The use of microsatellite genotyping for population studies in the pig with individual and pooled DNA samples

Abstract

In the present paper the results obtained from the genotyping of DNA microsatellite markers in 60 populations of European Pig are shown. The genotypings have been performed on individual and pooled DNA samples, standing out the technical efficiency of both methods in the characterisation of the European pig biodiversity. RESUMEN En el presente trabajo se muestran los resul-tados obtenidos del genotipado de marcadores microsatélites del ADN en 60 poblaciones de cerdos europeos. Estos genotipados han sido desarrollados sobre muestras de ADN individual y mezclado, destacándose la eficiencia técnica de ambos métodos para la caracterización de la biodiversidad de los cerdos europeos.

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M.A.M. Groenen, R. Joosten, M.-Y. Boscher, Y. Amigues, B. Harlizius, J.J. van der Poel, R. Crooijmans
The use of microsatellite genotyping for population studies in the pig with individual and pooled dna samples
Archivos de Zootecnia, vol. 52, núm. 198, 2003, pp. 145-155,
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Arch. Zootec. 52: 145-155. 2003.
THE USE OF MICROSATELLITE GENOTYPING FOR POPULATION
STUDIES IN THE PIG WITH INDIVIDUAL AND POOLED DNA
SAMPLES
EL USO DEL GENOTIPADO DE MICROSATÉLITES PARA ESTUDIOS DE POBLACIONES
EN EL CERDO CON MUESTRAS DE ADN INDIVIDUAL Y MEZCLADO
Groenen, M.A.M.1, R. Joosten1, M-Y Boscher2, Y. Amigues2, A. Rattink1, B. Harlizius1,
J.J. van der Poel1 and R. Crooijmans1
1Animal Breeding and Genetics group. Wageningen University. Marijkeweg 40. 6709 PG Wageningen. The
Netherlands.
2Laboratoire d'analyses génétiques pour les espèces animales (LABOGENA). Domaine de Vilvert. Jouy
en Josas. 78352 cedex. France.
ADDITIONAL KEYWORDS
Molecular genetics. Genotyping. Characterisation.
PALABRAS CLAVE ADICIONALES
Genética molecular. Genotipado. Caracterización.
SUMMARY
In the present paper the results obtained from
the genotyping of DNA microsatellite markers in
60 populations of European Pig are shown. The
genotypings have been performed on individual
and pooled DNA samples, standing out the
technical efficiency of both methods in the
characterisation of the European pig biodiversity.
RESUMEN
En el presente trabajo se muestran los resul-
tados obtenidos del genotipado de marcadores
microsatélites del ADN en 60 poblaciones de
cerdos europeos. Estos genotipados han sido
desarrollados sobre muestras de ADN individual
y mezclado, destacándose la eficiencia técnica
de ambos métodos para la caracterización de la
biodiversidad de los cerdos europeos.
INTRODUCTION
Quantitative assessment of genetic
diversity within and between popu-
lations is an important tool for decision
making in genetic conservation plans.
The most widely used method to
quantify this genetic diversity is by
genotyping a selection of unrelated
individuals from the populations under
investigation. In principle any marker
for which there has been described
genetic variation can be used for such
studies. In earlier studies markers that
were commonly used were mainly
based on blood groups and easily
identifiable enzymes in the blood (Van
Zeveren et al., 1990; Rohrer et al.,
1997). However, for technical reasons,
these markers were not very well suited
for large-scale population studies with

Archivos de zootecnia vol. 52, núm. 198, p. 146.
GROENEN
ET AL.
large numbers of markers and indivi-
duals. The development of DNA based
markers in the last two decades has
revolutionised the possibilities to moni-
tor genetic diversity of populations by
making it feasible to screen large
numbers in a relatively short time. One
type of marker that has been intensely
used for population studies in the last
10 years, are the so-called microsate-
llite or single sequence repeat markers.
These markers are very abundant,
show a high degree of polymorphism
and can be analysed by means of PCR,
enabling a high degree of automation
of the genotyping analysis. In pigs
microsatellites have already been used
in a number of studies to address the
biodiversity in commercial as well as
rare breeds (Van Zeveren et al., 1995;
Martínez et al., 2000; Laval et al.,
2000).
MATERIAL AND METHODS
TYPING PROTOCOL FOR INDIVIDUAL GENO-
TYPING OF MICROSATELLITES
The PCR reactions were performed
in a total volume of 12 ml containing 80
ng of genomic DNA, 1.5 mM MgCl2,
50 mM KCl, 10 mM Tris.HCl pH=8.3,
1 mM Tetramethylammoniumchloride
(TMAC), 0.1 percent triton X-100, 0.01
percent gelatin, 200 mM dNTP, 0.25
Unit Goldstar polymerase (Eurogentec
S.A., Belgium), 2.3 pmoles of each
primer and covered with 10 ml of mine-
ral oil (Sigma). The PCR protocol was
as follows: 5 min denaturation at 95°C;
36 cycles of [30", 95°C/30", 50-60°C/
30", 72°C] finally followed by 2 minutes
at 72°C. PCR reactions for the
different microsatellite markers were
pooled and a mixture of up to 1ml
pooled PCR products was added to 3.2
ml loading buffer (which contained the
GENESCAN-350 TAMRA internal
standard and formamide; final concen-
tration of 80 percent). The sample was
denatured at 95°C and loaded on a 6
percent denaturing polyacrylamide gel
(sequagel 6; National Diagnostics) on
an ABI 373A sequencing machine (12
cm well-to-read; loading 4 ml). PCR
products of different markers from
DNA of the same animal were pooled
in such a way that each marker signal
on the ABI automated sequencers has
a peak height of about 1000. The
fragment sizes were calculated relative
to the GENESCAN-350 TAMRA with
the GENESCAN fragment analysis
software (Perkin Elmer, Applied
Biosystems Division). Genotyping was
performed using the Genotyper 2.0
software.
TYPING PROTOCOL FOR GENOTYPING OF
MICROSATELLITES ON POOLED DNA
SAMPLES
Genotyping of pooled DNA was
performed essentially as described for
individual typings with the following
modifications:
(1)Microsatellite markers were
pooled in such a way to avoid overlap
of alleles, even if markers differed for
the fluorescent dye. This means that in
general 3 or 4 markers are analysed on
the same gel.
(2)Longer gels (36 cm well-to-read
distance) in combination with a
different gel matrix (long ranger 5.75
percent acrylamide, 7 M Urea) were
used to increase the resolution of the
individual peaks.
(3)In stead of allele frequencies,

Archivos de zootecnia vol. 52, núm. 198, p. 147.
MICROSATELITE GENOTYPING OF PIG POPULATIONS
peak frequencies were calculated
based on the area under the peaks
(Using the GENOTYPER 2.0 soft-
ware). Previous results on the analysis
of pooled chicken DNA revealed that
it was more reliable to use peak
frequencies, rather then to correct for
stutter bands.
RESULTS AN DISCUSSION
MICROSATELLITE MARKER PANEL SELECTED
FOR GENOTYPING
Previously we have described the
selection of 27 microsatellites to be
used as a standard panel for population
studies in the pig (Laval et al., 2000).
These markers were chosen based on
their quality, size, polymorphism and
location on the porcine genome
(Archibald et al., 1995; Rohrer et al.,
1994, 1996) as proposed by the FAO
(Barker et al., 1998). Quality was
based mainly on the absence of any
known null alleles, the sharpness of the
peaks on automatic sequencers and
robustness of the amplification
reaction. Markers were chosen to
maximise the genome coverage
provided by these 27 markers. All pig
chromosomes, except chromosome 18
were represented by this marker set.
Furthermore, large chromosomes often
were represented by two different
markers. When two markers were on
the same chromosome they were
chosen at a minimum distance of at
least 30 cM. The third criterion used
for the selection of the markers was
the size of the amplified product and
the fluorescent label of the amplification
product. This eventually enabled us to
design 3 different sets with 9 markers
each that could be used for multiloading
and simultaneous analysis on ABI
automatic sequencers. To avoid overlap
between adjacent markers labelled with
the same fluorescent dye, markers were
combined in such a way that the size
difference between the smallest allele
of the larger marker was at least 30 bp
longer then the largest allele of the
smaller marker. The 27 markers and
their distribution over these three sets
are shown in table I (set I-III).
Within the EU PigBioDiv project,
we decided to increase the coverage
of the markers across the pig genome.
Eventually 23 markers were added to
the original selection of 27 markers,
resulting in a total of 50 markers divided
over 6 sets that could be analysed
simultaneously on ABI automatic
sequencers (see table I). Although
the same criteria were applied for
selecting the markers as those that
were used for selecting set I to III, it
was no longer possible to use the
criterion for a minimum distance of 30
cM between the markers. Never-
theless, the majority of the markers
were located at least 20 cM apart.
MICROSATELLITE GENOTYPING ON INDIVI-
DUAL SAMPLES
As previously observed in the EU
funded PigMaP pilot project on
diversity, microsatellite genotyping
must be organised so that each marker
is typed in only one laboratory, to avoid
problems in allele calling. This is
particularly true if the analysis is
performed on gel based systems such
as on the ABI373 and ABI377. As a
consequence, it is recommended that
when data from different facilities must
be combined, each facility genotypes

Archivos de zootecnia vol. 52, núm. 198, p. 148.
GROENEN
ET AL.
Table I. Pig microsatellite markers selected for populations studies. The number of alleles
and the allele size range are based upon the PiGMaP (Archibald et al., 1995) and USDA
(Rohrer et al., 1996) reference populations. Distance in cM indicates the distance between
that particular marker and the preceding marker in the table. Set refers to the markers that
are analysed simultaneously on ABI automatic sequencers. (Marcadores microsatélites de cerdo
seleccionados para los estudios de poblaciones. El número de alelos y el rango de tamaño de los alelos
se basan en las poblaciones de referencia del PIGMAP (Archibald
et al.
, 1995) y el USDA (Rohrer
et
al.
, 1996). Las distancias en cM indican las distancias entre ese marcador particular y el marcador
precedente en la tabla. Las series se refieren a los marcadores que son analizados simultáneamente
con secuenciadores automáticos ABI).
Marker Chr arm Set #Alleles Allele size range Distance1 (cM)
Min Max
CGA 1p I 12 250 320 -
S0155 1q III 6 150 166 45
SW1828 1q VI 6 95 105 24
SW240 2p III 8 96 115 -
S0226 2q II 9 181 205 20
SW72 3p III 8 100 116 -
SW902 3q IV 7 195 214 40
S0002 3q III 7 190 216 45
S0227 4p II 10 231 256 -
S0301 4p V 6 254 266 20
S0217 4q IV 5 145 165 45
S0097 4q IV 8 208 248 50
S0005 5q III 10 205 248 -
IGF1 5q III 7 197 209 30
SW2406 6p V 7 220 256 -
SW1067 6q VI 7 144 175 50
SW122 6q II 10 110 122 15
S0228 6q II 12 222 249 20
S0025 7p IV 9 104 120 -
SW632 7q I 9 159 180 100
S0101 7q I 6 197 216 30
SW2410 8p VI 9 104 120 -
S0225 8q II 8 170 196 82
S0178 8q II 4 110 124 45

Archivos de zootecnia vol. 52, núm. 198, p. 149.
MICROSATELITE GENOTYPING OF PIG POPULATIONS
Table I . Pig microsatellite markers selected for populations studies. (Marcadores microsatélites
de cerdo seleccionados para los estudios de poblaciones). (continuación).
Marker Chr arm Set #Alleles Allele size range Distance1 (cM)
Min Max
SW911 9p I 9 153 177 -
SW174 9q IV 5 123 133 90
SW830 10p VI 7 176 205 -
S0070 10q IV 7 265 299 60
SW951 10q II 5 125 133 35
SW2008 11p IV 6 95 105 -
S0386 11q III 10 156 174 55
S0143 12p VI 5 148 166 -
S0090 12q II 4 244 251 75
SWR1941 13 VI 7 202 223 -
S0068 13 I 10 211 260 45
SW769213 VI 7 106 140 55
S0215 13 I 9 135 169 5
SW857 14 III 6 144 160 -
SW295 14 V 8 109 139 30
S0355 15 I 14 243 277 -
SW1111 15 V 6 165 183 25
SW936 15 I 13 80 117 50
SW742 16 V 9 193 224 -
S0026 16 III 8 92 106 30
SWR1004217 V 5 147 167 -
SW24 17 I 8 96 121 5
SW1023 18 V 5 84 117 -
SW787 18 IV 8 153 165 25
SW2476 Xq V 5 88 106 -
S0218 Xq II 8 164 184 35
1Distance on the USDA map.
2Marker SW769 (Chr 13) and SWR1004 (Chr 17) are included though they are both located at a distance
of only 5cM from the next marker.

Archivos de zootecnia vol. 52, núm. 198, p. 150.
GROENEN
ET AL.
the same individuals for different
markers. It is possible that the increased
standardisation on the capillary based
sequencers (ABI 3100, 3700, 3730)
will allow for an easier transferability
of the data across different laboratories,
eliminating the need to take this
approach in the future.
Individual genotyping has been done
for 60 breeds (considering the French
and British samples of Meishan as
two different breeds) for all 50 markers
described in table I. Genotyping of
sets I, II, III and VI was performed at
Labogena on an ABI3700, whereas
genotyping of sets IV and V was done
at the Wageningen University (WU)
on an ABI373 sequencer. Although
overall failure rates vary slightly
between the different typing labora-
tories this seems to be somewhat higher
at WU. The data shown in figure 1
suggest that the DNA quality is the
major factor influencing the success
Figure 1. Overview of the failure rates for the populations typed by Labogena (sets I, II, III
and VI) and Wageningen University (sets IV and V). The percentage of the animals that were
not genotyped is indicated on the y-axis. Populations are grouped along the x-axis according
to the participant responsible for the blood collection and DNA isolation. (Una revisión de las
tasas de fallo para las poblaciones tipificadas en Labogena (series I,II,III y VI) y la Universidad de
Wageningen (Series IV y V). El porcentaje de animales que no fueron tipificados están representados
en el eje de las Y. Las poblaciones se agrupan a lo largo del eje de las X de acuerdo al participante
responsable del muestreo de sangre y el aislamiento de ADN).
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
Set 4, 5
Set 1,2, 3, 6

Archivos de zootecnia vol. 52, núm. 198, p. 151.
MICROSATELITE GENOTYPING OF PIG POPULATIONS
rate for typing as this is very dependent
upon the origin of the samples. This is
also in agreement with the DNA typing
results using AFLP (see Plastow et
al., 2003, in this Proceeding). For
example, the average failure rate of
the genotypes for sets IV and V was
14.9 percent but for individual
populations this varied from 0.1 percent
for the Italian Duroc line 1 (ITSSDU01)
to 65.3 percent for the Spanish Negro
Canario (ESSSNC01). This clearly
demonstrates the key importance of
the procedures for collecting, handling
and storing of the blood and isolation of
the DNA. Based on other projects the
most critical factor is the collection of
the blood. We also observed clear
differences in the performance of the
different markers used, in particular
those in sets IV to VI. As has been
outlined above, the markers with the
highest quality had already been used
for sets I to III and it was known that
the overall quality of sets IV to VI was
somewhat lower. These factors pro-
bably are responsible for the difference
seen between the two laboratories.
For the markers in sets IV and V,
failure rates varied from 6.5 percent
for SW2008 to 32 percent for S0301.
However, it has to be realised that
these numbers are slightly biased
because the failure rate is also
influenced by the quality of the DNA
samples and markers that are less
robust are more susceptible to poor
DNA quality.
MICROSATELLITE GENOTYPING ON DNA
POOLS
Genotyping of microsatellites on
pooled samples will result in a drastic
reduction in the number of genotypes
that have to be performed and thus in
a concomitant reduction in time and
money involved. DNA typing in pools
has been successfully applied in chicken
(Khatib et al., 1995; Crooijmans et al.,
1996) and human (Pacek et al., 1993;
LeDuc et al., 1995) but had not yet
been tested in pigs. Within the EU
PigBioDiv project we tested whether
it also would be feasible to use this
approach for pig microsatellites. For
this approach, DNA from a number of
animals (1 population) is combined and
genotyping is performed on the pooled
sample. The PCR signal of the indivi-
dual alleles is directly correlated with
the frequency of that particular allele.
The frequency of a particular allele is
calculated based upon the area under
the peak of that allele relative to the
total of the area under the peak for all
alleles.
An aliquot of 200 ng of DNA from
all the individual animals from each of
the breeds was combined and analysed
as a pool with all the 50 microsatellites
of set I to VI. PCR reactions for the
pools were essentially the same as for
individual typings, with a total of 80 ng
of the pooled DNA per reaction. Pre-
viously, it was observed that abundant
alleles (high signal) can result in some
read through in the other dyes
complicating the analysis of the other
markers. Therefore, for the pooled
analysis, the 50 markers were combined
into 15 sets containing 3 to 4 markers
to avoid overlap of alleles of different
markers. The total number of breeds
analysed using the pools was 72, since
in addition to the 60 original breeds it
included one Italian breed (MR01) and
11 breeds that were used previously in
the PiGMaP pilot project (Laval et al.,

Archivos de zootecnia vol. 52, núm. 198, p. 152.
GROENEN
ET AL.
2000). Although all 50 markers were
analysed on the pooled samples,
eventually, we were only able to
generate useful genotyping data for 20
microsatellites. This in part is due to
the problems with the quality of the
DNA for some of the samples but the
main reason is the fact that the markers
in sets I to VI were selected for
genotyping on individual samples. For
genotyping of microsatellite markers
on pooled DNA samples, the markers
have to fulfil additional criteria
(Crooijmans et al., 1996) which are
not met by the majority of the markers
in sets I to VI. In particular the degree
of stuttering, the sharpness of the peaks
on the automatic sequencer and the
absence of alleles that differ only by 1
bp are additional important criteria for
using pooled DNA samples. If these
criteria are not met, it is often not
possible to identify individual alleles
and the allele frequency based on the
area under the peak is not calculated
correctly. In figure 2 and 3 respectively
examples are shown of markers that
are suitable (S0217 and SW787) and a
marker that is not suitable (S0101) for
analysis on DNA pools. In figure 3,
the genotyping results are shown for
two populations with similar allele
Figure 2. Example of typing results on DNA pools for successful microsatellite markers. The
results for two populations for the markers S0217 and Sw787 are shown. (Ejemplo de los
resultados de tipificación sobre los pools de ADN para los marcadores microsatélites satisfactorios.
Se muestran los resultados de dos poblaciones para los marcadores S0217 y Sw787).
Set 1,2,
3, 6
Sw7
S0217 SW787

Archivos de zootecnia vol. 52, núm. 198, p. 153.
MICROSATELITE GENOTYPING OF PIG POPULATIONS
frequencies. However, in one of the
populations shown, the peaks for alleles
210 and 212 are not recognised as
Figure 3. Example of typing results on DNA pools for an unsuccessful marker (S0101). The
arrows indicate alleles that are not correctly identified in the population at the top. (Ejemplo
de los resultados de tipificación para un marcador insatisfactorio (S0101). Las flechas, en la parte
superior, muestran los alelos que no son correctamente identificados en la población).
S0101
individual peaks by the GENOTYPER
software and as a result these are
included in the area for the peaks for

Archivos de zootecnia vol. 52, núm. 198, p. 154.
GROENEN
ET AL.
Table II. Pig microsatellites typed on pooled
DNA samples. The number of populations
that could be analysed successfully for these
markers is given. The total number of
populations genotyped was 72, considering
the French and British samples of Meishan
as two different populations. (Microsatélites
de cerdo tipificados sobre muestras mezcladas
de ADN. Se ofrece el número de poblaciones que
pudieron ser analizadas exitosamente con esos
marcadores. El número total de poblaciones
genotipadas fue 72, considerando las muestras
francesas e inglesas de Meishan como dos
poblaciones diferentes).
Marker Populations Analysed
SW911 69
S0002 58
S0070 62
S0090 61
S0097 56
S0217 56
S0218 52
S0227 68
SW1828 59
SW2008 68
SW2406 55
SW2476 51
SW72 67
SW787 67
SW830 67
SW857 62
SW902 66
SW936 68
SW951 68
SWR1004 49
alleles 211 and 213.
Table II gives an overview of the
markers for which it was possible to
analyse the majority of the populations.
In total 85 percent of the population-
marker combinations could be analysed
successfully for these 20 microsate-
llites.
Taking all of the results together,
the main outcome of the genotyping on
pooled DNA samples is that this method
seems to be feasible, but that it requires
a selection of a different (sub) set of
microsatellites. Furthermore, it is to be
expected that the use of capillary
sequencers rather then gel based
sequencers will further improve the
resolution, and thus will make genoty-
ping on pooled samples a feasible cost-
effective alternative for an initial scan
of the populations.
ACKNOWLEDGEMENTS
We acknowledge Dennis Milan for
his help in defining the panel of the 23
additional microsatellites and Louis
Ollivier and Graham Plastow for
critically reading the manuscript. This
research was financially supported by
the European Union (contract Bio4-
CT98-0188).
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