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- firms the physical mapping of the porcine SLN gene to chro- mosome 9p24-(1/3p21) previously obtained 9 . 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. References 1 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 the porcine gene for subunit C of succinate dehydrogenase complex (SDHC ) to chromosome 4 A. Stratil*, G. Reiner ² , L. J. Peelman , M. Van Poucke and H. Geldermann ² *Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Libe ˇ chov, Czech Republic. ² Institute of Animal Husbandry and Breeding, Department of Animal Breeding and Biotechnology, University of Hohenheim, Garbenstrasse 17, Stuttgart, Germany. Ghent 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: flavoprotein, 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 1q21 1,2 . To amplify a fragment of the porcine SDHC gene by PCR primers (Pair 1) were selected from the human sequence of exons 5 and 6 3 (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. of Allele frequencies Breed animals Allele 1 (A) Allele 2 (G) Large White 35 0Æ90 0Æ10 Landrace 22 0Æ91 0Æ09 Duroc 30 0Æ58 0Æ42 Belgian Landrace 17 0Æ71 0Æ29 Hampshire 20 0Æ85 0Æ15 Pie ´ train 23 0Æ87 0Æ13 Meishan 14 0Æ00 1Æ00 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

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Page 1: Linkage and radiation hybrid mapping of the porcine gene for subunit C of succinate dehydrogenase complex (SDHC ) to chromosome 4

(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

Page 2: Linkage and radiation hybrid mapping of the porcine gene for subunit C of succinate dehydrogenase complex (SDHC ) to chromosome 4

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

Page 3: Linkage and radiation hybrid mapping of the porcine gene for subunit C of succinate dehydrogenase complex (SDHC ) to chromosome 4

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