 samples)

DO NOT
SPILL

Figure 3: Representative electropherogram from ABI PRISM® Genetic Analyzer

The following data is based on a total of 349 nasal samples collected according
to the DNA Genotek protocol PD-PR-099 and stored at room temperature until
processed.
SNP analysis with Illumina Bovine3K• 21 nasal samples were collected by 2 individuals from Holstein cattle.
• 0.5 mL aliquot of the nasal samples was purified according to the DNA Genotek
purification protocol PD-PR-083 and the purified DNA was quantified byfluorescence, evaluated by agarose gel electrophoresis, and analysed on theIllumina Bovine3K assay.
SNP analysis with Illumina BovineSNP50 BeadChip:• 38 nasal samples were collected by 7 individuals and were collected from thefollowing breeds of cattle: Holstein, Jersey, Limousin and Charolais.
• 0.5 mL aliquot of the nasal samples was purified according to the DNA Genotek
purification protocol PD-PR-083 and the purified DNA was quantified byfluorescence, evaluated by agarose gel electrophoresis, and analysed on theIllumina BovineSNP50 BeadChip.
Microsatellite analysis with ABI PRISM Genetic Analyzer:• 290 nasal samples were collected by Holstein UK members from Holstein cattle.
• 100 µL aliquot of the nasal sample was processed according the DNA Genotek
purification protocol PD-PR-00220 and using Bovine Genotype™ Panel 1.2 and2.2 (Finnzyme) with ABI PRISM 3100 Genetic Analyzer.

Discussion• Performagene provides a quick and easy-to-use method for collecting large
amounts of high quality DNA from cattle.
• Performagene facilitates the transition of parentage analysis from microsatteliteto SNP genotyping by performing reliably on both methods as illustrated by thehigh call rates.
• Samples collected using Performagene can be used with either microsattelite or
SNP analysis for accurate parentage determination.

DNA Genotek would like to extend their appreciation to Holstein UK and Weatherby’s for assisting in the collection and analysis of the Microsatellite data
presented in this poster. DNA Genotek would also like to thank Illumina for conducting and providing the Bovine3K data analysis.

Superior samples
Proven performance
Performagene™ is a trademark of DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc.
All other brands and names contained herein are the property of their respective owners.

Patent (www.dnagenotek.com/legalnotices)
MK-00195 Issue 1/2013-05