for 30 s, and 72 �C for 30 s, preceded by 94 �C for 10 min.

Fifty nanograms of DNA was used for each reaction. The PCR

products were analysed by electrophoresis on 2% agarose gels

in 1.0· TBE buffer and stained with ethidium bromide.

Somatic hybrid cell and radiation hybrid panel assignments: Initial

bovine chromosomal assignment for the PTGDS gene was

obtained by PCR analysis using a somatic hybrid cell panel of

31 hamster/bovine and mouse/bovine hybrid somatic cell lines

described previously2. The bovine PTGDS gene was assigned on

BTA1 with significant concordant value of 97% to the previ-

ously mapped3 reference marker BM6438 (Fig. 1). A whole-

genome radiation hybrid (WGRH5000) panel4 composed of 90

selected radiation hybrid cell lines was used to determine the

specific chromosomal location for the PTGDS gene. PCR results

were scored as present (1), absent (0), or ambiguous (2). All

PCRs were performed twice and scored independently. RHM-

APPER software5 was utilized in assignment of PCR product to

a chromosome and identification of the nearest marker in the

bovine WGRH5000 map6. The LOD score of 10 was used as the

threshold for significant linkage. Two-point LOD scores showed

that PTGDS is linked to CSSM32 with a LOD score of 11.2 for a

h-value of 0.21.

Acknowledgements: We would like to thank Mrs Jan Elliott and

Ms Elaine K. Owens for providing the somatic cell and radiation

hybrids. This work was supported by FAPESP, Sao Paulo,

Brazil, Grant number 97/13403-1 for the project support to

M.E.J. Amaral.

References1 Urade Y. et al. (2000) Vitam Horm 58, 89–120.

2 Womack J. E. et al. (1986) J Hered 77, 2–7.

3 Bishop M. D. et al. (1994) Genetics 136, 619–39.

4 Womack J. E. et al. (1997) Mamm Genome 8, 854–6.

5 Slonin D. et al. (1997). J Comput Biol 4, 487–504.

6 Band M. R. et al. (2000) Genome Res 10, 1359–68.

Correspondence: M E J Amaral (eamaral@bio.ibilce.unesp.br)

Linkage mapping of SNPs in the porcinerelaxin gene

K. Wimmers, S. Chomdej, K. Schellanderand S. Ponsuksili

Institute of Animal Breeding Science, University of Bonn,

Endenicher Allee 15, 53115 Bonn, Germany

Accepted 21 May 2002

Source/description: Relaxin is a peptide hormone that is struc-

turally similar to insulin. It is involved in cyclic development of

follicles, maintenance of pregnancy and timing of parturition in

females as well as testicular functions in males. Relaxin

responsiveness has also been shown in mammary gland, skin and

small intestine1. The porcine relaxin gene, RLN, comprises two

exons that are separated by a 5.5 kilobase intron2. Primers were

designed from the published RLN sequence (GenBank accession

number J02792)1 and used to amplify overlapping fragments of

about 300–500 bp in size covering part of 5¢-untranslated region

(UTR), exon 1, part of intron 1, exon 2 and part of 3¢-UTR.

Screening for polymorphism of amplicons derived from pigs of five

breeds (Hampshire, Duroc, Pietrain, German Landrace, Berlin-

Miniature Pig) revealed two single nucleotide polymorphism

(SNPs) in exon 1 and intron 1, respectively.

Primer sequences:

Forward primer 1: 5¢-TGA AAC GCC TGG AGC AGA AG-3¢.Reverse primer 2: 5¢-GTT TCC AGC TGA GGC TCT TC-3¢.Reverse primer 3: 5¢-CAG CCA ATT AGG TCT CGA GC-3¢.

Polymerase chain reaction (PCR) conditions: The PCR amplifica-

tion was performed in 10 ll reaction volume using 25 ng of

genomic DNA, 0.2 lM of each primer, 50 lM of each dNTP,

0.5 U of Taq polymerase (Sigma Aldrich, Taufkirchen, Ger-

many), and reaction buffer with 1.5 mM MgCl2. Thermocycling

was performed using the following touchdown programme:

initial denaturation for 5 min at 95 �C, 10 cycles of 30 s at

95 �C, 30 s at 65–60 �C (–0.5 per cycle), and 30 s at 72 �C,

followed by 35 cycles of 15 s at 95 �C, 15 s at 60 �C and 30 s

at 72 �C and 5 min final extension at 72 �C.

Polymorphism, genotyping: A transversion (C > A) at position 1

of codon 8 (nt 22) in the first exon leading to a amino acid

exchange (L8I) was detected. For genotyping 228 bp PCR

products obtained with primers 1 and 2 were subjected to

single strand conformation polymorphism (SSCP) analysis by

diluting the products 1 : 1 with loading buffer (95% forma-

mide, 10 mM NaOH, 0.25% bromophenol blue and 0.25%

xylene cyanol), denaturing at 95 �C for 5 min, chilling on ice

and loading on 12% polyacryamide gels (acryla-

mide : bisacrylamide 49 : 1). Gels were run at 12 W for

2:30 hours at room temperature in 0.5· TBE. Banding pat-

terns were detected by silver staining.

Another transverion (T > G) was found at position 9 of the

intron altering the recognition site of CfoI. For genotyping of

this SNP PCR products of 331 bp in length obtained with

primers 1 and 3 were incubated with CfoI at 37 �C overnight.

Fragments of 289 and 42 bp in length were detected in agarose

gels for allele ‘t’ and of 209, 80 and 42 for allele ‘g’.

Mendelian inheritance, linkage mapping and allele frequen-

cies: Mendelian inheritance of both polymorphic sites was

monitored in 395 individuals of 21 families of the three

generation F2-DUMI resource population (Fig. 1). Linkage

analysis was performed using CRI-MAP package (version 2.4)

against four microsatellite markers of chromosome 1. The

multipoint linkage map was established using the BUILD and

FLIPS options (SW1515 – 33.0 cM – SW1851 – 31.4 cM –

S0155 – 7.6 cM – RLN – 56.5 cM – SW1301; sex-averaged

distances are given in Kosambi centimorgan). Two-point

linkage analysis revealed a recombination fraction of 0.08

� 2002 International Society for Animal Genetics, Animal Genetics, 33, 312–327

323Brief notes

between RLN and S0155 (LOD ¼ 18.3). The position of RLN

gene is in good agreement with the published physical

assignment to Sscr1q28–293.

Within the grandparental generation of the DUMI resource

population all Berlin-Miniature Pig grandparents (n ¼ 5) were

homozygous for allele ‘c’ at the SNP in exon 1, four Duroc

grandparents were homozygous (AA) one grandmother was

heterozygous at that site. In the parental generation (F1), two

boars were homozygous (CC) and 12 sows were heterozygous

(AC). The SNP was found to segregate among pigs of three

commercial breeds with allele ‘c’ being the prominent one

(Table 1).

At the SNP in intron 1 all Duroc grandparents (n ¼ 5) were

homozygous (TT). Four Berlin-Miniature Pig grandparents

were homozygous (GG) and one was heterozygous. All F1

animals (n ¼ 14) were heterozygous. All 93 animals of the

commercial of breeds were found to be homozygous for allele

‘t’ (Table 1).

References1 Haley J. et al. (1987) J Biol Chem 262, 11940–6.

2 Min G. et al. (1996) Biol Reprod 55, 1243–52.

3 Ellegren H. et al. (1994) Genomics 24, 342–50.

Correspondence: K. Wimmers (wimmers@itz.uni-bonn.de)

Isolation, polymorphism identificationand linkage mapping of the porcinehaptoglobin locus

S. Ponsuksili, K. Schellander and K. Wimmers

Institute of Animal Breeding Science, University of Bonn,

Endenicher Allee 15, 53115 Bonn, Germany

Accepted 21 May 2002

Source/description: Haptoglobin is an acute phase protein. This

glycoprotein of the a-2 globulin fraction facilitates removal of free

haemoglobin from the circulation of vertebrates, preventing loss

of iron and protecting kidneys from damage by haemoglobin after

injuries. In the pig, haptoglobin has been identified as an excel-

lent marker for acute phase response in association with infec-

tions or pathological lesions1. However very little information is

available about the effect of haptoglobin gene polymorphisms on

immunological traits. The complementary DNA (cDNA) se-

quence of the haptoglobin porcine gene (HP) was determined by

5¢ and 3¢-RACE (SMARTTM RACE cDNA Amplification Kit,

Clontech, Heidelberg, Germany) starting with primers derived

from an expressed sequence tag (EST) (GenBank accession

number BF7136822). The cDNA is 1182 bp in length (GenBank

accession number AF492467) with 138 bp of 3¢ untranslated

region, exhibits 83% identity to the human haptoglobin a 1S. The

Figure 1 Mendelian inheritance of the SNPs in

exon 1 and in intron 1 of relaxin gene.

Allele frequency

SNP22 – exon 1 SNP9 – intron 1

Breeds No. of animals ‘a’ ‘c’ ‘g’ ‘t’

German Landrace 31 0.06 0.94 0.00 1.00

Large White 27 0.02 0.98 0.00 1.00

Pietrain 35 0.11 0.89 0.00 1.00

Table 1 Frequencies of SNP alleles within the

relaxin locus of commercial breeds.

� 2002 International Society for Animal Genetics, Animal Genetics, 33, 312–327

324 Brief notes