linkage and radiation hybrid mapping of the porcine gene for subunit c of succinate dehydrogenase...
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(hf � 0.29, hm � 0.20, LOD � 3.58); S0019 (hf � 0.49,
hm � 0.18, LOD � 3.56); S0095 (hf � 0.34, hm � 0.16,
LOD � 3.42); OTF11 (hf � 0.00, hm � 0.00, LOD � 3.31).
Multipoint sex-averaged map of porcine chromosome 9, con-
structed using options ALL, BUILD, CHROMPIC, FLIPS2±6,
placed SLN between markers PPP2ARB and CRYAB (PPP2ARB
± 24.7 cM ± SLN ± 3.1 cM ± CRYAB ±). This assignment con-
®rms the physical mapping of the porcine SLN gene to chro-
mosome 9p24-(1/3p21) previously obtained9.
Acknowledgements: This work was supported by the EC contract
BIO4-CT98-0237 (GENETPIG), by the Italian MURST ex 60%
funds and was associated with the PiGMaP international
genetic mapping collaboration.
References1 Odermatt A. et al. (1998) J Biol Chem 273, 12360±9.
2 Davoli R. et al. (1999) Gene 233, 181±8.
3 Altschul S.F. et al. (1997) Nucl Acids Res 25, 3389±402.
4 Wawrzynow A. et al. (1992) Arch Biochem Biophys 298,
620±3.
5 Odermatt A. et al. (1997) Genomics 45, 541±53.
6 Archibald A.L. et al. (1995) Mamm Genome 6, 157±75.
7 Green P. et al. (1990) Documentation for CRI-MAP, Version 2.4.
Washington University, School of Medicine, St. Louis, MO.
8 Fontanesi L. et al. (2000) Anim Genet 31, 287±8.
9 Davoli R. et al. (2000) Anim Genet 31, 400±3.
Correspondence: R. Davoli (e-mail: [email protected])
Linkage and radiation hybrid mapping of theporcine gene for subunit C of succinatedehydrogenase complex (SDHC )to chromosome 4
A. Stratil*, G. Reiner², L. J. Peelman³,M. Van Poucke³ and H. Geldermann²
*Institute of Animal Physiology and Genetics, Academy of
Sciences of the Czech Republic, LibeÏ chov, Czech Republic.²Institute of Animal Husbandry and Breeding, Department of
Animal Breeding and Biotechnology, University of Hohenheim,
Garbenstrasse 17, Stuttgart, Germany. ³Ghent University,
Faculty of Veterinary Medicine, Department of Animal
Nutrition, Genetics, Breeding and Ethology, Merelbeke, Belgium
Accepted 22 January 2001
Source/description: Complex II (succinate-ubiquinone oxidore-
ductase; EC 1.3.5.1) is an important enzyme complex in both
the tricarboxylic acid cycle and the aerobic respiratory chains of
mitochondria in eukaryotic cells and prokaryotic organisms.
Complex II has four subunits: ¯avoprotein, iron-sulphur protein
and two integral membrane proteins ± the large cytochrome b,
cybL or C, subunit of succinate dehydrogenase complex (SDHC)
and the small cybS or D subunit (SDHD). None of these subunits
is encoded by the mitochondrial genome. The human gene for
subunit C (SDHC) was assigned to chromosome 1q211,2. To
amplify a fragment of the porcine SDHC gene by PCR primers
(Pair 1) were selected from the human sequence of exons 5 and
63 (EMBL accession numbers AF039593; AF039594). The
fragment (~3 kb) was sequenced and from the sequence a new
pair of PCR primers (Pair 2) was designed.
Primer sequences:
Pair 1: Forward: 5¢-CTTGTCTTCCCTCTCATGTAT-3¢Reverse: 5¢-AACCACTCCAGACTGGTATAG-3¢Pair 2: Forward: 5¢-AACCCTGAAGCCAGACCATACA-3¢Reverse: 5¢-AGCTCTGATGCGGAAGTTACA-3¢.
PCR conditions/cloning/sequencing: Polymerase chain reaction,
using primers of Pair 1, was performed in 25 ll reactions
Table 1 Allele frequencies at the porcine SLN locus.
No. ofAllele frequencies
Breed animals Allele 1 (A) Allele 2 (G)
Large White 35 0á90 0á10
Landrace 22 0á91 0á09
Duroc 30 0á58 0á42
Belgian Landrace 17 0á71 0á29
Hampshire 20 0á85 0á15
Pie train 23 0á87 0á13
Meishan 14 0á00 1á00
Figure 1 PCR±RFLP at the SLN locus. The genotypes are indicated
at the top of each lane. M, DNA molecular weight VIII (Roche
Diagnostics, Mannheim, Germany); U, undigested PCR fragment.
110 Brief notes
Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 105±121
containing 100 ng genomic DNA (this had to be of a good
quality), reaction buffer, 1.5 mM MgCl2, 200 lM each dNTP,
10 pmol each primer and 1 U of LA polymerase (Top-Bio,
Prague, Czech Republic). Ampli®cation conditions were 2 min
at 95 °C followed by 35 cycles of 53 °C (45 s), 68 °C (2 min)
and 94 °C (45 s), with a ®nal extension at 68 °C (7 min). A
major strong fragment (~3 kb) and two weak shorter fragments
were observed on 0.8% agarose gel. The major fragment was
cloned and subcloned (plasmid pUC18; Escherichia coli DH5a),
using SureClone Ligation Kit (Pharmacia Biotech AB, Uppsala,
Sweden) and sequenced on an ALFexpress Sequencing System
(Pharmacia Biotech). The sequence corresponded to parts of
exon 5 and 6 and intervening intron of the human SDHC gene.
The porcine sequence (without the primer Pair 1 sequences)
has been deposited in the EMBL database under accession no.
AJ300475.
The PCR, using primers of Pair 2 (that were designed from
the porcine sequence) was carried out in 25 ll reactions using
100 ng genomic DNA, reaction buffer, 1.5 mM MgCl2, 200 lM
each dNTP, 10 pmol each primer and 0.6 U Taq polymerase.
After initial 2 min denaturation step at 95 °C the PCR was
performed at 60 °C (45 s), 72 °C (1 min) and 94 °C (45 s) for
33 cycles, with a ®nal extension at 72 °C (7 min). A single
fragment of 758 bp was observed on agarose gel. The sequence
of the cloned fragment was as expected.
Polymorphism/Mendelian inheritance/allele frequencies: In PCR
fragments, ampli®ed both with the use of primers of Pair 1 and
Pair 2, polymorphism was observed after restriction with HinfI.
For routine typing Pair 2 primers were preferred. The poly-
morphic site is within intron 5; however, we have not
attempted to identify the exact restriction site and base
replacement. Two alleles were observed ± A (fragments
422 + 240 + 96 bp) and B (422-bp fragment was cut to
~330 + 90 bp). All three genotypes are shown in Fig. 1.
Codominant inheritance was con®rmed in the Hohenheim
Meishan ´ Pietrain pedigree4. Allele frequencies in eight breeds
of pigs are presented in Table 1.
Linkage mapping: Using the CRI-MAP software package5 the
SDHC gene was mapped in the Hohenheim Meishan ´ Pietrain
pedigree4 to a chromosome 4 linkage group6. The distances for
the region of interest (in Kosambi cM; sex average) were as
follows: MYC-31.3-V-ATPase-6.3-ATP1B1-5.5-ATP1A2-2.9-
SDHC-6.5-PKLR-7.8-EAL-4.4-AMPD1-0.0-NGFB-4.3-TSHB.
Radiation hybrid mapping: Radiation hybrid mapping was
performed using the INRA-University of Minnesota porcine
Radiation Hybrid panel (IMpRH)7,8. A panel of 118 hybrid
clones were screened by PCR, using the Pair 2 primers. The
results were analysed by the IMpRH mapping tool at the IMpRH
server (http://imprh.toulouse.inra.fr). A radiation hybrid map
was built using the RHMAP3.0 statistical package. Analyses
were performed under the equal retention probability model.
Using the RH2PT program, two-point distances were calculated
between all markers. Linkage groups were de®ned using a lod
score threshold of 4.8. Multipoint analyses were then performed
using RHMAXLIK. In Fig. 2, a clone from chromosome 4 is
shown to which SDHC was mapped. The most signi®cantly
linked marker (2pt analysis) was SW589 (15 cR;
LOD � 18.74).
Acknowledgements: We are grateful to Drs Martine Yerle and
Denis Milan (INRA, Castanet-Tolosan, France) for making
available the RH panel. We would like to thank Marie DatlovaÂ
Figure 1 Agarose gel electrophoresis (2%) showing genotypes of
porcine SDHC following digestion of the 758 bp PCR fragment with
Hinf1. The genotypes (AA, AB, BB) are given at the top of each lane.
M, 1000±100 bp marker; PCR, undigested PCR fragment.
Table 1 Frequencies of the HinfI alleles at the porcine SDHC gene.
Allele
Breed n A B
Large White 14 0.75 0.25
Landrace 12 0.33 0.67
Pietrain 6 0.92 0.08
Czech Meat Pig 15 0.50 0.50
Black Pied Prestice 7 0.71 0.29
Hampshire 6 0.75 0.25
Duroc 2 0.50 0.50
Meishan 8 0.19 0.81
Figure 2 Radiation hybrid mapping of the porcine SDHC to a
chromosome 4 clone. Distances are in cR.
111Brief notes
Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 105±121
and Ing. Gabriela PursÏova for excellent technical assistance.
This work was supported by Grant Agency of the Czech Re-
public (Grant no. 523/00/0669).
References1 Online Mendelian Inheritance in Man OMIM (TM). (2000)
Johns Hopkins University, Baltimore, MD. MIM Number:
602413: October 31 World Wide Web URL: http://
www.ncbi.nlm.nih.gov/omim/.
2 Hirawake H. et al. (1997) Cytogenet Cell Genet 79, 132±8.
3 Elbehti-Green A. et al. (1998) Gene 213, 133±40.
4 Geldermann H. et al. (1996) J Anim Breed Genet 113, 381±7.
5 Green P. et al. (1990) Documentation for CRI-MAP, Version
2.4. Washington University School of Medicine, St. Louis,
MO.
6 BlazÏkova P. et al. (2000) Anim Genet 31, 416±8.1
7 Yerle M. et al. (1998) Cytogenet Cell Genet 82, 182±8.
8 Hawken R.J. et al. (1999) Mamm Genome 10, 824±30.
Correspondence: A. Stratil (e-mail: [email protected])
Consensus and comprehensive linkage mapsof bovine chromosome 17
T. S. Sonstegard*, C. Bendixen², G. L. Bennett³,E. Kalm§, S. M. Kappes±, H. A. Lewin**, S. Lien²²,V. H. Nielsen², I. Olsaker³³, S. Schmutz§§, H.Thomsen§, C. P. Van Tassell* and N. Xu§
*USDA-ARS ANRI GEML, BARC-East, Beltsville, MD, USA.²Department of Animal Breeding and Genetics, Research Centre
Foulum, Tjele, Denmark. ³USDA-ARS, U.S. Meat Animal Research
Center, Clay Center, NE, USA. §Institut fuÈ r Tierzucht und Tierhal-
tung der Christian-Albrechts-Universitaet zu Kiel, Hermann-Rode-
wald-Str., Kiel, Germany. ±USDA, ARS, NPS, Beltsville, MD, USA.
**Animal Sciences, University of Illinois, Edward R. Madigan
Laboratory, Urbana, IL, USA. ²²Department of Animal Science,
Agricultural University of Norway, Norway. ³³Department of
Morphology, Genetics and Aquatic Biology, Norwegian College of
Vet. Medicine, Oslo, Norway. §§Department of Animal Science,
University of Saskatchewan, Saskatoon, Canada
Accepted 28 January 2001
Introduction: Comprehensive linkage maps have been con-
structed with the purpose of integrating existing genetic data
from several populations1,2,4,9. This workshop report, presented
under the of auspices of the International Society for Animal
Genetics (1998±2000), summarizes construction of consensus
and comprehensive linkage maps for bovine chromosome 17
(BTA17). Six laboratories contributed marker genotypes for
analysis that tallied to 19 443 informative meioses generated
from 41 marker loci. Eighteen loci were typed by at least two
laboratories and 17 of these loci were used to construct a
consensus linkage map. The sex-averaged consensus map
covered 98.9 cM. All 41 loci were subsequently used to con-
struct a comprehensive map. The sex-averaged comprehensive
map was 103.8 cM. Average distance between loci in the
comprehensive map was 2.53 cM.
Linkage analysis: Six genotype data sets generated from 58 bo-
vine pedigrees were submitted to the Beltsville Agricultural
Research Center, Beltsville, MD, USA in a standardized format
for analysis using CRIMAP V. 2.43. Marker genotypes were
submitted from the Canadian beef cattle reference herd (http://
skyway.usask.ca/~schmutz/), a Danish Holstein cattle popula-
tion7, the genome project of the German Cattle Breeders Fed-
eration (ADR)8, the University of Illinois reference/resource
families6, the US Meat Animal Research Center reference
population5 and the Norwegian cattle map population10. The
meioses numbers submitted by each laboratory were 859,
1017, 5406, 1983, 8300 and 1878, respectively. The number
of marker loci submitted by each laboratory were 5, 5, 9, 12,
34 and 14, respectively. A total of 19 443 informative meioses
from 38 microsatellite loci, two gene-associated polymor-
phisms, and an erythrocyte antigen type were represented in
the combined data containing a total of 30 047 marker geno-
types. Each data set was analysed independently using the
TWOPOINT, FLIPS and CHROMPIC options. Genotypic data
were then combined into a single data set using the MERGE
option. The consensus linkage group was constructed using the
BUILD option (LOD � 3.0) followed by FLIPS5 analysis to test
alternative marker orders. For the comprehensive map, mark-
ers were added using the BUILD option (LOD � 1.0) followed
again by FLIPS5 analysis. Markers not positioned by this cri-
teria were added to the linkage group using the ALL option. The
FLIPS5 analysis was repeated until the best ordering was
obtained. Map ®gures, number of meioses per marker (*.loc
®les), TWOPOINT and FIXED output ®les can be accessed at the
http://aipl.arsusda.gov/maps.
Consensus map: Sixteen of the 17 microsatellite markers and one
erythrocyte antigen marker typed by two or more laboratories
were used to produce a sex-average consensus map spanning
98.9 cM (Fig. 1). For the microsatellite marker URB048, a map
position could not be determined using the criteria established
for consensus map construction. This marker is positioned on
the comprehensive map. The female map was 110.4 cM in
length and the male map was 97.0 cM (data not shown).
Comprehensive map: Marker genotypes from 41 loci were ana-
lysed to produce a comprehensive map of BTA17 (Fig. 1). Two
markers (BMS2780 and BMS2780b) were haplotyped, because
recombination between marker genotypes generated from these
two different primer pairs ¯anking the same microsatellite locus
was not detected. The length of the sex-averaged map was
103.8 cM (Fig. 1), while the female and male maps were 109.2
and 102.8 cM, respectively (data not shown). The average
marker interval was 2.53 cM, and the largest intermarker in-
terval of 8.7 cM was found between RM323 and BM1233. The
order producing the highest log-likelihood is presented.
References1
1 Beever J.E. et al. (1996) Anim Genet 27, 69±75.
2 Casas E. et al. (1999) Anim Genet 30, 375±7.
112 Brief notes
Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 105±121