IOWA 2002 ANNUAL REPORT TO NRSP-8
Submitted by Max F. Rothschild and Christopher K. Tuggle
January 11, 2003

I. Project: NRSP-8: Swine Genome Committee

II. Cooperating Agencies and Principal Investigators

1. Agencies and Departments Cooperating: Iowa Agriculture Experiment Station and Animal Science Department, Iowa State University

2. Leaders of the Project: Max Rothschild (leader) and Christopher Tuggle (co-leader)

3. Cooperating Investigators: Jack Dekkers, Elizabeth Huff-Lonergan, Lloyd Anderson, Tom Baas, Yuandan Zhang, Zhiliang Hu, Daniel Ciobanu, Iowa State University; Daniel Pomp, University of Nebraska; M. Bento Soares and Tom Casavant, University of Iowa; Randy Prather, University of Missouri; William Beavis, National Center for Genomic Resources; and many PiGMaP collaborators.

III. Objectives

Objective 1: Develop high resolution comparative genome maps aligned across species that link agricultural animal maps to those of the human and mouse genomes.

Objective 2: Increase the marker density of existing linkage maps used in QTL mapping and integrate them with physical maps of animal chromosomes.

Objective 3: Expand and enhance internationally shared species genome databases and provide other common resources that facilitate genome mapping.

IV. General Project Plan

1. Mapping of Type I Comparative Loci (Objective 1).

   A significant interest of the group continues to be characterization and mapping of candidate genes for improvement of the linkage map as well as investigate type I loci for their role in quantitative genetics of economically important traits in the pig. We are using several general approaches. These include mapping cDNAs that have been sequenced in the pig or in other closely related species. These will be selected on the basis of expected physiological role and on the basis of their position on the human map. Using DNA from a panel of somatic cell hybrids provided by Martine Yerle and Joel Gellin (Toulouse) or the radiation hybrid panel supplied by the University of Minnesota, we will physically map Type I loci. We will amplify genes using species-specific PCR and DNA from each hybrid cell line, and data obtained will be sent to Toulouse for analysis. Genes that have been genetically mapped will be of high priority, as physical mapping of linked genes will tie together the genetic and physical maps. We have used and will continue to use the radiation hybrid panel and FISH techniques for physical mapping.

2. Developing and Mapping Expressed Sequence Tagged Sites (ESTs) (Objective 1)

   We have completed a three-year collaborative EST project with individuals from the University of Iowa, University of Missouri, University of Nebraska and NCGR to produce 20,000 ESTs and to map a significant number of these genes (about 700). Final numbers are 21,499 ESTs submitted to, that represent 10,574 different genes, and 703 genes mapped on the IMpRH.

3. Reference Pedigrees (Objective 1).

   Families exist for linkage research and QTL research. The first is a moderate sized group (330) of F2 offspring resulting from three generation gene mapping families have been born and growth and carcass data and DNA has been collected. These F2 animals come from original crosses involving Meishan x Duroc, Meishan x Hampshire, Meishan x Landrace, Minzhu x Landrace and Minzhu x Hampshire. These have been used for linkage studies and QTL research for growth and some meat traits.

4. Candidate Genes for Reproduction, Growth, Health and Quality Meat Traits (Objective 2)

   Several interesting genes that may affect a variety of traits in the pig are being chosen. Polymorphisms are first identified and the genes are then mapped. Association analyses have produced useful results for several genes.

5. QTL Identification (Objective 2)

   A large QTL genome scan for meat quality traits using a Berkshire x Yorkshire family was developed. A total of 2 Berkshire grandsires and 9 Yorkshire grand dams were used to produce 6 sires and 28 dams in the F1 generation. Phenotypes and DNA from 525 animals are being used. A total of nearly 160 genetic markers have been added to the map. Imprinted QTL are being investigated also.

6. Database development (Objective 3)

   Database development is continuing through the help of the US Pig Genome Coordination program.

V. Work Progress

1. A. Mapping of Type I Comparative Loci (Objective 1).

   Several candidate genes have been mapped this year using linkage and physical mapping. These are being placed in the regions of known QTL. Further identification and mapping of several other genes is under way.

2. Developing and Mapping Additional Expressed Sequence Tagged Sites (ESTs) (Objective 1)

   Our large collaborative EST project is with individuals from the University of Iowa, University of Missouri, University of Nebraska and NCGR. It is designed to produce 20,000 ESTs and to map a large number of these. We have sequenced and submitted to Genbank 21,499 ESTs from a number of normalized (placenta) and non-normalized (placenta, ovary, anterior pituitary, conceptus, fetus, and hypothalamus) cDNA libraries. Through clustering analysis, these sequences represent 10,574 different genes. Using BLAST analysis, we find 29% of these clusters do not have a significant hit (score < 200) to known EST and gene sequences. Further, approximately 44% of these clusters are novel relative to existing porcine ESTs and genes. Using newly developed software to identify pig ESTs with orthology to well-mapped human genes, we have mapped 703 ESTs to the physical map using the Toulouse SCHP and/or the IMpRH panel.

3. Generation of Reference Pedigrees (Objective 2)

   The original Chinese by American families have been developed and DNA and phenotypes on about 350 animals have been collected. Genotyping has proceeded on several chromosomes. DNA can be shared with interested parties. We have added many new genes on the map.

4. Candidate Genes for Economic Traits (Objective 2)

   Several genes continue to be investigated for their role in important economic traits in the pig. This includes additional validation research. New research has concentrated on genes affecting meat Quality and we have evidence for CAST affecting tenderness and HGMA1 affecting fat deposition. We are also investigating over 10 other genes for their role in traits of economic importance in the pig.

5. QTL Identification (Objective 2)

   A total of 525 animals have been slaughtered and 40 traits have been measured including many meat quality measures. We have now placed about 35 (total over 160) markers and genes on the map and genotyped all animals. In addition to the previously reported QTL we are now testing for imprinting and several such imprinted QTL have been identified. To follow-up F5 animals are being bred to produce F6 animals for a possible second set of phenotyped individuals. Analysis to find the underlying genes associated with the QTL has revealed genes causing two of the QTL (chromosome 2 and 15).

6. Database development (Objective 3)

7. Database development is continuing. An EST database has been developed and is quite useful.

   A QTL database is under development.

VI. Additions to the Project

None.

VII. Applications of Findings

1. A large number of genes continue to be identified and mapped by ISU researchers. An emphasis has been made (and will continue to be made) on genes that improve the comparative map as well as in connecting the genetic and physical pig genome maps.

2. Several new genes that may be important QTL are being mapped. This includes MC4R which is a major gene for feed intake, backfat and growth and also meat quality. In addition, new mutations in the PRKAG3 appear to be very important.

3. QTL for several meat quality traits have been discovered (Table 1). Additional fine mapping is underway and positional candidate genes are being considered.

4. Sequence analysis indicates we have identified over 10,000 genes of which approximately 4,500 to 5,000 are novel in the pig.

5. Comparative mapping between human and pig chromosomes corroborates chromosome painting results in that approximately 85-90% of loci map to expected locations, but also demonstrate that pig gene order cannot be predicted from the order of human genes within conserved syntenic groups.

VIII. General Project Plan

1. Objective 1
   1. Experiment A. Accelerate polymorphism identification and linkage mapping of comparative anchor loci.

   2. Experiment B. Continue physical mapping of genes using French somatic cell hybrid panel. Use of French Radiation Hybrid panel for high resolution physical mapping of new genes.

2. Objective 2
   1. Experiment D. Continued identification of polymorphisms and mapping of interesting reproduction, growth, meat quality and performance genes.
   2. Experiment E. Continue QTL research using several genes and markers. Continue to study role of candidate genes in traits of economic performance in the pig.
   3. Experiment F. Begin to study expression and function of additional new genes.

3. Objective 3
   1. Experiment G. Continue database development.

VIII. Publications

A. Publications during the year or not previously reported.

Bidanel, J. P. and M.F. Rothschild (2002) Current Status of Quantitative Trait Locus Mapping in Pigs. Pig News and 23 (2):39N-53N

Chaiwong, N., J.C.M. Dekkers, R.L. Fernando and M.F. Rothschild. 2002. Introgressing multiple QTL in backcross breeding programs of limited size. Communication Number 22-08. Procedings 7th World Congress on Genetics Applied to Livestock Production, August 19-23, Montpellier, France

Ciobanu, D. C., J. Bastiaansen, M. Malek, J. Helm, J. R. Woollard, G. S. Plastow, and M. F. Rothschild. 2002. New alleles in the PRKAG3 gene associated with low glycogen content in pig skeletal muscle and improved meat quality. Proceedings Plant and Animal Genome Conference, January 14-18, 2002, San Diego

Ciobanu, D. C., J. Bastiaansen, M. Malek, J. Helm, J. Woollard, G. S. Plastow, and M. F. Rothschild. 2001. Evidence for new alleles in the protein kinase AMP-activated, subunit gene associated with low glycogen content in pig skeletal muscle and improved meat quality. Genetics 159:1151-1162.

Ciobanu, D.S., S.M. Lonergan, J.W.M. Bastiaanses, M. Malek, E.J. Huff-Lonergan, G.S. Plastow and M. Rothschild. 2002. Evidence for new alleles in the Calpastatin gene associated with meat quality traits. Communication Number 11-10. 7th World Congress on Genetics Applied to Livestock Production, August 19-23, Montpellier, France

Ciobanu, D. S., S. Lonergan, J. Bastiaansen, J. R. Woollard, M. Malek, E. Huff-Lonergan, G. S. Plastow, and M. F. Rothschild. 2002. Discovery of new genetic markers affecting meat quality in pigs. Proceedings Plant and Animal Genome Conference, January 14-18, 2002, San Diego

Ciobanu, D.C., Y. Zhang and M.F. Rothschild. 2002. Rapid Communication. Mapping of the Ca+2 ATPase of fast twitch 1 skeletal muscle sarcoplasmic reticulum gene to porcine chromosome 3. J Anim Sci 80: 1386- 1387.

Emnett, R., S. Moeller, K. Irvin, M. Rothschild, G. Plastow and R. Goodwin. 2002. An Investigation into the genetic controls of pork quality: I Association studies with leptin receptor, melanocortin 4 receptor and peroxisome proliferator activated receptor-gamma. Ohio State University Department of Animal Sciences Research Reviews pg 84

Emnett, R., S. Moeller, K. Irvin, M. Rothschild, G. Plastow and R. Goodwin. 2002. An Investigation into the genetic controls of pork quality: I Association studies with heart fatty acid binding protein 1 and calpastatin. Ohio State University Department of Animal Sciences Research Reviews pg 85 Emnett, R., S. Moeller, K. Irvin, M. Rothschild, and E. Grindfleck. 2002. Physical assignment of adipocyte determination and differentiation factor-1 and pyruvate dehydrogenase E1-alpha in the pig. Ohio State University Department of Animal Sciences Research Reviews pg 86 Grapes, L. R. Fernando and M.F. Rothschild. 2002. Analysis of methods for fine mapping quantitative loci using linkage disequilibrium. Communication Number 21-19. Proceedings 7th World Congress on Genetics Applied to Livestock Production, August 19-23, Montpellier, France

Grapes, L., and M. F. Rothschild. 2002. BMP15 maps to the X chromosome in swine. Animal Genetics 33:158-159.

Grindflek, E., G. Plastow, and M. F. Rothschild. 2002. Mapping and investigation of the porcine major insulin sensitive glucose transport (SLC2A4/ GLUT4) gene as a candidate gene for meat quality and carcass traits. J Anim Breeding Genet. 119:47-55.

Holz, D.R., J. Helm, Y.D. Zhang and M.F. Rothschild. 2002. Rapid Communication. Linkage and physical mapping of the porcine basic firbroblast growth factor (FGF2) gene. J Anim Sci 80: 1384- 1385. Huff-Lonergan, E., T.J. Baas, M. Malek, J.C.M. Dekkers, K. Prusa, and M.F. Rothschild. 2002. Correlations among selected pork quality traits. Journal of Animal Science 80:617-627.

Isler, B.J., K. Irvin, M.F. Rothschild and G.J. Evans. 2002. Examination of the relationship between the prolactin receeptor gene and reproductive tract components in swine. Ohio State University Department of Animal Sciences Research Reviews pg 87

Kim, K. S., L. L. Anderson, C. K. Tuggle, and M. F. Rothschild. 2002. Mapping and functional analysis of the porcine ghrelin gene - A candidate gene for growth and appetite traits. Proceedings Plant & Animal Genome X meeting, January 14-18, 2002, San Diego, CA.

Kim, K.S., L. L. Anderson, J. M. Reecy, N. T. Nguyen, G. S. Plastow and M. F. Rothschild. 2002. Molecular genetic studies of porcine genes for obesity. Proceedings International Congress on Obesity. San Paulo, Brazil. August 25.

Kim, K. S., D. Ciobanu, G. Plastow, and M. F. Rothschild. 2001. Mapping of the porcine agouti-related protein (AGRP) gene to chromosome 6. Anim. Genet. 32:316-317.

Kim, K.S., H. Thomsen, J. Bastiaansen, Y. Zhang, J. C. M Dekkers, G. S. Plastow and M. F. Rothschild. 2002. Investigation of candidate genes for the growth and fatness QTL on pig chromosomes 1, 7, and 13 in a Berkshire x Yorkshire family and commercial populations. Proceedings of International Society of Animal Genetics, Gottigen, Germany, August 17.

Kim, K. S., Y. D. Zhang, D. Yim, Y. B. Kim, and M. F. Rothschild. 2002. Rapid Communication: Linkage mapping of the porcine immunoreceptor DAP10 and NKG2D. J Anim Sci 80: 1377- 1378.

Lee, H., J. C. M. Dekkers, M. Malek, R. L. Fernando, M. Soller and M. F. Rothschild. 2002. Comparison of approaches for determining significance threshold values for QTL mapping. Genetics 161: 905-914.

Ramos, A.M., J.M. Helm, Y.D. Zhang, T.Rangel-Figueierdo and M.F. Rothschild. 2002. Linkage and physical mapping of the porcine thyroglobulin (TG) gene. Animal Genetics 33:228-229.

Rothschild, M. F. 2001. Breakthrough genetic tests open new opportunities for pig producers and breeders. Pig World, October.

Rothschild, M.F. 2001. La genetica abre nuevas opportunidades para los productores y criadores de porcino. Albeitar (Spain) 51:10-11.

Rothschild, M.F. 2002. The role of genomics in the swine industry. Proceedings 17th Congress of the International Pig Veterinary Society, June 2-5, Ames, IA. Vol 1: pg 25-34

Rothschild, M.F. 2002. Patenting of innovations in Animal Breeding and Genetics. Proceedings 7th World Congress on Genetics Applied to Livestock production. Plenary Talk 03. Montpellier, France, August 18-23.

Rothschild, M.F. 2002. The role of genomics in the pig industry. Proceedings of the PIC China producers conference, Sept 20.

Rothschild, M.F. 2002. Transferring Biotechnology to Swine Genetic Programs: From Lab to Pork. Spanish Swine Breeders Conference. November 5, Barcelona, Spain

Rothschild, M.F. 2002. Approaches and limitations to measuring genetic diversity. EEC sponsored Conference on Swine Genetic Diversity, November 7, Cordoba, Spain.

Rothschild, M.F. and S. Newman. 2002. Intellectual property rights in animal breeding and genetics. CABI Press. 272 pages

Sherwood, J, D.Ciobanu, and M.F. Rothschild. 2002. Rapid Communication. Mapping of the Beta Tropomyosin (TPM2) gene to pig chromosome 1. J Anim Sci 80: 1379- 1380.

Shi, X.-W., Y.D. Zhang, M.F. Rothschild and C.K. Tuggle. 2002. Rapid Communication. Genetic linkage and physical mapping of the porcine cholesteryl ester transferase protein (CETP) gene. J Anim Sci 80: 1390- 1391. Sun, H.S., C.K. Tuggle, A. Goureau, C.J. Fitzsimmons, P. Chardon, P. Pinton, and M. Yerle. 2002. Precise mapping of breakpoints in conserved synteny between human chromosome 1 and pig chromosomes 4, 6, and 9. Anim. Gen. 33:91-96.

Sun, H.S., L. L. Anderson, T.-P. Yu, K.S. Kim, J. Klindt, and C. K. Tuggle. 2002. Neonatal Meishan pigs show POU1F1 genotype effects on plasma GH and PRL concentration. Anim. Reprod. Sci. 69:223-237.

Stumbaugh, A.N., T. J. Stabel, C. J. Fitzsimmons, C. K. Tuggle. 2002. TaqMan Real-Time RT-PCR detection of NRAMP1 transcripts in porcine Salmonella choleraesuis infected tissues. Proceedings Plant & Animal Genome X meeting, January 14-18, 2002, San Diego, CA, Abstract 773, page 271.

Thomsen, H., J.C.M. Dekkers, H.K. Lee and M. F. Rothschild. 2002. Characterisation of quantitative trait loci for growth and meat quality in a breed cross in swine. Communication Number 15-05. Proceedings 7th World Congress on Genetics Applied to Livestock Production, August 19-23, Montpellier, France

Tuggle , C. K., R. S. Prather, M. B. Soares, T. Casavant, D. Pomp, M. F. Rothschild, W. Beavis. Gene Discovery and Functional Genomics in the Pig. Proceedings the National Swine Improvement Federation, St. Louis, MO, December 6-7, 2001, p. 12

Woollard, J. R. and M. F. Rothschild. 2001. Assignment of the porcine calpain-10 gene (CAPN10) to chromosome 15q23-26. Animal Genetics 32:390-393.

Yim, D., H.-B. Jie, J. Sotiriadis, K. S. Kim, S-C. Chin, H-B. Jie, M. F. Rothschild and Y. B. Kim. 2001. Molecular cloning and expression pattern and chromosomal mapping of CD69. Immunogenetics 54:276-281.

Yu, T.-P., H. S. Sun, S. Wahls, I. Sanchez-Serrano, M. F. Rothschild, and C. K. Tuggle. 2001. Cloning of the full length pit PIT1 (POU1F1) cDNA and a novel alternative PIT1 transcript, and functional studies of their encoded proteins. Animal Biotech. 12(1): 1-19.

Zhang, Y.D., K. Garwood,W.D. Beavis, C.K. Tuggle, and M.F. Rothschild. 2002. Porcine Expressed Sequence Tags (ESTs): Comparative Analysis and Database Development. Proceedings Plant & Animal Genome X meeting, January 14-18, 2002, San Diego, CA, Abstract 811, page 280.

B. Publications planned.

Chaiwong, N., J.C.M. Dekkers, R.L. Fernando and M.F. Rothschild. 2003. Introgressing Multiple QTL through Backcross Breeding Programs. Swine Research Reports. Iowa State University (in press).

Ciobanu, D.C., S.M. Lonergan, M. Malek, J.R. Woollard, E.J. Lonergan and M.F. Rothschild. 2003. New Alleles in the Calpastatin Gene Associated with Improved Tenderness in Pigs. Swine Research Reports. Iowa State University (in press)

Fernando, R.L., B. R. Southey, J.C.M. Dekkers, M.F. Rothschild and M. Soller. 2003. Controlling the proportion of false positives (PFP) in a multiple test situation. Genetics (submitted).

Kim, K.S., N. T. Nguyen, J. M Reecy, L. L. Anderson, and M. F. Rothschild. 2003. Molecular genetic studies of porcine genes for obesity. Swine Research Reports. Iowa State University (in press).

Malek, M., D. C. Ciobanu, and M. F. Rothschild. 2003. Genetic and physical mapping of ACACB, PPP1CC and GPR49 genes on porcine chromosomes 5 and 14 help to define a break point on human chromosome 12. Proceedings Plant & Animal Genome XI meeting, January 11-15, San Diego, CA. (in press)

Nguyen, N-T, K.-S. Kim, H. Thomsen, J. Helm and M. F. Rothschild 2003. Investigation of candidate gene for growth and fatness QTL on the pig chromosome 7. Proceedings Plant & Animal Genome XI meeting, January 11-15 San Diego, CA. (in press).

Otieno, C.J., S. Vleck, C. Jelks, K.-S. Kim, N. T. Nguyen and M. F. Rothschild. 2003. Mapping of diabetes-related genes in the pig. Proceedings Plant & Animal Genome XI meeting, January 11-15, San Diego, CA. (in press)

Thomsen, H., J. C. M. Dekkers, and M. F. Rothschild. 2003. Detection and characterization of QTL for growth and meat quality traits in the Berkshire-Yorkshire cross. Swine Research Reports. Iowa State University (in press)

Zhao, H., M. F. Rothschild, R. L. Fernando, J. C.M. Dekkers 2003. Tests of candidate genes in QTL mapping resource populations. Genetics (submitted).