J. Anim. Breed. Genet. ISSN 0931-2668

ORIGINAL ARTICLE A pig–human comparative RH map comprising 20 on pig 13q41 that harbours the ETEC F4ac receptor J. Ren1*, H. Tang1*, X. Yan1,3, X. Huang1, B. Zhang1,H.Ji1, B. Yang1, D. Milan2 & L. Huang1

1 Key Laboratory for Animal Biotechnology of Jiangxi Province and the Ministry of Agriculture of China, Jiangxi Agricultural University, Nanchang, 330045, China 2 Institut National de la Recherche Agronomique (INRA), Laboratoire de Ge´ ne´ tique Cellulaire, BP27, 31326, Castanet Tolosan Cedex, France 3 Department of Biology, Nanchang University of Science and Technology, Nanchang 330038, China

Keywords Summary Comparative map; ETEC F4ac receptor; SSC13q41. The enterotoxigenic Escherichia coli (ETEC) F4ac is a major cause of diar- rhoea in newborn and young pigs. The locus for the intestinal ETEC Correspondence F4ac receptor (F4acR) has been mapped to pig chromosome (SSC) Lusheng Huang, Key Laboratory for Animal 13q41 with known homology to human chromosome (HSA) 3q21 and Biotechnology of Jiangxi Province and the q29. However, the causative and mutation(s) remain unknown. Ministry of Agriculture of China, Jiangxi The aim of this study was to characterize gene-derived markers on Agricultural University, Nanchang, 330045, China. Tel: 0086-791-3805967; SSC13q41 for fine mapping of the F4acR locus, and construct a high- Fax: 0086-791-3900189; resolution pig–human comparative map to select positional candidate E-mail: [email protected] genes for F4acR. Pig-specific sequence-tagged site markers were devel- oped for 20 genes that are located in a 6.8-Mb region on HSA3q21 and *Both the authors contributed equally q29, and a total of 34 single-nucleotide polymorphisms (SNPs) were to this study. identified in 14 of 20 markers developed. Eighteen markers were mapped to SSC13q41, while the other two markers (PLXNA1 and Received: 13 November 2007; accepted: 18 May 2008 KLF15) were assigned to SSC13q32 and SSC7q13, respectively, by radia- tion hybrid mapping. This result showed that there was a small con- served segment on SSC7 corresponding to HSA3q21. A framework map comprising 18 markers on SSC13q41 was established, refining the synte- ny breakpoint on SSC13q41 to a region of 12.3 centiRay. The compara- tive radiation hybrid (RH) map revealed three interesting candidate genes for F4acR from the , viz. MUC4, MUC13 and MUC20. Linkage analysis with six marker polymorphisms revealed that MUC4 had the most significant linkage with the F4acR locus.

7 cM flanked by Sw207 and S0283, close to Muc4 on Introduction SSC13q41 (Joller et al. 2006). However, the causative Pig chromosome (SSC) 13q41 is of particular interest gene and mutation(s) remain unknown. to the pig industry because of the presence of econom- To date, comparative mapping data between pig ically important gene(s) encoding the enterotoxi- and humans have shown that SSC13q41 shares genic Escherichia coli (ETEC) F4ab ⁄ ac receptor(s), homologies with human chromosome (HSA) 3q21 which control susceptibility ⁄ resistance to ETEC and 3q29, and has conserved gene orders with the F4ab ⁄ ac-caused neonatal diarrhoea in pigs (Python same direction on HSA3q29 whereas the inverted et al. 2002, 2005; Jørgensen et al. 2003). More direction on HSAq21 (Sun et al. 1999; Van Poucke recently, the position of the ETEC F4ac receptor et al. 1999, 2001, 2003; Rink et al. 2002, 2006; (F4acR) locus was refined to a region of approximately Meyers et al. 2005). The synteny breakpoint in the

ª 2009 The Authors doi:10.1111/j.1439-0388.2008.00751.x Journal compilation ª 2009 Blackwell Verlag, Berlin • J. Anim.Breed. Genet. 126 (2009) 30–36 J. Ren et al. Comparative RH map of SSC13q41 pig was markedly refined from an initial 100-centi- RH mapping Ray(cR) region between zinc finger 148 and transferrin receptor (Van Poucke et al. 2001; Rink et al. Radiation hybrid mapping of MUC4 was performed 2002) to a region of 23 cR flanked by anonymous as described previously (Peng et al. 2007). For other markers CL408784 and CL343765 (Humphray et al. markers, the hybrid DNA of 90 clones of IMpRH7000 2007). However, its exact location remains panel (Yerle et al. 1998), hamster DNA (parallel con- unknown. Moreover, a majority of markers on trol), porcine genomic DNA (positive control) and SSC13q41 are anonymous markers; the actual genes water (negative control) were amplified in each within this region have been poorly defined. The assay. The PCR products were separated in 2% aga- aim of this study was hence to develop a set of pig- rose gels and visualized with ethidium bromide specific sequence-tagged site (STS) polymorphic staining. Each marker was analysed at least in dupli- markers in a target region close to S0283 on cate. Markers were scored as positive (1), negative SSC13q41 for fine mapping of the F4acR locus, and (0) or ambiguous (?) for each hybrid, and consensus construct a high-density, framework-supported RH vectors were initially subjected to the IMpRH web comparative map to select interesting positional can- server (http://imprh.toulouse.inra.fr/) to obtain max- didate genes for F4acR. imum two-point lod scores for regional assignments. The Carthagene software (de Givery et al. 2005) was then used to build a framework-supported RH map Material and methods for those markers. Linkage groups were defined with Marker selection and primer design a two-point Logarithm of the odds ratio (LOD) score Fifteen genes on HSA 3q21.1–21.2 and five genes on threshold of 6 and a distance threshold of 50 cR. A HSA 3q29 (Table 1) were selected for marker devel- 1000:1 framework map was constructed with a step- opment. Genes were targeted by their locations in wise locus-adding strategy under the haploid model the human genome (NCBI Build 36.1). Available of fragment retention and then checked by a flips porcine or other mammalian expressed sequence tag algorithm. After validation under the haploid model, (EST) sequences (Table 1) showing significant simi- the final framework map was recomputed under a ) larity with a significance threshold E value of e 5 diploid model, and the most likely marker order and were selected for primer designing through the distance between markers were estimated. Markers UCSC genome browser (http://genome.ucsc.edu/cgi- not included in the frameworks were projected on bin/hgGateway). Primers (Table 1) were designed the framework map one by one and the distance with the Primer Premier 5.0 software (Premier Bio- was considered as fixed between framework mark- soft, Palo Alto, CA, USA). ers. Comparative mapping was realized based on data in the human genome assembly (http:// www.ncbi.nlm.nih.gov/mapview/, Build 36.1), and a Amplification and sequencing map was drawn using the MapChart software

Porcine genomic DNA of a 240-day F2 animal in a (Voorrips 2002). Synteny break refers to adjacent White Duroc · Erhualian intercross was used as a porcine markers that do not match their predicted, template. Amplifications were performed in a rou- corresponding human markers. tine way with magnesium concentration of 1.5 mm and optimal annealing temperature and extension Linkage mapping of the F4acR locus time (Table 1). The PCR products were purified with the QIAquick DNA Purification Kit (Qiagen, Hilden, Six marker polymorphisms including MUC4 intro- Germany) and bidirectionally sequenced with the n17 g.243A>G (Peng et al. 2007), MUC13 c.935A>C respective polymerase chain reaction (PCR) primers. (Zhang et al. 2008), MUC20 g.191C>T (Ji HY, Ren J, The identities of amplicons were checked using the Yan XM, Huang X, Zhang B, Huang LS. unpublished blastn program via the NCBI BLAST server (http:// data), SLC12A8 g.159A>G, MYLK g.1673A>G and www.ncbi.nlm.nih.gov/BLAST/). Primers that failed KPNA1 g.306A>G (Huang et al. 2008) were geno- to produce porcine-specific products or porcine typed across the entire population of the White products apparently distinguished from hamster Duroc · Erhualian intercross as described in other products were discarded, and new primers studies. Pairwise linkage analysis was performed (Table S1) were designed from intronic sequences with crimap version 2.4 (Green et al. 1990). The obtained with the original EST primers for subse- option ‘TWOPOINT’ was used to find the linked quent RH mapping. markers with an LOD score higher than three. The

ª 2009 The Authors Journal compilation ª 2009 Blackwell Verlag, Berlin • J. Anim. Breed. Genet. 126 (2009) 30–36 31 32 Table 1 PCR primers and conditions for marker development SSC13q41 of map RH Comparative

a Tm ET Size Sequence identity with other Marker Gene name HSA3 (Mb) Forward primer (5¢–3¢) Reverse primer (5¢–3¢) (C) (s) (bp) Acc. no. sequences (acc. no.) Polymorphismc

KPNA1 Karyopherin alpha 1 q21.1 (123.62) tggagaacaagaagccaaaag cccgaagtaatgctcaataag 61 120 2500 EF443103 55 bp: 96% (Pan XM_516692) A ⁄ C46 ADCY5 Adenylate cyclase 5 q21.1 (124.49) atctggggcaatacsgtgaac aggtcatcatctcgcctttgc 56 120 2000 DQ885462 72 bp: 98% (Bos CO974530) A ⁄ G143 ,C⁄ G310 PTPLB protein tyrosine q21.1 (124.69) ttacataaaacatcctggc acacactggggttgagac 55 120 2100 EF443104 72 bp: 94% (Bos BG223786) C ⁄ T507 phosphatase-like member B MYLK Kinase related protein q21.1 (124.81) cgcatcattgacgaggactttg gaggaccttcagagaccccgc 64 60 1200 DQ885466 133 bp: 100% (Sus AJ684369) G ⁄ A1473 ,G⁄ A1567 , G ⁄ A1559 ,A⁄ G1473 CCDC14 Coiled-coil domain q21.1 (125.12) gagccaggaattactgcagag agtcacctcagcatcacgc 63 30 370 DQ885463 140 bp: 92% (Bos CF766925) A ⁄ G134 containing 14 RPON1 ropporin, rhophilin q21.1 (125.17) tgttatcctctgaccacgac gcacaatcatctctgtatcttc 57 60 850 EF443105 136 bp: 100% (Sus BI343536) G ⁄ A390 ,C⁄ G494 , associated protein 1 G ⁄ A496 ,T⁄ A572 KALRN Kalirin q21.2 (125.30) ctgtgtttctaagacttgactg tgttcaagaagagcatcaag 57 30 600 EF443102 106 bp: 100% (Sus BP148678) C ⁄ A172–3 ,A⁄ T509 , T ⁄ G579 ,G⁄ T582 , G ⁄ T590–1 ITGB5 Integrin beta 5 q21.2 (125.96) gtggatggtgacgagcagc ggaggctgtgctttgtttctac 62 90 1800 EF443101 107 bp: 100% (Sus BI467544) C ⁄ T536 205 293

ora compilation Journal MUC13 Mucin 13 q21.2 (126.11) tgagcaagatgagtgccccagt tagccaggcaggcacaagca 67 30 536 EF443099 186 bp: 100% (Sus BW976511) G ⁄ A ,A⁄ G , C ⁄ T371 ,C⁄ T501 HEG1 HEG homologue 1 q21.2 (126.17) ggtttccacagttgagwccac catgtgaagatggatataggc 60 30 200 EF443100 37 bp: 100% (Homo BC004539) G ⁄ A100 ,T⁄ A174 SLC12A8 solute carrier family 12, q21.2 (126.28) accaacttctttctacacccg gaaatacacaatggcagcaac 58 30 750 EF443107 128 bp: 89% (Homo AF389851) G ⁄ A159 ,C⁄ T397 member 8 ZNF148 zinc finger protein 148 q21.2 (126.43) gcctttagaacgaactatcac aggtacttctgtatgaaacgc 60 90 1500 EF443106 139 bp: 88% (Pan XM_516711) None ª ROPN1B ropporin, rhophilin q21.2 (127.17) gtgagggagcggtctgaacg aatctcctccgtgaagcagcc 67 120 2000 DQ885468 29 bp: 100% (Sus CJ038963) None 09BakelVra,Berlin Verlag, Blackwell 2009 associated protein 1B KLF15 Kruppel-like factor 15 q21.2 (127.54) ctcgtcgccgaaatgcccg gcacctgagccaccgctgtag 69 30 174 DQ885465 174 bp: 100% (Sus BP453728) None PLXNA1 Plexin A1 q21.2 (128.19) accctggctgctcggaatctg acaggttcacgggcaggtcac 65 90 1700 DQ885467 97 bp: 100% (Sus BX916678) T ⁄ G253 MUC20 Mucin 20 q29 (196.93) gagacaccagccacccgtag tgtgtcttcattggagtcaggc 67 30 430 EF443098 392 bp: 100% (Sus DN104443) G ⁄ A87 ,G⁄ A120 MUC4b Mucin 4 q29 (196.96) caggatgcccaatggctctac ccccgaagttgtgaaaggaag 65.5 30 538 DQ124298 158 bp: 99% (Sus BF075199) A ⁄ G87 ,A⁄ G120 SENP5 SUMO1 ⁄ sentrin-specific q29 (198.08) ttccacaacagaaaaacgacag tccttgtaaatcctcttccgc 62 60 1300 DQ885461 32 bp: 100% (Sus AY610504) None protease 5

• DLG1 Drosophila discs, large q29 (198.26) tattgggacctatgaaagacag ctttttccatttgctctcttg 60 30 400 DQ885464 82 bp: 100% (Sus AJ654156) None .Ai.Bed Genet. Breed. Anim. J. homologue 1 LMLN Leishmanolysin-like q29 (199.17) actgtctacgataaaagcattg agcttgtgggaaaagtttg 60 120 3000 –d 22 bp: 100% (Sus BE234189) None metallopeptidase M8 family

a

ª Extension time.

09TeAuthors The 2009 b 126 Information about MUC4 has been presented in Peng et al. (2007). c .Ren J.

20)30–36 (2009) Position refers to the GenBank accession number. dOnly a 51-bp distinct sequence was obtained due to sequencing difficulty, of which 22 nucleotides showed 100 identity to a porcine EST (BE234189) significantly homologous to human LMLN

gene. al. et J. Ren et al. Comparative RH map of SSC13q41 option ‘Fixed’ was used to establish the genetic map cies of all markers ranged from 21% to 34% with an and the recombination fractions were converted into average value of 25.8%, close to the 24% obtained genetic distances using the Kosambi mapping by Rink et al. (2006) for 140 EST and 62 microsatel- function. lite markers on SSC13. The most significantly linked marker of 14 mark- ers, namely KPNA1, ADCY5, PTPLB, MYLK, CCDC14, Results and discussion RPON1, KALRN, ITGB5, MUC13, HEG1, SLC12A8, Development of pig-specific STS markers ZNF148, RPON1B and LMLN, was an anonymous We designed 20 primer pairs from porcine or other marker S0075 with distances of 7–43 cR and LOD mammalian EST sequences to amplify porcine scores of more than 7. The deduced chromosomal genomic DNA. All amplicons were bidirectionally location of these markers is SSC13q41 because S0075 sequenced and showed significant identities to the has been physically mapped to this region porcine or other mammalian target exon sequences (Jørgensen et al. 2003). The mapping results of ADCY5 (Table 1), indicating that they are pig-specific STS and MYLK were consistent with the previous assign- markers. All intron sequences are flanked by the ment of the two genes to SSC13q41 by Fluorescent in consensus GT–AG splice site except for LMLN, situ hybridization (FISH) or RH mapping (Van Poucke MUC20 and KLF15 sequences. Only 51 nucleotides et al. 1999, 2001). Four markers including MUC20, were determined in the LMLN sequence because MUC4, SENP5 and DLG1 were also assigned to of sequencing difficulty. The 174-bp KLF15 and SSC13q41 because multipoint analysis localized these 369-bp MUC20 sequences are located in a single markers to a region flanked by SWR2189 and S0075 on exon. SSC13q41 (data not shown). A total of 34 single-nucleotide polymorphisms KLF15 showed significant linkage with SWR1210 (SNPs) were identified in 14 markers. These SNPs on SSC7 at distance of 50 cR (LOD = 6.43). As comprised 21 transversions and 13 transitions, SWR1210 was physically mapped to SSC7q13-14 resulting in no amino acid change (Table 1). Five (http://www.animalgenome.org/maps/marcmap.html), SNPs were found at positions 509–591 in the KALRN the chromosomal localization of KLF15 could be sequence (Table 1), indicating a mutation hotspot. SSC7q13-14. However, it has been shown that HSA3

As the template DNA was from an F2 hybrid of two completely corresponds to SSC13 (Meyers et al. distinct breeds (Duroc and Chinese Erhualian), the 2005; Rink et al. 2006; Humphray et al. 2007). KLF15 identified 34 SNPs could be interbreed polymor- is a member of the KLF gene family, so the discrep- phisms. However, the breed origin of each polymor- ancy could be caused by a new family member, a phism was not determined. These polymorphisms pseudogene or an unidentified comparative segment. are all located on SSC13q41 harbouring the ETEC We checked the identities of the KLF15 sequence F4ab ⁄ ac receptor locus (Python et al. 2002, 2005; against the human Build 36 genome database. This Jørgensen et al. 2003), therefore providing informa- sequence showed unique similarity to KLF15 in tive markers for fine mapping of the ETEC F4ab ⁄ ac humans (accession numbers NT_005612 and receptor locus using the White Duroc · Erhualian NW_921807, E-value of 2e-29, identity = 134 ⁄ 156), intercross or other experimental populations. No indicating that the discrepancy could not be caused SNP was found in six markers in this study, viz. by a new KLF family member or a pseudogene. ZNF148, ROPN1B, KLF15, SENP5, DLG1 and LMLN. Through the ENSEMBL genome browser (http:// www.ensembl.org/index.html), we found one BAC end sequence (bE383L19T7) of clone CH242-383L19 Regional assignment of markers showing significant similarity to an intron of Seven of 20 original EST primer pairs including human KLF15 with an E-value of 1.5e-12, and ADCY5, PTPLB, CCDC14, ITGB5, HEG1, ZNF148 and this BAC clone has been assigned to a contig MUC20 failed to produce scorable PCR products for on SSC7q13 (http://pre.ensembl.org/Sus_scrofa_map/ the RH panel typing. New primers were designed cytoview?mapfrag=CH242-383L19). These data con- from the obtained intron sequences for those mark- firmed that the location of KLF15 on SSC7q13 corre- ers. The 90 clones of the IMpRH7000 panel were then sponds to HSA3q21.2, indicating the presence of a genotyped for all 20 markers. Distances and LOD small unidentified comparative segment between scores between the 20 markers and their closest SSC7 and HSA3. markers on the first-generation RH map (Yerle et al. PLXNA1, adjacent to KLF15 at distance of only 1998) are presented in Table S1. Retention frequen- 0.6 Mb in the human genome, is most significantly

ª 2009 The Authors Journal compilation ª 2009 Blackwell Verlag, Berlin • J. Anim. Breed. Genet. 126 (2009) 30–36 33 Comparative RH map of SSC13q41 J. Ren et al. linked with SWC22 on SSC13q32 at distances of and 23 cR flanked by anonymous markers CL408784 59 cR (LOD = 5.11). The chromosomal localization and CL343765 (Meyers et al. 2005). of PLXNA1 was thus deduced to be SSC13q32. Using the framework map, we constructed the As ROPN1B, KLF15 and PLXNA1 are in a continuous comprehensive map comprising 18 markers on region on HSA3q21 and they correspond to different SSC13q41 (Figure 1). The distance between two regions in the pig genome, we can conclude that extremity markers (KPNA1 and DLG1) was estimated conserved synteny breaks occur between these genes at 166.5 cR. Considering the resolution of the IM- in a region less than 1.0 Mb in the human genome, pRH7000 panel of 53.2 kb ⁄ cR on SSC13 estimated by achieving higher map resolution when compared Rink et al. (2006), the physical length spanned by with the reported 2-Mb region on the previous the 18 markers is about 8.86 Mb, which is roughly human–pig comparative map (http://www.tou- similar to the 6.81 Mb distance of their orthologues louse.inra.fr/lgc/pig/RH/Carto/HT_7HSA3_L_8_V_H- in two blocks in the human genome. As shown in SA08_2003_PF_FT.HTM#). Figure 1, the gene orders on the comprehensive map and human physical map are generally conserved with the same direction in one block and the oppo- Construction of the RH map site direction in the other block. However, some Six of the 20 markers, viz. SENP5, LMLN, SLC12A8, microrearrangements were observed on the compre- HEG1, MUC13 and ADCY5, were placed in the 1000:1 hensive map. We could not confirm the existence of framework map (Figure 1). The six framework these microrearrangements in this region for our sta- markers (genes) include two conserved segments tistic strength was not sufficient to determine accu- showing conserved linkage with HSA3q29 and q21. rate orders of these closely spaced markers. Genes are oriented in the reverse order in one seg- Construction of a higher-resolution RH map using ment homologous to HSA3q21, and placed in the the IMpRH12000 panel (Yerle et al. 2002) may help same order in the other segment corresponding to obtain highly accurate orders of these markers and HSA3q29, which is in agreement with the previous pinpoint the truth. comparative maps (Jørgensen et al. 2003; Van Poucke et al. 2003; Meyers et al. 2005; Rink et al. 2006). The positional candidate genes for F4acR Moreover, this framework map enriched the marker density of the comparative map on SSC13q41, The F4acR locus has been narrowed to a region refining the conserved synteny breakpoint in pigs to flanked by Sw207 and S0283 on SSC13q41 (Joller et al. a region of 12.3 cR defined by LMLN and SLC12A8, 2006), and the positional candidate genes were most compared with the reported regions of 100 cR likely those encoding some transferrins or mucin-like between TFRC and ZNF148 (Van Poucke et al. 2003) sialoglycoproteins (Grange et al. 1998, 2002). S0283 is

Figure 1 Linkage and comparative map comprising 20 markers on SSC 13q41. Map shown (left, middle to right) are a genetic map showing linkage of six markers with the F4acR locus, a pig RH map of 13q41 and a human map of 3q21 and q29. Dis- tances are in centiMorgan (cM), centiRays (cR) and megabases (Mb), respectively. The framework markers are indicated in bold and other markers in italic. Two conserved segments are shown as shaded box on the pig RH map.

ª 2009 The Authors 34 Journal compilation ª 2009 Blackwell Verlag, Berlin • J. Anim. Breed. Genet. 126 (2009) 30–36 J. Ren et al. Comparative RH map of SSC13q41 very close to KALRN because a pig BAC clone positive Acknowledgements for S0283 contained KALRN (Jørgensen et al. 2003). Our RH mapping data showed that the synteny break We are grateful to Dr Martine Yerle (INRA, at SSC13q41 occurs in the region between LMLN and Castanet-Tolosan, France) for kindly providing the SLC12A8 containing the anchored marker of KALRN hybrid panels. We thank Zhiyan Zhang for his help (Figure 1). Considering the known homology of in drawing the comparative map. This study was Sw207 with HSA3q29 (Jørgensen et al. 2003), we pro- financially supported by National 863 Project of posed that the genomic region harbouring the F4acR China (2006AA10Z194) and Jiangxi Provincial Key locus could more likely correspond to HSA3q29 rather Research Project. than 3q21. In this study, we assigned three mucin genes, References MUC4, MUC13 and MUC20, to SSC13q41. The three mucin genes encoding membrane-bound O-glyco- de Givery S., Bouchez M., Chabrier P., Milan D., Schiex are interesting positional candidate genes T. (2005) CARTHAGENE: multipopulation integrated for F4acR. We have found strong association of an genetic and radiation hybrid mapping. Bioinformatics, intronic polymorphism in MUC4 with susceptibil- 21, 1703–1704. ity ⁄ resistance to ETEC F4ab ⁄ ac (Peng et al. 2007). Grange P.A., Erickson A.K., Anderson T.J., Francis D.H. More recently, we isolated the porcine MUC13 and (1998) Characterization of the carbohydrate moiety of MUC20 genes, and detected cosegregating susceptible intestinal mucin-type sialoglycoprotein receptors for and resistant haplotypes in MUC13 (Zhang et al. the K88ac fimbrial adhesin of Escherichia coli. Infect. 2008) and observed greatly significant associations Immun., 66, 1613–1621. between MUC20 haplotypes and susceptibility ⁄ resis- Grange P.A., Mouricout M.A., Levery S.B., Francis D.H., Erickson A.K. (2002) Evaluation of receptor binding tance to ETEC F4ab ⁄ ac in the White Duroc · Erhua- specificity of Escherichia coli K88 (F4) fimbrial adhesin lian intercross (Ji HY, Ren J, Yan XM, Huang X, variants using porcine serum transferrin and glycos- Zhang B, Huang LS. unpublished data). However, phingolipids as model receptors. Infect. Immun., 70, the causative mutation(s) that absolutely distinguish 2336–2343. susceptible animals from resistant animals remains Green P., Falls K., Crooks S. (1990) Documentation for unidentified. CRI-MAP, Version 2.4. Washington University School We performed linkage analysis for the F4acR locus of Medicine, St Louis, MO. with six polymorphisms identified in this study. All Huang X., Ren J., Yan X.M., Peng Q.L., Tang H., markers were significantly linked with the F4acR Zhang B., Ji H.Y., Yang S.J., Huang L.S. (2008) locus with LOD scores of higher than 10, and the Polymorphisms of three gene-derived STS on pig most significant linkage was observed between the chromosome 13q41 are associated with susceptibility MUC4 intron17 g.243A>G polymorphism and the to enterotoxigenic Escherichia coli F4ab ⁄ ac in pigs. Sci. F4acR locus (LOD = 40.18). The linkage significance China Ser. C, 38, 271–276. between markers flanking MUC4 and the F4acR Humphray S.J., Scott C.E., Clark R., Marron B., Bender locus decreased remarkably (Figure 1), indicating C., Camm N., Davis J., Jenks A., Noon A., Patel M., that MUC4 is most likely the gene encoding F4acR. Sehra H., Yang F., Rogatcheva M.B., Milan D., Characterization of functional and regulatory muta- Chardon P., Rohrer G., Nonneman D., Jong P., Meyers tions in MUC4 is under way. S.N., Archibald A., Beever J.E., Schook L.B., Rogers In summary, we developed pig-specific STS mark- J. (2007) A high utility integrated map of the pig ers for 20 genes distributed in a 6.8-Mb region on genome. Genome Biol., 8, R139. HSA3q21 and q29 and assigned them to the pig gen- Joller D., Jørgensen C.B., Bertschinger H.U., Bu¨ rgi E., ome using the RH mapping, revealing a small con- Stannarius C., Mortensen P.K., Cirera S., Archibald A., served segment between SSC7 and HSA3. The Genini S., Edfors-lilja I., Andersson L., Fredholm M., Vo¨ geli P. (2006) Refined linkage mapping of the Escher- established comparative RH map comprising the 20 ichia coli F4ac receptor gene on pig chromosome 13. In: genes not only refined the synteny breakpoint at Proc. 30th Int. Conf. Anim. Genet., 20–25 August, SSC13q41, but also revealed three mucin genes Porto Seguro, Brazil, B512. (especially ) as interesting candidate genes for MUC4 Jørgensen C.B., Cirera S., Anderson S.I., Archibald A.L., F4acR. The identified SNPs within these genes pro- Raudsepp T., Chowdhary B., Edfors-Lilja I., Andersson vide informative markers for fine mapping of the L., Fredholm M. (2003) Linkage and comparative map- F4acR locus in the White Duroc · Erhualian ping of the locus controlling susceptibility towards resource population.

ª 2009 The Authors Journal compilation ª 2009 Blackwell Verlag, Berlin • J. Anim. Breed. Genet. 126 (2009) 30–36 35 Comparative RH map of SSC13q41 J. Ren et al.

E. coli F4ab ⁄ ac diarrhoea in pigs. Cytogenet. Genome. Res., Van Poucke M., Yerle M., Tuggle C., Piumi F., Geneˆt C., 102, 157–162. Van Zeveren A., Peelman L.J. (2001) Integration of Meyers S.N., Rogatcheva M.B., Larkin D.M., Yerle M., porcine chromosome 13 maps. Cytogenet. Cell Genet., 93, Milan D., Hawken R.J., Schook L.B., Beever J.E. 297–303. (2005) Piggy-BACing the human genome II. A high- Van Poucke M., Yerle M., Chardon P., Jacobs K., Geneˆt resolution, physically anchored, comparative map of C., Mattheeuws M., Van Zeveren A., Peelman L.J. the porcine autosomes. Genomics., 86, 739–752. (2003) A refined comparative map between porcine Peng Q.L., Ren J., Yan X.M., Huang X., Tang H., Wang chromosome 13 and human . Cytogenet. Y.Z., Zhang B., Huang L.S. (2007) The g.243A>G Genome. Res., 102, 133–138. mutation in intron 17 of MUC4 is significantly associ- Voorrips R.E. (2002) MapChart: software for the graphi- ated with susceptibility ⁄ resistance to ETEC F4ab ⁄ ac cal presentation of linkage maps and QTLs. J. Hered., infection in pigs. Anim. Genet., 38, 397–400. 93, 77–78. Python P., Jo¨ rg H., Neuenschwander S., Hagger C., Yerle M., Pinton P., Robic A., Alfonso A., Palvadeau Y., Stricker C., Bu¨ rgi E., Bertschinger H.U., Stranzinger G., Delcros C., Hawken R., Alexander L., Beattie C., Vo¨ geli P. (2002) Fine-mapping of the intestinal recep- Schook L.B., Milan D., Gellin J. (1998) Construction of tor locus for enterotoxigenic Escherichia coli F4ac on a whole-genome radiation hybrid panel for high-reso- porcine chromosome 13. Anim. Genet., 33, 441–447. lution gene mapping in pigs. Cytogenet. Cell Genet., 82, Python P., Jo¨ rg H., Neuenschwander S., Asai-Coakwell 182–188. M., Hagger C., Bu¨ rgi E., Bertschinger H.U., Stranzinger Yerle M., Pinton P., Delcros C., Amal N., Milan D., Robic G., Vo¨ geli P. (2005) Inheritance of the F4ab, F4ac and A. (2002) Generation and characterization of a 12,000 F4ad E. coli receptors in swine and examination of four rads radiation hybrid panel for fine mapping in pig. candidate genes for F4acR. J. Anim. Breed. Genet., Cytogenet. Genome. Res., 97, 219–228. 122(s1), 5–14. Zhang B., Ren J., Yan X.M., Huang X., Ji H.Y., Peng Rink A., Santschi E.M., Eyer K.M., Roelofs B., Hess M., Q.L., Zhang Z.Y., Huang L.S. (2008) Investigation of Godfrey M., Karajusuf E.K., Yerle M., Milan D., Beattie the porcine MUC13 gene: isolation, expression, poly- C.W. (2002) A first-generation EST RH comparative morphisms and their strong association with suscepti- map of the porcine and human genome. Mamm. Gen- bility to enterotoxigenic E. coli F4ab ⁄ ac. Anim. Genet., ome, 13, 578–587. 39, 258–266. Rink A., Eyer K.M., Roelofs B., Priest K.J., Sharkey- Brockmeier K.J., Lekhong S., Karajusuf E.K., Bang J., Supporting Information Yerle M., Milan D., Liu W.-S., Beattie C.W. (2006) Radiation hybrid map of the porcine genome compris- Additional Supporting Information may be found in ing 2035 EST loci. Mamm. Genome, 17, 878–885. the online version of this article: Sun H.-F.S., Ernst C.W., Yerle M., Pinton P., Rothschild Table S1 Regional assignments of 20 markers in M.F., Chardon P., Rogel-Gaillard C., Tuggle C.K. (1999) the first-generation RH map Human chromosome 3 and pig chromosome 13 show Please note: Wiley-Blackwell is not responsible complete synteny conservation but extensive gene- for the content or functionality of any supporting order differences. Cytogenet. Cell Genet., 85, 273–278. information supplied by the authors. Any queries Van Poucke M., Tonsten A., Mattheeuws M., Van Zeveren (other than missing material) should be directed to A., Peelmann L.J., Chowdhary B.P. (1999) Comparative the corresponding author for the article. mapping between human chromosome 3 and porcine chromosome 13. Cytogenet. Cell Genet., 85, 279–284.

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