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Animal Genetics, 1993 24,163-170
W Coppieters A Van de Weghe L Peelman A Van Zeveren Y Bouquet Department of Animal Nutrition, Production and Ethology, Faculty of Veterinary Medicine, University of Ghent, Heidestraat 19, B-9820 Merelbeke, Belgium. A Depicker Laboratory of Genetics, University of Ghent, Heidestraat 19, B-9820 Merelbeke, Belgium.
Characterization of porcine polymorphic microsatellite loci W Coppieters, A Van de Weghe, L Peelman, A Depicker, A Van Zeveren, Y Bouquet
Summary Recently, a European collaborative research
Twenty-seven (CA), and two (GA), microsatel- lite clones were isolated out of a size-selected genomic pig library. These were sequenced and the number of uninterrupted dinucleotides was found to range from 1 2 to 26. Flanking primers were chosen for 11 dinucleotide repeats and optimal conditions for polymerase chain reaction (PCR) amplifications were established. Different microsatellite loci were amplified sim- ultaneously by combining primer sets. Related and unrelated pigs were screened for length polymorphisms of the different microsatellite loci. The polymorphic information content (PIC) of these loci ranged between 0.62 and 0.83. Segregation studies in pig reference families established Mendelian inheritance. Locus SO022 was found to be X-linked.
Keywords: microsatellite, pig, genetic map
Introduction
Construction of high resolution genetic maps of commercially important animals is one of the main objectives in modern animal genetics. These genetic maps are a valuable tool in the localization of loci involved in quantitative traits by means of linkage analysis studies (Lander & Botstein 1989).
The realization of this objective needs a rich source of variable markers, as can be provided by molecular DNA techniques. Among the most frequently occurring polymorphic loci in many eukaryotic genomes are the dinucleotide repeat sequences, especially the (CA), stretches (Mies- feld et al. 1981; Hamada et al. 1982; Weber & May 1989; Litt & Luty 1989). In the pig genome the number of CA repeats was estimated between 65000 and 100000 copies, highly dispersed in a major part of the genome (Winter0 et al. 1992). Due to a variation in the number of repeats, they exhibit a length polymorphism, that can be demonstrated by PCR amplification (Johansson etal. 1992).
Correspondence: Wouter Coppieters.
Accepted 22 February 1993
project to map the pig genome, called PiGMaP was initiated (Archibald et al. 1990). In this paper we describe the characterization of several dinucleotide repeats from the pig genome. A pig DNA library containing small genomic inserts was constructed in a plasmid vector and the clones were stored in microtitre plates. Twenty- nine CA and GA repeats were isolated and sequenced and 11 were analysed for their poly- morphic content.
Materials and methods
Animals
A panel of 10 unrelated pigs (five Pietrain and five Belgian Landrace) was initially used to detect polymorphisms associated with micro- satellite loci. By typing the reference families of PiGMaP, Mendelian inheritance was estab- lished. The allele sizes both of the test panel and the founder animals of the PiGMaP reference families (11 Meishan, 11 Large White, three Pietrain, three Wild Boar and two Yorkshire) were used to estimate allele frequencies and PIC values (Botstein etal. 1980).
Construction of a library with short genomic inserts
Genomic pig DNA was isolated as described (Coppieters et al. 1990). A genomic library with short inserts was constructed as follows: 50 pg of pig genomic high molecular weight DNA was digested to completion with Sau3A and separated on a 3% preparative low melting point agarose gel (Nusieve GTG, FMC, Rockland, ME, USA). Fragments ranging from 250 to 500bp were recovered by phenol extraction (Maniatis et 01.1982) and cloned in plasmid pSP72 (Promega, Madison, WI, USA). Escherichia coli DH5u cells (BRL) transformants were transferred to microti- tre wells containing Terrific Broth with 50 pg/ml ampicillin (Tartof & Hobbs 1987). Three sequenced clones with a known number of CA repeats were included in each microtitre plate.
Filter replicas were made according to standard procedures (Maniatis et al. 1982).
163
164 Screening of the library Coppieters, Van de Weghe, Peelman, et al.
Synthetic poly (dA-dC).poly (dG-dT) or poly (dA-dG) .poly (dC-dT) (Pharmacia LKB, Uppsala, Sweden) was labelled to at least 10' cpm/pg by multipriming using radioactive C ~ ( ~ ' P ] ~ C T P (Feinberg & Vogelstein 1983).
After baking the filters were washed four times during 60 rnin in 6 x SSC, 0.1% SDS at 65°C. Prehybridization was performed during 2 h in 6 x SSC, 1% SDS, 5 x Denhardt's solution (O.l0/o polyvinylpyrrolidone, 0.1% BSA and 0.1% ficoll) and 50% formamide at 42°C in a rotating hybridization oven. Labelled probe was added at 1 ng/ml to the prehybridization buffer and the filters were hybridized overnight. Subse- quently the filters were washed for 30 min in 2 x SSC, 0.1% SDS at 65°C and twice for 30 min in 1 x SSC, 0.1% SDS at 65°C.
Autoradiograms were taken (Fuji HRH films, Tokyo, Japan) overnight with intensifying screens at -70°C.
DNA sequencing
Plasmid minipreparations of positive clones were prepared by a boiling method from a 5-ml culture (Holmes & Quigley 1981) and further purified on Quiagen columns (Diagen, Diissel- dorf, Germany). Plasmids were sequenced (Sanger et al. 1977) in both directions (T7 and SP6 promoter primers) with the T7 sequencing kit from Pharmacia LKB.
PCR amplification of microsatellite loci
Polymerase chain reaction (PCR) primers were chosen complementary to the sequences flanking the dinucleotide repeats with the primer analysis software package Oligo V.4 (Medprobe, Oslo, Norway).
PCR conditions for each primer pair were established by optimizing the Mg'+ concentra- tion and annealing temperature. Amplification was performed with an automated thermal cycler GeneAmp PCR System 9600 (Perkin Elmer Cetus, Emeryville, USA) in 1O-pl reactions (10mM Tris.HC1 pH 8.3; 5 0 m ~ KCl; 0.001% gelatine; 1 - 3 m ~ MgCl,; 2 0 0 ~ ~ dTTP, dATP, dGTP; 2.5mM dCTP; 2pCi CY[~'P]~CTP; 1 p~ of each primer; 0.25U Taq polymerase (Perkin Elmer Cetus); 20ng genomic DNA). A first denaturation step at 94°C for one min was followed by 24 cycles of 30s denaturation at 94"C, 30s annealing at the optimal temperature, 30 s polymerization at 72°C and a final polymerization step of 10 min at 72"C, at the end of the programme.
After addition of 6pl loading solution (96O/0 formamide) and heating for 2 min to 80"C, the
PCR products were analysed on a 60-cm-long, denaturating, wedge-shaped, 5% polyacryla- mide sequencing gel and detected by overnight exposure to Fuji HRH films. By using the plasmid microsatellite clones as a template for amplifi- cation, PCR products of a known size were produced and used as a size reference besides M13 sequencing reactions.
Multiplex PCR
Several multiplex PCR amplifications (Cham- berlain et al. 1988) were developed by combining different primer sets in one PCR reaction, titrat- ing the Mg" concentration and optimizing the annealing temperature. The initial combinations of our multiplex amplifications were based on the expected PCR product size and a comparable annealing temperature (maximum k 5°C). We added one new primer pair at a time and tested the combinations by trial and error.
Results
Screening of the library and sequence analysis
A total of 30 microtitre plates containing about 2800 genomic plasmid clones were screened for (CA), and (GA), repeats. Twenty-seven (CA), positive and 2 (GA), positive clones were iso- lated and sequenced. All clones contained dinu- cleotide sequences as given in Table 1. The repeats were categorized according to the rules given by Weber (1990). Twenty-five clones con- tained perfect dinucleotide repeats ranging from 1 2 to 26 repeats and three clones contained imperfect repeats. The GA positive clone DG3D5 contained two GA repeats, one short (8 repeats) perfect repeat, and one compound TAIGA perfect repeat, separated by about 150bp. Clone DHlF8 included also a poly T stretch of 32 bp.
The sequences of the microsatellite clones were compared to the porcine short interspersed element (SINE) PRE-1 (Singer et al. 1987) for homology. Eight of the 29 sequenced clones contain a segment homologous to the PRE-1 sequence and are indicated in Table 1. In four of these clones (DA3B8, DElF4, DE2H9, DH2E3) the dinucleotide repeat directly follows the 3' poly A tail of the PRE-1 element (data not shown).
Amplification of the microsatellite loci
Prime pairs (see Table 1). producing amplifi- cation products between 80 and 300bp, were chosen for 11 microsatellite sequences with the aid of the primer analysis software package Oligo. Primers complementary to PRE-1 homolo- gous sequences were avoided. PCR conditions
165 Pig microsatellites
Table 1. Cloned and sequenced pig microsatellites: primer sequences and PCR conditions
Annealing PiGMaP [Mg”] temperature locus
Clone Repeat Category Primer sequences (5’4’) (mM) (“C) name
DAlE6 (GT)23 Azi
DA1E7b (CA)13
DAlGl (AC),,
DA3Beb (GT)z3 DB1D3 (CA),,
DB2G3 (CA)zo
DB3E2 (CA),, DB3FI (AC)&A(AC), DClAlO (CA),, DE1F4b (TG),, DE2Hgb (CA),, DE3F7 (AC),,
DFlE2’ (GAIT4 DF2A5 [GT)l.5 DFPDl (CA)14TT(CA),
DGlB6 DGlCl DGlCll DG3D5”
DG3C5 DHlF8
DH2E3b DH3G11 D13B2b
DJlE12 (CA),,
Perfect
Perfect
Perfect
Perfect Perfect
Perfect
Perfect Imperfect Perfect Perfect Perfect Perfect
Perfect Perfect Imperfect
Perfect Perfect Perfect Compound Perfect Perfect Perfect
Perfect Perfect Perfect
Perfect
Perfect Perfect
TCTTT GAACT AAAAT AT AGACT C 2 TTCTCCAAACTCTGTCTCAC TGCCACACCTCCAGTTCAAC TTCAGCACAATGGGAAATGG CTAGGAGAAAATCTGAGGTT GTTTGAATGGAGGTGCTGTA
GCACAGTTGATGCTTCATGC GATCAAAAGTCCCCAATTCC TTCTTATTTCTCTGTGTCTT ATTGTTTCCCTTTCTTCTGA
GGTATACAAACAACCTAAGT TATGGCTACATAACAAAGTG
TCTCCCTTCCCTCCATCTCT CTCCATCAGCCAAAAACATT
AAAGAAGGAAAGAACTGATA ATGGAGGATAATGTGAAAAA
ATCAATCAGAGAAACAGGGT TCTTTGTAATCTGTCCTTCC
CTGGGCAGCTCTATAATATC TTACCCTTTCTACTCTGTGC AACCTTCCCTTCCCAATCAC CACAGACTGCTTTTTACTCC
2
2
3
2
2
2
2
2
4
1
50
55
60
65
50
50
50
50
65
60
50
SO023
SO027
SO017
SO018
so019
SO020
SO025
SO024
so021
so022
SO026
DJ3ETb (TG),TC(TG),TC(TG), Imperfect A7 (G’b Perfect
a (GA),-positives; contains a PRE-1 homologous sequence.
(see Table 1) were as described in ‘Materials and methods’.
At first, length variation of the dinucleotide repeats was examined on a panel of 10 unrelated animals. Only locus SO027 (clone DA1E7), con- taining a short repeat of 13 CA dinucleotides, did not show variation (data not shown). Then the reference families of the PiGMaP project were typed for the 10 polymorphic microsatellite markers. The polymorphisms are summarized in Table 2. For most microsatellite loci we observed, as expected, allelic size variation corresponding to a multiple of two bases, most
probably due to a variation in the copy number of the dinucleotide repeat. Two systems showed also one-basepair differences. For the SO024 microsatellite locus (clone DFZD1) we could identify eight alleles with a length polymor- phism in the range 166-184 bp by differences of a multiple of two nucleotides. One of the Meishan pigs contained also an allele of 171 bp, differing by only one basepair from the 172 allele. The amplification of the SO025 microsatellite locus (clone DE3F7) showed also one-basepair differ- ences between alleles. In total we observed four alleles with an uneven number and seven with an
Tab
le 2
. Pi
g m
icro
sate
llite
pol
ymor
phis
rns
(the
clo
ned
alle
le is
pri
nted
in
bold
)
Loc
us
SO02
3 SO
017
SO01
8 so
01
9
so0
20
SO
025
SO02
4 so
02
1
so02
2 SO
026
DJl
E12
C
lone
D
AlE
6 D
AlG
l D
BlD
3 D
B2G
3 D
B3F
1 D
E3F
7 D
FZ
Dl
DG
3D5
DI3
B2
Num
ber
of
type
d un
rela
ted
anim
als
30
39
40
39
37
29
37
32
39
0 24
Alle
le
Alle
le
Alle
le
Alle
le
Alle
le
Alle
le
Alle
le
Alle
le
Alle
le
Alle
le
size
A
llele
si
ze
Alle
le
size
A
llele
si
ze
Alle
le
size
A
llele
si
ze
Alle
le
size
A
llele
si
ze
Alle
le
size
A
llele
si
ze
Alle
le
(bp]
fre
quen
cy (
bp)
freq
uenc
y [b
p) f
requ
ency
(bp
) fr
eque
ncy
(bp)
fre
quen
cy (
bp)
freq
uenc
y (b
p) f
requ
ency
[bp
) fr
eque
ncy
(bp)
fr
eque
ncy
[bp)
fr
eque
ncy
106
0.03
10
4 0.
13
100
0.03
98
0.3
0 96 0
.32
94
0.07
8
6 0
.03
80 0
.02
76 0
.02
0 0.
05
PIC
valu
e 0.
75
Wei
ghte
d
num
ber
of
repe
atsb
aver
age
25.2
177
0.01
173
0.32
1
71
0.0
3 16
9 0.
30
167
0.01
16
5 0.
03
16
1 0
.01
157
0.22
1
51
0.0
8
0.72
15.6
272
0.03
264
0.16
26
2 0.
03
260
0-16
25
8 0.
01
256
0.03
25
4 0.
15
252
0.26
25
0 0.
06
248
0.08
24
6 0.
04
0.82
17.9
212
0.01
20
8 0.
08
206
0.41
20
2 0.
06
198
0.08
1
96
0.2
1 19
4 0.
04
19
0 0
.04
184
0.06
18
2 0.
01
0.74
18.4
121
0.14
119
0.51
11
7 0.
18
113
0.03
11
1 0.
15
0.62
14.3
12
1 0
.02
11
9 0
.02
11
6 0
.02
115
0.10
11
4 0.02
11
3 0
.45
11
2 0
.10
110
0.12
10
8 0.
03
10
6 0
.03
10
4 0
.09
0.73
21.4
184
0.01
18
2 0.
28
180
0.03
17
8 0.
03
176
0.11
17
4 0.
08
172
0.43
17
1 0.
01
166
0-12
0.76
18.1
212
0.02
20
6 0.
02
204
0.14
20
2 0.
06
198
0.02
196
0.34
19
4 0.
13
192
0.02
18
4 0-
09
182
0.08
17
4 0.
09
0.80
31.0
14
3 0
.19
139
0.14
13
7 0.
10
13
5 0
.21
13
3 0
.09
12
9 0
.02
127
0.07
1
21
0.1
7 11
9 0.
02
0.83
26.4
110
0.02
10
8 0.
15
106
0.02
10
4 0.
33
102
0.06
10
0 0.
08
98 0
.19
92
0.15
0.77
19.7
~~
a 20
mal
e an
d 1
9 fe
mal
e an
imal
s; th
e ze
ro a
llele
, seg
rega
ting
wit
h th
e Y
chr
omos
ome,
is n
ot ta
ken
into
acc
ount
whe
n ca
lcul
atin
g th
e al
lele
fr
eque
ncie
s.
Acc
ordi
ng to
Web
er (
1990
).
167 Pig microsatellites
even number of basepairs. By typing the PiGMaP reference families for segregation of the micro- satellite polymorphisms we obtained evidence for a zero allele at the SO023 locus (clone DAlE6) in three Meishan pigs, and a Y-linked zero allele for the SO022 (clone DI3B2) locus. The SO022 locus is clearly X-linked as all males carry a zero allele. This is demonstrated in Fig. 1: all male descendents show only one maternal allele and not the paternal allele; all female descendents show the paternal allele and one of the maternal alleles.
Allele frequencies
Because of the existence of a recessive zero allele for locus S0023, the allele frequencies for this locus are based only on animals for which data from family segregation analysis were available.
In this limited study different allele frequen- cies were observed for the different breeds. Moreover, some alleles were specific for a certain breed. In Fig. 2 the allele frequency distribution for locus SO017 and SO018 in the 11 Meishan pigs, the 11 Large White pigs and in the other breeds is given as a bar diagram.
Fig. 1. Analysis on a sequencing gel of the PCR amplification products of microsatellite locus SO022 on a PiGMaP reference pedigree. DNA fragment sizes (bp] are marked to the right. The allele 127 of the father is transmitted only to the female offspring.
Table 3. Multiplex microsatellite amplifications
Combination in
Loci quadruplex triplex duplex
SO01 7 SO018 SO024 so019 so020 SO023
[MgZ+l 2 Annealing 50
so021 SO025 so022
3 60
1.5 50
temperature (“CI
Multiplex PCR
The conditions for multiplex amplifications are given in Table 3. Multiplex PCR of up to four simultaneous amplifications was performed. In Fig. 3 a complex amplification with four different primer sets is shown. The use of wedge-shaped gels allows longer running times without loosing the short PCR fragments and ensures a good resolution for bigger PCR fragments.
Discussion
Based on the screening of the investigated geno- mic pig library (average insert size of 400 bp) the number of CA repeats in the pig is roughly estimated at 75 000 copies per haploid genome. The GA repeats are clearly less abundant and their number can be estimated approximately at 5000 per haploid genome. These figures are comparable with other estimations by Winter0 et al. (1992) and Johansson et al. (1992).
All primer pairs amplify unique loci of the pig genome. Polymorphism was observed with 10 of the 11 amplified microsatellite loci. They have a weighted average number of repeats (Weber 1990) above 14 and observed PIC values ranging from 0.62 to 0.83 as can be expected for longer microsatellites. However, these data should be considered with caution since they are based on a limited number of observations and on a very heterogeneous sample of unrelated animals of seven different breeds for which certain alleles seem to be breed specific. Only the DAlE7 microsatellite clone (locus S0027) containing a short (13 CA repeats) dinucleotide repeat showed no polymorphism. This is in agreement with the conclusions of Weber (1990) that the informativeness of sequences with about 11 to 15 repeats is variable.
For the SO024 and SO025 locus alleles of even and uneven length were observed. Most probably a deletion or an insertion of an uneven number of bases in the flanking sequence of the dinucleo- tide repeat can explain these observed allelic size variations.
The zero allele at the SO022 microsatellite locus segregated together with the Y chromo- some in all the reference families studied. There- fore this locus is most probably located on the X chromosome and has no homologous locus on the Y chromosome. The zero allele at the SO023 locus on the other hand could be caused by a mutation in one of the primer binding sites.
Four of the sequenced microsatellites were directly flanked with a pig SINE-sequence. This restricted the choice of specific priming oligo- nucleotides for these loci. The 3’ regions of SINE
168 Coppieters, Van de Weghe, Peelman, et 01.
50017
0.8
0 . 7
0 . 6
0.5
0 . 4
0.3
0.2
0.1
0
A I le les
50018
0.4
0.3
0.2
0.1
0 272 2% 262 260 258 256 254 252 250 248 246
A I ie les Melshan 124 i a r o e White Other breeds
Fig. 2. Bar diagram of the allele frequencies of locus SO017 and SO018 in Meishan, 11 Large White pigs and in the other typed breeds (respectively 28 and 29 individuals for t h e SO017 and SO018 locus).
169 Pig microsatellites
sequences have previously been reported to be rich in tandem repeats of short sequences as well as in human (Epstein et al. 1990) as in pig (Davies et al. 1992).
Microsatellites can be used for DNA-based individual identification and paternity diag- nosis. Based on the observed allele frequencies the exclusion probability for the quadruplex microsatellite typing described here is estimated as 0.96 (Wiener et al. 1930; Jamieson 1965). This exclusion power can be reached by a single PCR test performed on a very restricted blood sample and the interpretation of the typing data is very straightforward. Because of the technical evolu- tion in automatic DNA sequencing, microsatel- lite typing can be automated completely, up to the interpretation of the results by the develop- ment of the appropriate software (Ziegle et al. 1992). The abundance of microsatellite loci and their high informativeness will accelerate the development of a genomic linkage map'of the pig. One can therefore expect that in the near future this map will cover a major part of the genome.
Fig. 3. Analysis on a sequencing gel of a multiplex PCR of four microsatellite loci, on a PiGMaP reference pedigree. DNA fragment sizes (bp) are marked to the right and the different loci are marked to the left of the gel.
This will permit tracing and study of quantitative trait loci (QTL) by linkage analysis (Lander & Botstein 1989). Therefore the development of multiplex amplification and automatization of microsatellite typing will offer the opportunities to screen big resource populations within a reasonable time.
Acknowledgements
This work was supported by the EC Bridge Programme Biot N. 0187-c. We thank Linda Impe and Dirk Vanassche for excellent technical assis- tance. We also thank Dr A. Archibald, AFRC IAPGR (UK), Dr M. Groenen, Wageningen Agri- cultural University (The Netherlands), Dr L. Andersson, Swedish University of Agricultural Sciences (Sweden), Dr H. Geldermann, Uni- versity of Hohenheim (Germany) and Dr J. Gellin, INRA (France) for supplying the DNA of the PiGMaF' reference families.
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