Fluorescence in situ hybridization (FISH) analysis: Q-banded

metaphase spreads were prepared from sheep embryonic fibro-

blasts, prephotographed and karyotyped according to the in-

ternational standard (ISCNDA 1989). Purified plasmid DNA

(1 lg) containing an approximately 4800-bp PCR fragment of

ovine IL2 gene including all exons and introns (GeneBank

accession number AF287479) was labelled with biotin-16-

dUTP using a nick-translation system kit (Gibco BRL Life

Technologies, Rockville, MD, USA). After ethanol precipitation,

the pellet was dissolved in 10 ll hybridization solution (50%

formamide, 10% dextrane sulphate, 2· SSC, pH 7.0) containing

7 lg salmon sperm DNA and 3 lg sheep genomic DNA. Pre-

annealing was performed at 37 �C for 20 min, hybridization

was performed over night. The posthybridization treatment

followed standard protocol6. Hybridization signals were visual-

ized using avidin-conjugated fluorescein (avidin-FITC) and an

Olympus AH-2 epifluorescence microscope (Marietta, GA, USA).

Digitized images were analysed with the program Photoshop 5.5

(Adobe Systems Incorporated, San Jose, CA, USA). After the

digitizing and analysing of 15 metaphase cells, 12 metaphase

spreads produced doublet symmetrical spots in the centre of

OAR17, q23 (Fig. 2). These results are in accordance with the

linkage mapping data of ovine IL2 and correspond to the

physical location of the bovine IL2 on BTA 173.

Comments: The differences in the distance between the markers

OarFCB0048 and OarCP0016 described in this study in com-

parison to the SMC map (Fig. 1c) may be caused by using dif-

ferent mapping populations which can result in variable

recombination rates7.

Acknowledgements: We thank Tanja Gans and Manuela Uebel

for assistance in linkage mapping work. Gesine Luhken is

supported by the Deutsche Forschungsgemeinschaft (Gradu-

iertenkolleg Molekulare Veterinarmedizin, project 455).

References1 Montgomery G.W. & Sise J.A. (1990) NZ J Agri Res 33,

437–41.

2 Mezzelani A. et al. (1995) Mamm Genome 6, 629–35.

3 Chowdhary B.P. et al. (1994) Cytogenet Cell Genetics 65,

166–8.

4 Hayes H. et al. (1991) Cytogenet Cell Genet 57, 51–5.

5 Prinzenberg E.-M. et al. (1999) Anim Biotechnol 10, 49–62.

6 Pinkel D. et al. (1988) Proc Natl Acad Sci USA 85, 9138–42.

7 Thomsen H. et al. (2001) Mamm Genome 12, 724–8.

Correspondence: Gesine Luhken (gesine.luehken@agrar.uni-giessen.de)

SNPs in the porcine INHA gene and linkagemapping to SSC15

S. Hiendleder*,†, G. Reiner‡, H. Geldermann‡

and V. Dzapo*,†

*Department of Animal Breeding and Genetics, Justus-Liebig-

University, Giessen, Germany. †Central Biotechnical Unit Strahlen-

zentrum, Justus-Liebig-University, Giessen, Germany. ‡Institute for

Animal Husbandry and Animal Breeding, Division of Animal

Breeding and Biotechnology, University of Hohenheim, Stuttgart,

Germany

Accepted 26 February 2002

Source/description: a-Inhibin (INHA) is a subunit of the

dimeric glycoprotein hormone inhibin which is involved in

the reproductive axis. In the female, inhibins participate in

the regulation of pituitary follicle-stimulating hormone (FSH),

follicular maturation and steroidogenesis1. Mutations in the

porcine INHA gene have previously been detected by South-

ern blot analysis2. Porcine INHA complementary DNA

(cDNA) sequence (GenBank no. X03265) was used to design

primers.

Primer sequences:

Forward: CAC ATA TGT ATT CCG GCC.

Reverse: CCG TCT CGT ACT TGA AAG.

Polymerase chain reaction (PCR), sequencing and restriction digest

conditions: Reactions contained 100 ng of DNA, 10 pmol of

each primer, 200 lM dNTPs, 0.5 units of Pwo Polymerase

(Hybaid, Heidelberg, Germany), 1 mM MgSO4 and 2.5 ll PCR

buffer (100 mM Tris–HCl, pH 8.8, 250 mM KCl) in a final vol-

ume of 25 ll. The cycling protocol was 1.5 min at 94 �C, 35

cycles of 94 �C for 30 s, 52 �C for 1.5 min and 72 �C for

1.5 min, with a final extension at 72 �C for 5 min. The PCR

fragments were sequenced by standard procedures on a LICOR

4200 (MWG Biotech, Ebersberg, Germany). Digestion with

Hin6I (Hybaid, Heidelberg, Germany) was carried out according

to the manufacturers recommendations.

M GG AG GG AG AG AG AA AG AG AG

Figure 1 PCR–RFLP analysis of INHA polymorphism with Hin6I. The DNA

fragments were separated on a 2% agarose gel.

247Brief notes

� 2002 International Society for Animal Genetics, Animal Genetics, 33, 224–248

Polymorphism: Comparison of seven sequences derived from

Wild Boar, Large White, Meishan and Pietrain identified two

alleles (GenBank no. AY028465 and AY028466) with five

single nucleotide polymorphisms (SNPs) at bp 91 (G/A), 131

(C/T), 139 (A/G), 175 (G/A) and 184 (G/A) of the 731-bp PCR

fragment. Further comparisons with two cDNA sequences

(GenBank no. X03265 and M13980) showed additional SNPs

at bp 33 (G/A, Arg/His), 47 (A/G, Thr/Ala) and 730 (G/A). The

mutation at bp 184 destroys a Hin6I restriction site generating

a single fragment of 262-bp instead of 182 and 80-bp frag-

ments in addition to constant 245, 139 and 80-bp fragments

from the PCR product. The 262-bp fragment occurred with a

frequency of 0.24 in 29 unrelated individuals from the Duroc,

German Landrace, Large White and Meishan breeds, and Wild

boar. Five Meishan were homozygous for the 182 and 80-bp

fragments.

Mendelian inheritance: Segregation was consistent with

codominant inheritance (Fig. 1) and was recorded for 329

individuals of a three-generation resource pedigree3.

Chromosomal location: Typing of the Hin6I polymorphism in

three-generation resource families comprising of 329 individ-

uals3 and linkage analysis with four other markers (S0148,

EAG, Sw15, Sw2053) on SSC15 showed significant linkage to

Sw15 (two point lod score 3.26) and Sw2053 (two point lod

score 12.2). INHA was thus placed at 84 cM, distal of Sw2053.

This is consistent with chromosomal assignments of INHA to

SSC15 by somatic cell hybrid analysis4 and radiation hybrid

mapping5.

Comments: QTL for ovulation rates have been reported on

SSC15 at 51 cM6, 79 cM7 and 107 cM8, respectively. All but

one SNP are amenable to PCR–restriction fragment length

polymorphism (RFLP) analysis. The INHA SNPs therefore pro-

vide candidate gene markers for further QTL studies.

Acknowledgements: We thank H. Schomber for excellent tech-

nical assistance. Financial support from the H. Wilhelm

Schaumann Stiftung zu Hamburg to S.H. is gratefully

acknowledged.

References1 Knight P.G. (1996) Front Neuroendocrin 17, 476–509.

2 Hiendleder S. et al. (1995) Anim Genet 26, 131–2.

3 Geldermann H. et al. (1996) J Anim Breed Genet 113, 381–7.

4 Rettenberger G. et al. (1996) Mamm Genome 7, 275–9.

5 Robic A. et al. (1999) Mamm Genome 10, 565–8.

6 Rathje T.A. et al. (1997) J Anim Sci 75, 1486–94.

7 Rohrer G.A. et al. (1999) J Anim Sci 77, 1385–91.

8 Wilkie P.J. et al. (1999) Mamm Genome 10, 573–8.

Correspondence: Stefan Hiendleder (s.hiendleder@gen.vetmed.

uni-muenchen.de)

248 Brief notes

� 2002 International Society for Animal Genetics, Animal Genetics, 33, 224–248