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


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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,
Universidad de Córdoba
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Archivos de Zootecnia,
ISSN (Printed Version): 0004-0592
Universidad de Córdoba
Non-Profit Academic Project, developed under the Open Acces Initiative
Arch. Zootec. 52: 145-155. 2003.
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
2Laboratoire d'analyses génétiques pour les espèces animales (LABOGENA). Domaine de Vilvert. Jouy
en Josas. 78352 cedex. France.
Molecular genetics. Genotyping. Characterisation.
Genética molecular. Genotipado. Caracterización.
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.
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.
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.
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.,
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
Genotyping of pooled DNA was
performed essentially as described for
individual typings with the following
(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.
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.
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.
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.
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
, 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.
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.
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).
Set 4, 5
Set 1,2, 3, 6
Archivos de zootecnia vol. 52, núm. 198, p. 151.
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.
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
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.
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
S0217 SW787
Archivos de zootecnia vol. 52, núm. 198, p. 153.
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).
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.
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-
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.
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-
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... Several meaningful population statistics and comparisons can be deduced from allele frequency data. Although individual genotyping is the most accurate method to estimate allele frequencies in populations, it has been shown that pooled sampling methods [9,16,20,[42][43][44], and references therein can perform very adequately as well, at a fraction of the cost of labor and consumables. ...
... DNA was isolated from individual samples using standard phenol-chloroform protocols [32]. Pools of DNA were made by adding equal amounts of DNA for each of the individuals to a single vial for each of the lines [9,16]. ...
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Full-text available
Microsatellite diversity in European and Chinese pigs was assessed using a pooled sampling method on 52 European and 46 Chinese pig populations. A Neighbor Joining analysis on genetic distances revealed that European breeds were grouped together and showed little evidence for geographic structure, although a southern European and English group could tentatively be assigned. Populations from international breeds formed breed specific clusters. The Chinese breeds formed a second major group, with the Sino-European synthetic Tia Meslan in-between the two large clusters. Within Chinese breeds, in contrast to the European pigs, a large degree of geographic structure was noted, in line with previous classification schemes for Chinese pigs that were based on morphology and geography. The Northern Chinese breeds were most similar to the European breeds. Although some overlap exists, Chinese breeds showed a higher average degree of heterozygosity and genetic distance compared to European ones. Between breed diversity was even more pronounced and was the highest in the Central Chinese pigs, reflecting the geographically central position in China. Comparing correlations between genetic distance and heterozygosity revealed that China and Europe represent different domestication or breed formation processes. A likely cause is a more diverse wild boar population in Asia, but various other possible contributing factors are discussed.
... These repeated sequences that comprise microsatellites, of which the most common and studied is the repeat (CA) n / (GT) n, are flanked by other sequences of nucleotides known and named by primers. Thus, microsatellites are easily identified and used as markers, making them easy to amplify by PCR (Groenen et al., 2003). ...
... The microsatellites are shown to be well conserved among closely related species and the primers used to amplify a given sequence sometimes can also amplify analogous sequences in other species. This type of molecular marker demonstrates good reproducibility, with high precision and with low efforts of genotyping individuals in a population with relatively easy detection techniques, analysis, and automation, in addition to the high polymorphism displayed, providing a lot of information (Vignal et al. 2002;Martinez, 2001;Groenen et al., 2003;Yang et al., 2013). ...
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An in-depth study of characterization and evaluation of selection strategies in the Lusitano horse breed was conducted to identify factors affecting the genetic variability of the breed and provide baseline information for the establishment of a sustainable genetic improvement program. Pedigree records collected in 53417 animals born from 1824 to 2009 were used. The mean generation interval was 11.33±5.23 and 9.71±4.48 years for sires and dams, respectively. For animals born between 2005 and 2009, the mean number of equivalent generations was 11.20±0.71 and the average inbreeding was 11.34±7.48%. The rate of inbreeding per year was 0.173±0.070, and the corresponding effective population size was about 28. The effective number of founders, ancestors and studs was 27.5, 11.7 and 5.4, respectively. These results reflect a strong emphasis placed on a few sire-families and raise concerns regarding the conservation of genetic diversity for the future. Mixed model procedures were used to estimate genetic parameters, fixed effects and genetic trends for morpho-functional traits in Lusitano horses by uni- and multivariate animal models. Morphological traits included were partial scores attributed to more than 18000 horses at the time of registration in the studbook and included the classification of head/neck, shoulder/withers, chest/thorax, back/loin, croup, legs and overall impression, plus a final score (FS) and a score for gaits (GA) and the measurement of height at withers (HW). For functionality, the traits considered were scores obtained in dressage (WEDT) and maneability (WEMT) trials of working equitation (WE, about 1500 records by 200 horses), and classical dressage (CD, about 12130 records by nearly 760 horses). Fixed effects considered in the analyses of morphology, GA and FS were stud, year, sex, inbreeding and age. For functionally traits, the fixed effects were event, level of competition, sex, inbreeding and age. Heritability (h2) estimates for all partial morphological scores ranged between 0.12 and 0.18, except for legs (0.07), and were 0.18 for FS, 0.61 for HW and 0.17 for GA. For performance, h2 was 0.32 for WEDT and CD and 0.18 for WEMT. The genetic correlations among partial components of morphology were positive but widely different (0.08 to 0.77). The favourable genetic relationships existing between morphology and performance indicate that morphology and gaits traits can be used to enhance selection response when the improvement of WE or CD is intended. The magnitude of inbreeding depression was small for all the traits analyzed. The estimated breeding values for morphology, gaits and WE presented a large variability, indicating that selection can be effective, but the genetic trend observed over the last few years was positive but moderate for all traits. The assessment of genetic diversity and population structure obtained by either pedigree data or microsatellite markers was compared. The same pedigree database was used and, in addition, data on either 6 or 8 microsatellite markers genotyped in more than 19000 horses, from 1998-2007. Genealogical inbreeding was a better predictor of molecular inbreeding than the opposite, but it had a modest correlation with multilocus heterozygosity (6% of its variability). Still, the rates of inbreeding per generation estimated by the two methods were very similar. Genetic distances among the major studs producing Lusitano horses were comparable when they were estimated from pedigree or molecular information, with a correlation between FST distances of 0.82, and similar dendrograms were obtained in both cases. Overall, estimates derived from a reduced number of microsatellites or from pedigrees are poorly correlated when considered at the individual level, but parameters derived from pedigree are better predictors of molecular-derived indicators. However, when considered at the breed-level, the estimated diversity parameters, time trends and population substructure are very similar when genealogical data or microsatellite markers are considered.
... This was achieved by sampling 50 individuals from different breeds and lines, and determining diversity at DNA level. The emphasis was on standard DNA marker technologies, such as simple sequence repeat (so-called MS) and amplification of fragment length polymorphism (AFLP), and on the use of high throughput genotyping devices (for details of the project see: Groenen et al., 2003;Ollivier et al., 2003;Plastow et al., 2003). The essential results can be found in SanCristobal et al. (2006a) for MS, SanCristobal et al. (2006b) and for AFLP, and for an overall analysis of genetic diversity, cumulating MS (PiG-MaP and PigBioDiv breeds) and AFLP (PigBioDiv breeds only) information. ...
... Detecting significant LD therefore needs narrowly spaced genetic markers, not available until recently. With an average map distance of 35 cM between neighbouring markers among the 50 MS selected in PigBioDiv (see Table 1 in Groenen et al., 2003), the MS data collected in this study could not be expected to allow any precise evaluation of LD extent in the pig. One of the earliest studies of LD in pigs actually used 15 MS, spaced 5 cM on average, and was able to show significant LDs on two pig chromosomes (Nsengimana et al., 2004). ...
The second edition of this book contains chapters that discuss modern pig genetics, including taxonomy and evolution, domestication, coat colour variation, morphological traits and hereditary diseases, immunogenetics, cytogenetics, genomics, behaviour genetics, reproduction, transgenics, developmental genetics, pig genetic resources, performance traits, carcass and meat quality genetics, genetic improvement, pigs as models for biomedical sciences, pig breeds and genetic nomenclature. This book is intended for those who study or work with pigs.
... Este tipo de marcador molecular apresenta boa reprodutibilidade, com elevada precisão e com baixos esforços de genotipagem dos indivíduos numa população, com técnicas relativamente fáceis de detecção, análise e automação, para além do alto polimorfismo exibido, fornecendo muita informação (Vignal et al., 2002;Pires e Ginja, 2004;Groenen et al., 2003). ...
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A study was conducted with the Malhado de Alcobaça (MA) swine population, aiming at its morphologic, demographic, productive and genetic characterization, as a basis for the possible recognition (or not) of this population as a distinct breed. The MA population, historically known as Porco Sintrão, Torrejano, da Granja, da terra, etc., is currently reduced to a single herd (Selecpor, SA.), with 170 sows and 11 boars. The MA animals have a spotted colour, concave head shape, long ears, elongated body shape and tall limbs. For morphologic characterization, 14 different measurements of height, width and perimeter were collected in 50 sows and 10 boars, based on standard recommendations, and from those, different zoometric indexes were calculated. Average mature weight was 212±24 kg for females and 248±25 kg for males. Correlations between different measurements were calculated, and an equation for prediction of mature weight in MA pigs was developed, based on the 7 more significant variables (r2 = 0,90). When compared with those collected in Bísaro pigs, several zoometric variables were significantly different. For productive characterization, individual and pedigree records collected in the farm over 16 years were used. The full data base included records on about 70 boars and 1100 sows, with approximately 3000 farrowing records. Overall mean values for number of pigs born alive and weaned/litter were 9,1±2,6 and 8,3±2,2, respectively, while the means for gestation length, age at first farrowing and longevity were 116±1,4 days, 15,0±2,5 months and 2,0±1,5 years, respectively. Heritability estimates were 0,05 for litter size and 0,10 for number weaned/litter. The average number of generations known in this population is approximately 4, and average inbreeding is 8,8% for pigs born in 2003-2004, with a rate of inbreeding/year of 0,76%. About 50% of the current genetic pool is accounted for by the genetic contributions of only 5 founder animals. For genetic characterization, a set of 27 polymorphic genetic markers (microsatellites), recommended by FAO/ISAG for studies on swine genetic diversity, were used. A sample of 249 animals from 7 populations (MA, Bísara, Alentejana, Duroc, Landrace, Pietrain and Large White) was used, with 23-50 animals represented per breed. DNA extraction was performed with Chelex® and Proteinase-K, followed by 3 PCR multiplexes, each with 9 markers, using fluorescence-labelled primers. The PCR products were analysed by capillary electrophoresis (ABI310 sequencer), followed by interpretation of results with Genescan and Genotyper. For statistical analyses, standard population genetics procedures were followed, using the Genetix, Cervus, Genepop and Populations packages. Of the 27 microsatellite loci studied, two were discarded from the analyses because of poor amplification and reading. The total number of alleles detected for the 25 microsatellite loci in the seven populations was 261, with a range per locus between 6 (S0090) and 20 (S0005), while the average number of alleles (NMA) per marker ranged between 3,17 (S0227) and 8,33 (S0068). For the Duroc breed, locus S0101 was monomorphic. Average observed and expected heterozygosities for the set of markers used were 0,621 and 0,667, respectively. Among populations, NMA ranged between 3,77 (Duroc) and 7,18 (Landrace), with observed heterozygosities ranging between 0,468 (Duroc) and 0,645 (Landrace). The mean PIC for the set of markers used was 0,685 and, in all populations, several loci were not in Hardy-Weinberg equilibrium. The degree of genetic differentiation among populations (GST) was 0,1844. Fixation indexes, calculated based on 22 markers, were 0,0673 for FIS, 0,2393 for FIT and 0,1844 for FST, with the highest FIS values observed in the Alentejana and Bísara breeds. Genetic distances were estimated among populations (DS, DRey e DA) and individuals (DAS), for construction of phylogenetic trees, using the Neighbour-Joining methodology. The largest DS was observed between MA and Duroc (0.7616) and the smallest between MA and Landrace (0,2313). For the different distances calculated, the Duroc and Alentejana were consistently separated from the other breeds, while MA and Landrace were always close to each other. Bayesian methods were used to allocate individuals to breeds, with a correct classification to the breed of origin ranging between 61% in Alentejano and 88% in MA. To study the mutation in the RYR1 locus (which confers stress susceptibility), a new technique for detection of SNPs was developed in the Molecular Genetics Laboratory of “Estação Zootécnica Nacional”. The identification of the point mutation was performed by SNaPshot, followed by capillary electrophoresis in an automated sequencer. Frequency of the recessive allele (n) was 0 in Duroc, 0,275 in Landrace, 0,34 in MA, 0,75 in Pietrain and lower than 0,10 in the other breeds. The proportion of heterozygous carriers was 0,15 in Landrace, 0,40 in Pietrain and 0,60 in MA. In populations with frequencies of the recessive allele lower than 0,10, recessive homozygous (nn) individuals were not detected, but their occurrence was 0,55 in Pietrain, 0,20 in Landrace and 0,04 in MA.
... The set of markers, not linked to the sex, is known for a good quality of the loci and a low typing error rate. For more details on the microsatellites used, see Appendix 7 in FAO (FAO 2011) and Groenen (2003). The probabilities of identity between two random individuals and two siblings were estimated to be 7.9×10 -11 and 1.5×10 -4 respectively using Genalex. ...
The wild boar (Sus scrofa scrofa) is a peculiar species. It is an appreciated game species for hunters, a nightmare for farmers and a subject of debate for the society in general. The tenfold increase of the population over the last decades in France and all over Europe, despite increased hunting pressure, generated great human-wildlife conflict. The wild boar is responsible for great economic losses due to vehicle collision, diseases transmission and damaged crops and ecosystems. Improving management strategies becomes a prime interest to avoid such conflicts, or at least keep them under control. Obtaining information on the species is a first step toward good management strategies. The objective of my work is, in a first part, to characterize the mating system of the wild boar and to identify some parameters, especially hunting, influencing the reproductive processes. The second part focus on the investigation of the influence of the mating system on wild boar life history traits. My researches are based on the study of several populations contrasting in their hunting practices and on longitudinal data of a highly monitored population. The study is based on data collected on wild boars killed by hunting. Genotypes were obtained for pregnant females and their litter and paternity analyses were realized to measure the number of fathers in a litter and estimate multiple paternity rates (proportion of litter sired by more than one father). I was able to show that the mating system is mainly promiscuous (several males mate with several females) contrasting with the polygyny (a dominant male monopolizing a group of females) usually described in this species. Moreover, reproductive processes, estimated by the number of mates of a female and the multiple paternity rates, are influenced by hunting variations in a population. I also showed that number of fathers has positive effect on female fecundity. High rates of multiple paternity together with high genetic diversity were found in a heavily hunted population, suggesting multiple paternity may buffer yearly bottlenecks. However, the increase of number of fathers is not associated with increase of within-litter variation
... Hal ini diperkuat oleh hasil penelitian Tascon et al. (2000), Arranz et al. (2001), Rendo et al. (2004), Alvarez et al. (2004 yang telah melakukan analisa mikrosatelit pada sekelompok kecil ternak kambing. Pada babi, mikrosatelit telah banyak digunakan pada penelitian biodiversitas baik pada bangsa komersial maupun bangsa liar (Groenen et al., 2003). Penggunaan analisa mikrosatelit sebagai uji keturunan pada ternak Kuda sudah secara rutin digunakan diberbagai laboratorium dan balai penelitian. ...
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Microsatellite analysis in biotechnology of animal reproduction (A Review) ABSTRACT. In year 1970 was found nucleotide sequence which have repeated sequence of nucleotide. with high polymorh and using PCR could be amplified. That sequenz of nucleotide called Microsatelite. Microsatelite consist of 1 – 6 repeated nucleotide, which is CA repeated as mostly a repeated DNA in the animal (Tautz and Renz, 1984). Based on difference of long and amount of repeated nucleotide, there are three kind of DNA satelite, midi-, mini- and microsatelite (Matiat and Vergmaud, 1982). Microsatelite analysis was used to analyze of paternity and identity of animal, which was done as a conventional analysis with blood group analysis. The advantage of microsatelite analysis compare to blood group system are the exclution probability was high (EXP 99.9%), needs small sampel (tissues, sperm or follicel of hair), could be use for all animal without special age and possible for died animal.
... During these projects, genotyping facilities and associated databases were established. Many new polymorphic markers were developed and evaluated (Groenen et al., 2003; Ollivier 2009). Based on these evaluations, the Food and Agriculture Organization of the United Nations (FAO) and the International Society for Animal Genetics have recommended specific set of markers for assessing biodiversity of pigs (FAO 2004; Hoffmann et al., 2009). ...
... This condition as same as researches of [9]; [2]; [35]; [1] which were analyzed by Goat. By Pigs, microsatellite was used in biodiversity commercial and wild animals [15]. ...
In year 1970 was found nucleotide sequenz which have repeated sequenz of nucleotide. with high polymorh and using PCR could be amplified. That sequenz of nucleotide called Microsatelite. Microsatelite consist of 1 – 6 repeated nucleotide, which is CA repeated as mostly a repeated DNA in the animal [39]. Based on difference of long and amount ofrepeated nucleotide, there are three kind of DNA satelite, midi-, mini-and microsatelite [28]. Microsatelite analysis was used to analyse of paternity and identity of animal, which was done as a conventional analysis with blood group analysis. The advantage of microsatelite analysis compare to blood group system are the exclution probability was high (EXP 99.9%), needs small sampel (tissues, sperm or follicel of hair), could be use for all animal without special age and possible for died animal.
... The new project provides potentially for a wider range of alleles which may make this an interesting approach. The lower potential cost is also of significant interest for diversity studies in general, although the first project established that there are still some problems with data interpretation that require further exploration (Groenen, 2003). ...
... The new project provides potentially for a wider range of alleles which may make this an interesting approach. The lower potential cost is also of significant interest for diversity studies in general, although the first project established that there are still some problems with data interpretation that require further exploration (Groenen, 2003). ...
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We present a new method that allows rapid determination of allele frequencies at loci exhibiting length polymorphism. In this method a fluorescence-labeled PCR primer is used to amplify the polymorphic region from pooled DNA samples originating from a large number of individuals. The fluorescent PCR products are separated by gel electrophoresis on an automatic DNA sequencer and the relative amount of the PCR products are determined. The distribution of the PCR products obtained from the alleles present in the pooled samples directly corresponds to the allele frequency in the population in question. The allele frequencies at a short tandem repeat locus in the von Willebrand factor gene and at the D1S80 locus were determined in the Finnish population. We found that the allele frequencies determined by quantitative analysis of PCR products from pooled DNA samples and by analyzing individual samples were in good agreement.
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For 17 microsatellite markers, allele frequencies were determined in nine highly selected commercial broiler and six highly selected commercial layer lines using pooled blood samples from 60 animals. The average number of marker alleles was 5.8 over all lines, 5.2 over broiler lines, and 3.0 over layer lines. The average number of marker alleles within a line was 2.9, 3.6, and 2.0 for all, broiler, and layer lines, respectively. Over all 15 lines, the average percentage of heterozygosity was 42, whereas the heterozygosity in the broiler lines was 53% and in the layer lines only 27%. In broiler lines, 50% of the marker-line combinations showed a heterozygosity above 60%, whereas this was only 5% in layer lines. Estimation of allele frequencies with microsatellite markers was first assessed in pooled and individual samples before usage in the commercial lines. Allele frequencies for 19 microsatellite markers were estimated in chicken pooled blood samples and compared with allele frequencies from individual typed animals. Similar results were obtained when pooled blood samples (heterozygosity of 35.3%) or individual typed animals (heterozygosity of 34.2%) were used. The method to determine allele frequencies using pooled blood samples is faster, cheaper, and as reliable and repeatable as determining allele frequencies using individual typings.
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Accurate quantification of relative allele frequencies in pooled DNA samples can be carried out for microsatellite markers having a dinucleotide repeat unit, conditional on the absence of overlapping "shadow" bands. This provides a basis for extending DNA pooling to this useful class of DNA marker. Expressions for the standard error of densitometric estimates of allele frequencies from pooled samples are presented, and their statistical application is illustrated in a variety of situations. This enables DNA pooling to be utilized in situations requiring the testing of statistical hypotheses concerning differences in allele frequencies between populations, or samples.
Genetic variability within four Belgian pig breeds, (Landrace (BL), Negative (BN), Piétrain (P), and Large White (LW)) was investigated using seven microsatellite loci. Differences in allele frequencies indicate each breed's particular identity. Computation of polymorphism information contents (PIC), theoretical heterozygosities, effective numbers of alleles, and probabilities of encountering, at random, two identical individuals in each breed revealed a variability, decreasing in the order LW, BN, BL, and P. Five types of genetic distance between the breeds were also calculated. Two‐by‐two breed comparisons indicated some distances to be rather important, confirming the breeds' morphological differences. BL and BN show the closest resemblance, while P is the most distant breed. The chances of encountering, at random, two identical individuals among two breeds are small, and confirm the conclusions resulting from the genetic distances. The efficiency of the microsatellites in parentage control is high. Zusammenfassung 7 Microsatellitsystemen studiert in 4 Belgische Schweinerassen Anhand von 7 Microsatellitsystemen wurde die genetische Variabilität innerhalb 4 Belgischer Schweinerassen (Landrasse [BL], Negativ [BN], Pietrain [P] und Large White [LW] untersucht. Unterschiede in Allelhäufigkeiten deuten auf die besondere Identität jeder einzelnen Rasse. Berechnung von Polymorphismus, Informationsinhalt (PIC), theoretischer Heterozygotiewerte, wirksamer Anzahl von Allelen und Wahrscheinlichkeit zweier identischer Individuen in jeder Rasse brachte Hinweise auf abnehmende Variabilität in der Reihenfolge von LW, BN, BL und P. Ausserdem wurden 5 Arten genetischer Distanzen zwischen den vier Schweinerassen berechnet. Bei paarweisem Vergleich zeigten diese Distanzen sich als ziemlich gross, so dass damit die morphologischen Differenzen zwischen den Rassen bestätigt werden. BL und BN haben die engste Verwandtschaft und P ist am weitesten entfernt von BL, BN und LW. Die Wahrscheinlichkeit, zufällig zwei identische Tiere in zwei Rassen zu finden, fehlt praktisch und bestätigt die Schlussfolgerungen, die sich aus den genetischen Distanzen ergeben. Die Microsatelliten sind sehr zweckmässig anwendbar in der Abstammungskontrolle.
An analysis of 25 microsatellite loci in 210 animals has been used to define the genetic structure of the Iberian pig, traditionally classified into several varieties. In addition, a sample of 20 Duroc pigs was used as an outgroup for topology trees. Inter-variety genetic variation was estimated by unbiased average heterozygosity and the number of alleles observed. Significant deviations from the Hardy–Weinberg equilibrium (HWE) were shown for 19 loci across the whole population. By contrast, equilibrium deviation within varieties was much lower. Genetic variation measures, genetic distance values and a neighbour-joining tree were used to estimate subdivision. In addition, an individual tree was constructed to contrast the assignation of animals into varieties. Despite the low bootstrap values obtained in the varieties neighbour-joining tree, the degree of genetic variation found was sufficient to support the division of the Iberian pig into varieties, although in some cases the traditional classification cannot be accepted. These results have shown the value of this marker panel in the study of intra-breed genetic structures.
Polymorphic microsatellite markers are widely used in molecular analyses. The range of allele sizes and the allele frequencies within a population are important characteristics of the marker. Their determination previously has involved genotyping a large number of individuals. We have developed a technique for defining these characteristics by coamplification of many samples in a DNA pool. Groups of 32 and 42 DNA samples were genotyped and results were compared with those from individual genotype determinations. To improve the accuracy in the estimation of allele frequencies, arithmetic removal of stutter bands was carried out and the consistency of each marker was characterized. This approach was also applied to a group of 94 individuals. All of the work has been done using nonradioactive methods. Potential applications of this technique are in population genetics, high throughput genotyping, and loss of heterozygosity studies.
We report the most extensive genetic linkage map for a livestock species produced to date. We have linked 376 microsatellite (MS) loci with seven restriction fragment length polymorphic loci in a backcross reference population. The 383 markers were placed into 24 linkage groups which span 1997 cM. Seven additional MS did not fall into a linkage group. Linkage groups are assigned to 13 autosomes and the X chromosome (haploid n = 19). This map provides the basis for genetic analysis of quantitative inheritance of phenotypic and physiologic traits in swine.
We report the highest density genetic linkage map for a livestock species produced to date. Three published maps for Sus scrofa were merged by genotyping virtually every publicly available microsatellite across a single reference population to yield 1042 linked loci, 536 of which are novel assignments, spanning 2286.2 cM (average interval 2.23 cM) in 19 linkage groups (18 autosomal and X chromosomes, n = 19). Linkage groups were constructed de novo and mapped by locus content to avoid propagation of errors in older genotypes. The physical and genetic maps were integrated with 123 informative loci assigned previously by fluorescence in situ hybridization (FISH). Fourteen linkage groups span the entire length of each chromosome. Coverage of chromosomes 11, 12, 15, and 18 will be evaluated as more markers are physically assigned. Marker-deficient regions were identified only on 11q1.7-qter and 14 cen-q1.2. Recombination rates (cM/Mbp) varied between and within chromosomes. Short chromosomal arms recombined at higher rates than long arms, and recombination was more frequent in telomeric regions than in pericentric regions. The high-resolution comprehensive map has the marker density needed to identify quantitative trait loci (QTL), implement marker-assisted selection or introgression and YAC contig construction or chromosomal microdissection.