Animal Genetics, SHORT COMMUNICATION

Assignment of the gene for porcine insulin-like growth factor 1 (IGF1) to chromosome 5 by

1994 25,37-39

linkage mapping A K Winter@, M Fredholm, L Andersson

A K Wintere M Fredholm Department of Animal Genetics, The Royal Veterinary and Agricultural University, Biilowsvej 13, 1870 Frederiksberg C, Copenhagen, Denmark

L Andersson Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Box 7055, S-75007 Uppsala, Sweden

Summary

Investigation of published sequence data from the porcine insulin-like growth factor 1 (IGF1) gene, resulted in the detection of a microsatellite in the first intron of the gene. Polymerase chain reaction (PCR) primers flanking the (CA),, repeat were constructed. Polymorphism and Mendelian segregation were documented in a three-gener- ation pedigree and allele frequencies were determined in 74 unrelated animals from four different breeds. Seven alleles were encountered. Linkage analysis was performed in a large pedi- gree established for gene mapping. Linkage between the IGFl microsatellite and an anony- mous microsatellite marker, S0005, was detected. Furthermore, IGFl and SO005 was found to be linked to the porcine submaxillary gland mucin (MUC) gene, previously assigned to chromosome 5. The results presented here extend the linkage group on pig chromosome 5 and are in accordance with conserved synteny between human chromosome 1 2 , cattle chromo- some 5, mouse chromosome 10 and pig chromo- some 5.

Keywords: microsatellites, pigs, IGFl, linkage analysis, conserved synteny

The rapid advances in gene mapping technolo- gies have led to development of gene maps in many mammalian species. To facilitate the pro- gress in this development, and to exploit information obtained across species, two types of anchor loci have been recognized as essential to the genetic map of a species (O’Brien & Graves 1991): Type I anchor loci, which are evolutionary conserved loci (usually structural genes) and Type I1 anchor loci which are species-specific, highly polymorphic markers. In this study both types of anchor loci have been employed result- ing in an extension of the linkage map on porcine chromosome 5.

Correspondence: A K Wintere.

Accepted 28 August 1993

Genetic variation at the porcine insulin-like growth factor 1 gene (IGF1) was investigated by analysis of the microsatellite sequences which have been reported to be present in the first intron of the IGFl gene (EMBL accession number X64400; Kirkpatrick 1992). The polymorphism in the IGFl microsatellite and the anonymous microsatellite SO005 (Fredholm et al. 1993; Gen- bank accession number M97232) was used for linkage analysis resulting in the assignment of the two loci to a previously reported linkage group (Ellegren et al. 1993) comprising the loci for interferon y (IFNG), diacylglycerol kinase (DAGK) and the anonymous microsatellite S0092. Furthermore, linkage to the porcine submaxillary gland mucin (MUC) gene, that has been assigned to chromosome 5 by in situ hybrid- ization and linked to the above-mentioned link- age group by restriction fragment length polymorphism (RFLP) analysis (Johansson et al. 1993) was established.

The sequence of the porcine IGFl gene was obtained by computer search in the EMBL data- base version 1 (EMBL accession number X64400). An (AC),, repeat was identified in the first intron at positions 961 to 1002. Primers flanking the repeat unit were designed to amplify a 290bp fragment: A, 5’ACCCTTGAGAGGG-

GATTCTGCTGAGCTG 3’. Optimal PCR conditions for the primers were determined as previously described (Winter0 et al. 1992).

For routine typing of the IGFl locus, micro- satellites were amplified with 32P end-labelled primer, 1 . 5 m ~ MgC12 and an annealing temperature of 65°C. For routine typing of the SO005 locus 1.5mM MgClz and an annealing temperature of 59°C was used. PCR products were size fractionated by electrophoresis on denaturing polyacrylamide gels. Mendelian seg- regation was confirmed by testing a backcross family with a total of 45 animals (results not shown).

IGFl allele frequencies were determined in 74 unrelated animals (no parents or grandparents in common) from four purebred swine breeds (n = 2 1 , 21, 11 and 2 1 individuals from the Duroc,

37

TATTGCTAGGC 3’: B, 5‘GGGCAAA-

38 Cytogenet ic m a p Winter@, Fredholm, Human rshromosome I ?

Andersson ~-

12

13 1

"1 , ..

13 IGFl

Cytogenet ic map Pig c h r o m o s o m e 5

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I ?

I 1

11

12

33

IFNG

MUC'

Genet ic m a p Pig rhroniosomt. 5

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so005

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Fig. 1. Cytogenetic map of human chromosome 12 and cytogenetic and genetic map of pig chromosome 5. This shows that IGFl and IFNG are close together on the q-arm in humans, but far apart in pigs as determined by linkage analysis and in situ localization. In contrast, IGFl and LDBH are on each side of the centromere in humans but on the distal part of the q-arm in pigs

Danish Landrace, Hampshire and Yorkshire breeds respectively). As shown in Table 1, a total of seven different allelic variants were detected with the frequencies differing significantly between breeds. The allele frequencies are clearly different from those reported previously for US pig population (Kirkpatrick 1992), indi- cating a certain genetic divergence at this locus between breeds. Frequencies of the SO005 alleles are published elsewhere (Fredholm ei al. 1993).

Linkage analysis was performed in a reference pedigree developed for gene mapping by crossing two European wild pigs (males) with eight domestic pigs (females) of a Large White breed (Swedish Yorkshire). Twenty-six F1 animals (4 males and 22 females) were selected and intercrossed to generate an F2 generation comprising 200 individuals (Johansson et al. 1992). Linkage analysis was carried out with the lod score method using the LINKAGE package version 5.1 (Lathrop & Lalouel 1988), on a Sparcstation-I1 (Sun Microsystems). The IGF1 and SO005 loci were tested for linkage against more than 100 polymorphic markers so far typed

Table 1. IGFl (microsatellite) allele frequencies in four different breeds (n is the number of individuals genotyped)

Allele size Duroc Landrace Hampshire Yorkshire (b) (n = 21) (n = 21) ( n = 11) (n = 21)

278 0.05 280 0.02 286 0.36 0.57 0.09 0.21 288 0.31 0.12 0.14 0.02 290 0.14 0.26 0.68 0.43 292 0.19 0.02 0.09 0.24 294 0.05

Table 2. Summary of two-point lod score analyses among four genetic markers on pig chromosome 5

Loci 8 2

IGF1-IFNG 0.329 4.6 IGF1-SO005 0.212 23.0 IGF1-MUC 0.103 361.6

S0005-IFNG 0,165 191.7 S0005-MUC 0.1 76 23.2 IFNG-MUC* 0.272 5.7

* Previously reported by Johansson et al. 1993.

in the Swedish pedigree (Ellegren et al. 1993; Leif Andersson et al., unpublished). Both IGFl and SO005 could be firmly assigned to porcine chromosome 5 by highly significant lod scores to IFNG and MUC; the last two loci were recently mapped to this chromosome by in situ hybridiz- ation and linkage analysis (Johansson et al. 1993). A summary of the two-point lod score analysis among the four loci is given in Table 2 , A multipoint analysis using ILINK provided clear evidence for the order IFNGS0005-MUC-IGF1 (odds 3 x l o6 against the next most likely order). The genetic map together with map distances in Kosambi cM based on these data are depicted in Fig. 1.

A conserved synteny between human chromo- some 12, cattle chromosome 5, mouse chromo- some 10 and pig chromosome 5 has previously been reported (Threadgill et al. 1990; O'Brien & Graves 1991; Johansson et al. 1993). The assign- ment of the porcine IGF1 gene to the distal part of chromosome 5q is consistent with this model since the corresponding genes are located on human chromosome 12q23, cattle chromosome 5

39 Assignment of porcine IGFl

and mouse chromosome 10 (O’Brien & Graves 1991).

The previously conserved syntenies have been based on the chromosomal localizations of mito- chondrial citrate synthase (CS) (human chromo- some 12pll-qter, cattle chromosome 5p, mouse chromosome 10 and pig chromosome 5p), pepti- dase B (PEPB) (human chromosome 12q21, cattle chromosome 5, mouse chromosome 10 and pig chromosome 5 (inconsistent)), interferon y (INFG) (human chromosome 12q24.1, cattle chromosome 5, mouse chromosome 10 and pig chromosome 5) and lactate dehydrogenase B (LDHB) (human chromosome 12~12.1-12.2, cattle chromosome 5, mouse chromosome 6 , and pig chromosome 5). However, the fact that IGFl and IFNG are far apart on pig chromosome 5 but close to each other in man and mouse, implies that a chromosomal rearrangement must have occurred since the divergence from a common ancestor. In support of this is the localization of LDHB to 12~12.2-p12.1 in humans and to the distal part of the q-arm on pig chromosome 5 (Ryttman et al. 1986). The similarity between man and mouse suggests on the basis of parsi- mony, that the rearrangement is more likely to have occurred in the porcine evolutionary lineage.

Acknowledgements

The authors wish to thank Mette Sorensen and Lene Stangerup for excellent technical assis- tance. The work was funded by the Animal Biotechnology Research Center, the Nordic Joint Committee of Agricultural Research (NJCAR) and the BRIDGE programme of the Commission of the European Communities.

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