Mapping, Polymorphisms and Use for Quantitative Trait Loci Identiﬁcation for Growth and Carcass Traits in a Meishan · Pie´Train Intercross

SHORT COMMUNICATION doi:10.1111/j.1365-2052.2006.01480.x Porcine OGN and ASPN: mapping, polymorphisms and use for quantitative trait loci identiﬁcation for growth and carcass traits in a Meishan · Pie´train intercross

A. Stratil*, M. Van Poucke†, H. Bartenschlager‡, A. Knoll*,§, M. Yerle¶, L. J. Peelman†, M. Kopecˇny´* and H. Geldermann‡ *Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libeˇ chov, Czech Republic. †Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium. ‡Department of Animal Breeding and Biotechnology, Institute of Animal Husbandry and Breeding, University of Hohenheim, Garbenstrasse 17, D-70593 Stuttgart, Germany. §Department of Animal Morphology, Physiology and Genetics, Mendel University of Agriculture and Forestry, Zemeˇ deˇ lska´ 1, 613 00 Brno, Czech Republic. ¶INRA, Laboratoire de Genetique Cellulaire, 31326 Castanet-Tolosan, France

Summary The porcine orthologues of human chromosome HSA9q22.31 genes osteoglycin (OGN) and asporin (ASPN) were mapped to porcine chromosome SSC3 using linkage analysis and a somatic cell hybrid panel. This mapping was refined to SSC3q11 using fluorescence in situ hybridization. These results confirm the existence of a small conserved synteny group between SSC3 and HSA9. Polymorphisms were revealed in both genes, including a pentanucleotide microsatellite (SCZ003)inOGN and two single nucleotide polymorphisms (AM181682.1:g.780G>T and AM181682.1:g.825T>C) in ASPN. The two genes were included in a set of markers for quantitative trait loci (QTL) mapping on SSC3 in the

Hohenheim Meishan · Pie´train F2 family. Major QTL for growth and carcass traits were centred in the ASPN–SW902 region.

Keywords asporin, ﬂuorescence in situ hybridization mapping, linkage analysis, osteoglycin, quantitative trait loci, somatic cell hybrid mapping.

The osteoglycin or mimecan (OGN; formerly known as OIF) to SSC3 by radiation hybrid (RH) mapping (Rink et al. and asporin (ASPN) genes belong to the small secreted leu- 2002; Mikawa et al. 2004). More recently, Meyers et al. cine-rich proteoglycans (SLRP) family, which are important (2005) mapped four porcine loci (one expressed sequence for collagen fibrillogenesis, cellular growth, differentiation tag and three BAC-end sequences) orthologous to HSA9 and migration (Tasheva et al. 2004). The two genes, to- sequences to SSC3 by RH mapping, and they assumed that gether with two other members of the SLRP family, extra- these loci were located on SSC3q11 near the centromere cellular matrix protein 2 (ECM2) and osteoadherin (OMD), using cytogenetic information. In this article, we provide form a cluster that is located on human chromosome further evidence that a small syntenic segment homologous HSA9q22.31 (Henry et al. 2001) between TPM2 and to HSA9 is located on SSC3q11 within a quantitative trait TGFBR1 (http://www.ensembl.org/Homo_sapiens/). TPM2 loci (QTL) interval for growth and carcass traits. Polymor- and TGFBR1 were previously mapped to porcine chromo- phisms identified in the two genes were added to a set some SSC1 (Kopecˇny´ et al. 2002, 2004), in agreement with of markers and used for QTL mapping of SSC3 in a heterologous chromosome painting (Goureau et al. 1996). Meishan · Pie´train (M · P) intercross. Based on these results, the location of OGN and ASPN on The Hohenheim M · P family used for linkage analysis SSC1 would be expected. and QTL mapping was previously described by Geldermann However, single porcine loci that were orthologous to et al. (2003). Unrelated pigs of eight breeds (Large White, human chromosome 9 sequences were previously assigned Landrace, Czech Meat Pig, Pie´train, Black Pied Prestice, Hampshire, Duroc and Meishan) were used to estimate Address for correspondence frequencies of the OGN and ASPN alleles. Genomic A. Stratil, Institute of Animal Physiology and Genetics, Academy of fragments specific for OGN (2007 bp) and ASPN (1493 bp) Sciences of the Czech Republic, 277 21 Libeˇ chov, Czech Republic. were obtained by polymerase chain reaction (PCR) using E-mail: [email protected] primers presented in Table S1 and standard PCR conditions. Accepted for publication 5 May 2006

2006 The Authors, Journal compilation 2006 International Society for Animal Genetics, Animal Genetics, 37, 415–418 415 416 Stratil et al.

PCR fragments from Pie´train and Meishan pigs were se- Chromosome-wide QTL mapping was performed using a quenced either directly (ABI PRISM 310 Genetic Analyzer; least-squares method developed for the analyses of crosses Applied Biosystems, Foster City, CA, USA) or after cloning between outbred lines (Haley et al. 1994). The present into plasmids. analysis was based on 10 genetic markers (SW72, S0206, The sequences of the fragments of OGN (AM181681) and ASPN, OGN, SW902, SW828, SW314, APOB, SW349 and ASPN (AM181682) were orthologous to human OGN SW2532; Beeckmann et al. 2003 and this study) that were (partial exons 6 and 7 and the intervening intron) and included in the linkage map with a total map length of

ASPN (partial exons 4 and 5 and the intervening intron). 135 cM. From the marker data for each F2 animal the A polymorphic composite pentanucleotide microsatellite probabilities for the inheritance of the alleles from each (SCZ003) with repeat unit TAACA/TAACG was found in founder breed in steps of 1 cM were calculated as additive intron 6 of OGN by sequencing. It was scored on an ABI and dominant components. These components were inclu- PRISM 310 Genetic Analyzer (Applied Biosystems). In the ded in a generalized linear model procedure with further ASPN fragment, two single nucleotide polymorphisms continuous (age at slaughter) and discontinuous (2-month (SNPs) (g.780G>T and g.825T>C) could be resolved by PCR classes as seasonal factor, sex, litter number) independent restriction fragment length polymorphism (RFLP) using variables to calculate additive and dominance effects at a Hin1II (NlaIII) and TaqI respectively. Digestion with TaqI given position of a putative QTL for each trait. yielded either the uncut fragment of 1493 bp (allele C)or Codominant inheritance of SCZ003 (in OGN) and SNP the cut fragments of 847 and 646 bp (allele T). g.825T>C (in ASPN) alleles was confirmed in the Hohen- Linkage mapping of OGN (with SCZ003) and ASPN (with heim M · P three-generation pedigree (Geldermann et al. the TaqI PCR-RFLP) was performed using the CRI-MAP soft- 2003). Allele frequencies in unrelated pigs are presented in ware package (Green et al. 1990). For cytogenetic mapping Table 1. These genes were mapped by linkage analysis to using a porcine–rodent somatic cell hybrid panel (Yerle chromosome 3 as follows (distances in Kosambi cM; sex et al. 1996), PCR was performed with primers (Table S1) averaged values): SW72 – 24.9 – S0206 – 13.7 – ASPN – 0.6 specific for OGN (OGN-F3, R3) and ASPN (ASPN-F2, R2). – OGN – 6.9 – SW902 – 16.7 – SW828 – 30.1 – SW314 – The results were analysed using the tools available at http:// 8.4 – APOB – 16.0 – SW349 – 17.7 – SW2532. Both genes www.toulouse.inra.fr/lgc/pig/pcr/pcr.htm and the statisti- were also mapped to SSC3 using the somatic cell hybrid cal rules defined by Chevalet et al. (1997). panel, and more specifically to SSC3p11, with a probability The BAC ends of porcine genomic clone 253B1 (Rogel- for regional mapping of 0.78 and 0.72, a correlation of 0.86 Gaillard et al. 1999) contain homologous sequences to a and 0.80 and error risk lower than 0.1% for OGN and ASPN human clone (AL137848), which harbours OGN and ASPN, respectively. As this assignment to SSC3 was unexpected, we and an overlapping neighbour (AL157827). The presence of confirmed the location on SSC3 using FISH mapping of the ASPN in BAC 253B1 was confirmed by PCR (using primers BAC clone containing ASPN on porcine metaphase spreads. ASPN-F2, R2; Table S1). The isolated BAC DNA was then The analysis of fifteen metaphase spreads allowed us to refine used as probe for fluorescence in situ hybridization (FISH) the mapping of ASPN to SSC3q11 (Fig. S1). mapping. Briefly, 100 ng of total BAC DNA was labelled by ASPN and OGN were linkage mapped in the Hohenheim

incorporation of biotinylated 16-dUTP using random pri- M · PF2 family between markers S0206 and SW902 on ming. Hybridization was carried out for 15 h at 37C. SSC3. This region corresponds to the QTL interval for sev- Standard protocols were used for post-hybridization wash- eral carcass traits signiﬁcant at the P < 0.05 genome-wide ings and revelation of ﬂuorescent signals. level (Beeckmann et al. 2003). The analysis of polymor-

Table 1 Allele frequencies for microsatellite SCZ003 in porcine osteoglycin and for single nucleotide polymorphism g.825C>T (TaqI) in porcine asporin.

SCZ003 g.825C>T

Breed n 259 269 274 279n C T

Large White 16 0.031 0.813 0.125 0.031 14 0.179 0.821 Landrace 14 0 0.821 0.179 0 17 0.147 0.853 Czech Meat Pig 14 0 0.786 0.214 0 14 0.214 0.786 Pie´ train 23 0 0.435 0.391 0.174 23 0.522 0.478 Black Pied Prestice 7 0 0.643 0.214 0.143 7 0.357 0.643 Hampshire 7 0 0.786 0.214 0 6 0.167 0.833 Duroc 14 0 0.964 0.036 0 9 0.056 0.944 Meishan 8 0 0 0 1.000 12 1.000 0

2006 The Authors, Journal compilation 2006 International Society for Animal Genetics, Animal Genetics, 37, 415–418 Mapping of porcine OGN and ASPN and use for QTL identiﬁcation 417

Table 2 Signiﬁcant QTL effects on SSC3.

Trait cM F-ratio VF2(%) a ±SE d ±SE

Loin and neck meat weight (kg) 43.2 12.0*** 6.7 )0.286 ± 0.090 0.528 ± 0.134 Shoulder meat weight without external fat (kg) 43.2 10.4*** 5.8 )0.072 ± 0.049 0.324 ± 0.073 Daily gain (110–210 days) (g/day) 46.1 10.3*** 5.7 )28.872 ± 10.112 52.804 ± 14.358 Carcass length (cm) 44.2 9.2** 5.1 )0.675 ± 0.472 2.868 ± 0.695 Meat area on Mld at 13th/14th rib (cm2) 39.2 8.8** 4.9 )0.787 ± 0.401 2.221 ± 0.581 Ham meat weight without external fat (kg) 42.2 8.5** 4.7 )0.143 ± 0.099 0.581 ± 0.147 Half carcass weight (kg) 46.1 7.2* 3.9 )0.785 ± 0.522 2.638 ± 0.742 Ham weight including bones and external fat (kg) 44.2 6.2* 3.3 )0.152 ± 0.142 0.716 ± 0.209 Weight of head (kg) 46.1 6.1* 3.2 )0.081 ± 0.063 0.300 ± 0.090 Food conversion ratio (kg/kg) 32.9 6.0* 3.2 0.232 ± 0.075 )0.191 ± 0.116 Fat-to-meat ratio (area at 13th/14th rib) (cm2/cm2) 35.9 5.9* 3.1 0.017 ± 0.016 )0.082 ± 0.025 Weight of bacon meat relative to lean cuts weight (%) 46.1 5.6* 2.9 0.401 ± 0.124 )0.172 ± 0.176

Signiﬁcant at *P < 0.05 chromosome-wide threshold; **P < 0.05 genome-wide threshold; ***P < 0.01 genome-wide threshold. QTL, quantitative trait locus; SSC3, Sus scrofa chromosome 3; a, additive effect (positive/negative signs indicate the superior/inferior trait values inherited from the paternal resource group); d, dominance effect (positive for higher values of heterozygous individuals than the mean of homo- zygotes; negative for lower values); VF2(%), percentage of F2 phenotypic variance explained by the QTL; Mld, Musculus longissimus dorsi.

acknowledge technical assistance of Marie Datlova´ and Libusˇe Koberova´. This research was supported by the Czech Science Foundation (grant nos 523/03/0858 and 523/06/ 1302) and IRP IAPG (no. AV0Z50450515).

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