SHORT COMMUNICATION doi:10.1111/j.1365-2052.2006.01489.x Rearranged order between pig and in a quantitative trait loci region on SSC3

M. R. Mousel, D. J. Nonneman and G. A. Rohrer USDA-ARS, US Meat Animal Research Center, Clay Center, NE, USA

Summary A quantitative trait locus (QTL) for ovulation rate on 3 that peaks at 36 cM has been identified in a Meishan-White composite resource population with an additive effect of 2.2 corpora lutea. As part of an effort to identify the responsible gene(s), typing of additional on the INRA-University of Minnesota porcine radiation hybrid (IMpRH) map of SSC3 and comparative analysis of gene order was conducted. We placed 52 known genes and expressed sequence tags, two BAC-end sequences and one microsatellite (SB42) on a framework map that fills gaps on previous RH maps. Data were analysed for two-point and multipoint linkage with the IMpRH mapping tool and were submitted to the IMpRH database (http://imprh.toulouse.inra.fr/). Gene order was confirmed for 42 loci residing in the QTL region (spanning c. 17 Mb of human sequence) by using the high-resolution IMpRH2 panel. Carthage`ne (http://www.inra.fr/internet/departments/MIA/T/CarthaGene) was used to estimate multipoint marker distance and order using all public markers on SSC3 in the IMpRH database and those typed in this study. For the high-resolution map, only data for markers typed in both panels were used. Comparative analysis of human and porcine maps identified conservation of gene order for SSC3q and multiple blocks of con- served segments for SSC3p, which included six distinct segments of HSA7 and two segments of HSA16. The results of this study allow significant refinement of the SSC3p region that contains an ovulation rate QTL.

Keywords ovulation rate, radiation hybrid mapping, swine.

Swine reproduction is controlled by an unknown number of radiation hybrid (IMpRH) and IMpRH2 panels were used to genes that, if identified, could realize a large potential for create a high-resolution comparative map underlying the increased fertility, litter size and profit to producers. Genome putative QTL peak on SSC3. scans for porcine reproductive quantitative trait loci (QTL) The UCSC browser gateway (http:// have been conducted by Rohrer et al. (1999), Wilkie et al. genome.ucsc.edu/cgi-bin/hgGateway) was used to identify (1999), Cassady et al. (2001) and Rathje et al. (1997). regions on human 2 (18–96 Mb), 7 (4– Rohrer et al. (1999) identified a suggestive ovulation rate 101 Mb) and 16 (0.3–31 Mb). Porcine expressed sequence QTL on the p-arm of porcine chromosome 3, which had a tags were selected at approximately 1-Mb intervals based peak at 36 cM, a 95% confidence interval (CI) of 3–70 cM on human chromosome locations, and Primer3 (http:// and an additive effect of 2.2 corpora lutea. This chromo- frodo.wi.mit.edu/cgi-bin/primer/primer3.cgi; Rozen & some region currently has insufficient markers to accurately Skaletsky 2000) was used to design primer pairs distinguish human chromosomal homology and gene order. (Table S1). A well-characterized radiation hybrid panel is publicly The IMpRH (7000 rad; 118 clones) and IMpRH2 available for pigs (Yerle et al. 1998), and a recently devel- (12 000 rad; 90 clones) panel DNA samples were provi- oped higher-resolution panel also exists (Yerle et al. 2002). ded by the University of Minnesota and INRA respectively In this study, the INRA-University of Minnesota porcine (Yerle et al. 1998, 2002). Typing of the RH clones was performed using standard methods. Each locus was scored Address for correspondence as present or absent, and ambiguous loci were re-ana- G. A. Rohrer, USDA-ARS, US Meat Animal Research Center, Clay lysed. Data were analysed for two-point and multipoint Center, NE, USA. linkage with the IMpRH mapping tool and submitted E-mail: [email protected] to the IMpRH database (http://imprh.toulouse.inra.fr/). Accepted for publication 23 April 2006 Carthage`ne (http://www.inra.fr/internet/departments/MIA/

Ó 2006 International Society for Animal Genetics, No claim to original US government works, Animal Genetics, 37, 403–406 403 404 Mousel et al.

Human draft genomic sequence (Mb)

Framework of USDA linkage map 0.0 SDK1 I LMTK2 (cM) .50 SW2021 1.0 ZNF655 MCM7 1.5 MOSPD3 2.0 3.1–4.1 0 SW274 PERQ1 2.5 EP0 EPHB4 97.4 3.0 SLC12A9 3.5 Bl181480 4.0 FIS1 CUTL1 4.5 10 SRCRB4D 5.0 BAZ1B 101.5 SW2021 5.5 CH242-263E18 6.0 GTF2I 0.0 7907 II CH242-4I17 75.7 .50 SW833 72.3 20 SW2429 HSA7 1.0 AUTS21 SW72 73.5 SW2429 1.5 AUTS22 68.2

CALN11 0.0 CALN12 III RSAFD1 30 .50 SBDS TPST1 1.0 CGRPRCP ASL 1 SB42 1.5 ASL 2 71.3 GUSB 66.1 2.0 BI185976 c16orf58 2.5 BCKDK 14373 40 PRRT2 3.0 FAM57A SB42 64.9 3.5 NUPR1 S0174 CLN3 31.4 4.0 SULT1A3 0.0 PRKCB1 IV 0.5 S0174 50 1.0 CRYM 1.5 DNAH3 SW1432 2.0 2.5 MIR16 V 0.0 TNP2 19.4 60 0.5 SW1432 HSA16 11.3 1.0 PMM2 SW816 1.5 0.0 SW816 ALG1 VI 0.5 HMOX2 14595 130 1.0 CREBBP 1.5 FLJ14154

2.0 TSC2 2.5 16015 DECR2 3.0 0.3

Figure 1 A comprehensive map of the ovulation quantitative trait locus region of SSC3 with 54 genes/expressed sequence tags in six linkage groups. On the left is a framework map of the USDA porcine chromosome 3 linkage group. At the centre is the porcine radiation hybrid (RH) map, and on the right is the relative order of the porcine genes compared with the human gene order (HSA7 diagrammed by grey boxes and HSA16 by white boxes). Markers that are bolded and italicized were previously placed on the INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel and were used as a scaffold and for gene orientation. Linkage groups I, II and III are results from analyses of data of IMpRH and IMpRH2 panels, while groups IV, V and VI are results using public markers run on the 118-clone IMpRH panel and markers mapped in this study. Numeric superscripts refer to different amplicons of the same gene that have been mapped to the RH panel. The same superscripts are used in Table S1, which contains primer sequences and PCR conditions for each amplicon.

Ó 2006 International Society for Animal Genetics, No claim to original US gov. works, Animal Genetics, 37, 403–406 Comparative map of pig chromosome 3 405

T/CarthaGene) was used to estimate multipoint marker dis- HSA7 from 72.3 to 73.5 Mb is inverted relative to Meyers tance and order using all public markers on SSC3 in the et al. (2005); however, the previous study only typed IMpRH database and those developed in this study. For the markers at 75.1 and 73.3 Mb so this realignment was not QTL region, data from both the IMpRH and IMpRH2 panels apparent in their results. Jiang et al. (2005) placed 116 were analysed simultaneously, and a single map is reported markers on SSC3, and their results generally agree with the (Fig. 1). assignment of gene order found in this article. As Jiang et al. Fifty-two of the 68 orthologous genes selected from (2005) used a different RH panel and these data are not HSA2, 7 and 16 were successfully mapped to SSC3 (Ta- publicly available, we are unable to determine the cause of ble S1). Of the 16 that did not map to SSC3, seven (10%) the inconsistencies observed between our results and theirs. mapped to six other chromosomes and nine (13%) did not Markers placed on the q-arm of SSC3 in this study corres- amplify consistently. The markers that mapped to other pond to 18.6–96.7 Mb on HSA2 which verifies previous chromosomes were sequenced and their identity verified. reports (Pinton et al. 2000; Jiang et al. 2005; Meyers et al. One gene, TAC1, was assigned to HSA7 at 97.0 Mb but 2005). mapped to SSC9. This gene actually flanks a break in con- These data refined the human–porcine comparative map served synteny and confirms the results of Meyers et al. and placed additional markers on SSC3 which are within an (2005). Four genes initially purposed to be located on HSA7 OR QTL. This high-resolution map will also facilitate at 64.5, 71.4, 71.6 and 79.1 Mb were mapped to SSC1, assembly of the porcine genome sequence of this important SSC6, SSC2 and SSC8 respectively with two-point LOD region of the pig genome. Unfortunately, there were rear- scores >12, and one gene on HSA16 at 25.2 Mb mapped to rangements of gene order, and two human chromosomes SSC1. Reassessment of the porcine sequences identified are represented within the QTL peak. These numerous re- incorrect assignments at the time of selection to their hu- arrangements in this broad QTL interval makes selection of man homologues for TC86769 (HSA6), PPP1R12C positional candidate genes difficult. A more precise local- (HSA19), TC97126 (HSA19), REPC (HSA4) and ZSCAN2 ization of the QTL is necessary to determine if the QTN is in (HSA15). An interesting observation for the first four genes a gene located on HSA7 or 16. (TC86769, PPP1R12C, TC97126 and REPC) is that they all had significant homology to a gene on HSA7 within a Acknowledgements breakpoint region. Therefore, these genes were probably replicated at one time, and one copy resides within an The authors express their appreciation to Bree Quigley and evolutionary break-point region as described by Jiang et al. Kris Simmerman for technical support and to Sherry Kluver (2005). A potential novel breakpoint or incorrect assign- for assistance with figure development. Mention of trade ment of ABCC6 at 16.2 Mb on HSA 16 was detected as it names or commercial products is solely for the purpose of mapped to SSC12 with LOD scores >12. providing information and does not imply recommendation, Forty loci were mapped using the IMpRH2 panel to verify endorsement or exclusion of other suitable products by the gene order underlying the ovulation rate (OR) QTL peak. US Department of Agriculture. Marker data for nine of these markers were publicly avail- able in the IMpRH panel, and the remaining 31 were References mapped in the IMpRH panel for this study. For three genes, two unique amplicons were mapped so the total number of Cassady J.P., Johnson R.K., Pomp D., Rohrer G.A., Van Vleck L.D., markers was 43. The mean retention frequency was 39.0% Spiegel E.K. & Gilson K.M. (2001) Identification of quantitative (range 23.3–60%) for the IMpRH2. Analysis of the IMpRH2 trait loci affecting reproduction in pigs. Journal of Animal Science and IMpRH data for these 43 markers revealed three link- 79, 623–33. age groups, containing 17, 7 and 19 markers respectively Jiang Z., Michal J.J., Melville J.S. & Baltzer H.L. (2005) Multi- alignment of orthologous genome regions in five species provides using a LOD of 5.0 or greater (Fig. 1). The inclusion of both new insights into the evolutionary make-up of mammalian panel data improved the ability to order closely linked genomes. Chromosome Research 13, 707–15. markers relative to the analyses of data from only the Meyers S.N., Rogatcheva M.B., Larkin D.M., Yerle M., Milan D., IMpRH panel (data not shown). Hawken R.J., Schook L.B. & Beever J.E. (2005) Piggy-BACing the Genes located on the p-arm of SSC3 were greatly rear- human genome II. A high-resolution, physically anchored, com- ranged compared with HSA7 and HSA16 (Fig. 1) which parative map of the porcine autosomes. Genomics 86, 739–52. agrees with previous reports. Most notably are the extreme Pinton P., Schibler L., Cribiu E., Gellin J. & Yerle M. (2000) Loca- similarities of the current results with those recently pub- lization of 113 anchor loci in pigs: improvement of the com- lished by Meyers et al. (2005). While our results are virtu- parative map for , pigs, and goats. Mammalian Genome ally the same, the current study has twice the number of 11, 306–15. markers/Mb for the QTL region studied and utilized the Rathje T.A., Rohrer G.A. & Johnson R.K. (1997) Evidence for quantitative trait loci affecting ovulation rate in pigs. Journal of IMpRH2 panel to provide greater resolution. With the Animal Science 75, 1486–94. additional markers, our results indicate a small region of

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Rohrer G.A., Ford J.J., Wise T.H., Vallet J.L. & Christenson R.K. Yerle M., Pinton P., Delcros C., Aranal N., Milan D. & Robic A. (1999) Identification of quantitative trait loci affecting female (2002) Generation and characterization of a 12,000-rad radia- reproductive traits in a multigeneration Meishan-White compo- tion hybrid panel for fine mapping in pig. Cytogenetics and Genome site swine population. Journal of Animal Science 77, 1385–91. Research 97, 219–28. Rozen S. & Skaletsky H.J. (2000) Primer3 on the WWW for general users and for biologist programmers. In: Bioinformatics Methods and Protocols: Methods in Molecular Biology (Ed. by S. Krawetz & S. Supplementary Material Misener), pp. 365–386. Humana Press, Totowa, NJ. The following supplementary material is available for this Wilkie P.J., Paszek A.A., Beattie C.W., Alexander L.J., Wheeler M.B. article online at http://www.blackwell-synergy.com: & Schook L. B. (1999) A genomic scan of porcine reproductive Table S1 SSC3 RH map markers with database informa- traits reveals possible quantitative trait loci (QTLs) for number of tion, start of HSA homologous loci, primer information, corpora lutea. Mammalian Genome 10, 573–8. Yerle M., Pinton P., Robic A. et al. (1998) Construction of a whole- closest microsatellite according to IMpRH mapping tool, genome radiation hybrid panel for high-resolution gene mapping annealing temperature, product size and map location. in pigs. Cytogenetics and Cell Genetics 82, 182–8.

Ó 2006 International Society for Animal Genetics, No claim to original US gov. works, Animal Genetics, 37, 403–406