Progress 12/01/00 to 12/31/03
Outputs
A total of 2,035 ESTs from immune tissues and placentae were used to construct the, as yet unpublished, Second Generation EST Radiation Hybrid Map confirms 98% coverage of the human genome. Currently ~ 60 breaks between chromosomes and ~70 breaks within regions of homology have been detected. A final number cannot be determined at this time since the orientation of a significant number of contigs in HGS Build 29 remains to be determined. Additionally, 4.9% of markers in the current map identify other homologous chromosomes in HGS Build 29 vs 5.4% in the 1st Generation EST Radiation Hybrid Map. The current porcine RH map extends beyond the genetic linkage map on 38 telomeres. 28.9% of all markers do not find orthologous sequences in the HGS. The human genome sequence assembly currently does not extend beyond the human RH map(s). The 2pt LOD score criterion for creating the 2nd generation porcine EST radiation hybrid map using RHMAP V3 was set at 6. Good statistical
support for local gene order was considered to be more valuable than creating large linkage groups and obtaining complete chromosome coverage through a minimum number of linkage groups. This is particularly important in those regions where the human genome sequence is not fully assembled or ambiguous. A total of 588 ESTs (~29%) do not identify sequences with significant homology in HGS Build 29. An additionally 4.9% ESTs (100 markers) identify significant matches on human chromosome fragments that do not correspond to the respective porcine chromosome based on bi-directional painting results. We are currently using Carthagene (http://www.inra.fr/bia/T/schiex/Export/WABI.pdf) to calculate an updated version of the 2nd Generation pig human comparative map (IMpRH7000Rad panel). Markers that introduced new homologies to the comparative map are reevaluated based on their multipoint linkage assignment (using the IMpRH mapping tool), rather than on their 2pt LOD value. These maps will be
transferred to the FTP site as they become available. An Excel file (Complete Info for 2035 ESTs.xls) containing all pertinent information (annotation, primers, location in HGS build 29, mapping information) is also available from the FTP site. All vectors generated by our group are public and will continue as such as markers are assigned. Build 30 of the HGS was released June 24th, 2002 and covers > 70% of the human genome in currently 1,736 contigs assembled, covering 2.87 billion base pairs. BLASTN comparisons against the contig assembly and dbEST have been completed. Radiation Hybrid Mapping Project - IMNpRH12000rad: A total of 1873 (92% 2035 ESTs on the IMpRH7000rad map) is the current number of framework ESTs currently assigned to all chromosomes on the IMNpRH12,000-rad panel.. The location of these ESTs is currently being finalized and will be available on our web sites (www.toulouse.inra.fr/lgc/lgc/, http://www.ag.unr.edu/ab/standard.htm), shortly. We have also assigned 700
microsatellites to the IMpRH12000rad panel.
Impacts
This project provided the first high resolution comprehensive map of the swine genome, linked to the swine genetic map. It provides an excellant platform for the new high resolution RH map of the swine genome. This, in turn, will provide rapid identification of candidate genes associated with production and product quality traits in pigs through marker (allele) assisted selection.
Publications
- No publications reported this period
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Progress 12/01/00 to 11/30/02
Outputs
The Second Generation EST Radiation Hybrid (RH7000rad) Map (2035 ESTs). A total of 2,035 ESTs from immune tissues and placentae were used to construct the, as yet unpublished, Second Generation EST Radiation Hybrid Map confirms 98% coverage of the human genome. Currently 60 breaks between chromosomes and 70 breaks within regions of homology have been detected. A final number cannot be determined at this time since the orientation of a significant number of contigs in HGS Build 29 remains to be determined. Additionally, 4.9% of markers in the current map identify other homologous chromosomes in HGS Build 29 vs 5.4% in the 1st Generation EST Radiation Hybrid Map. The current porcine RH map extends beyond the genetic linkage map on 38 telomeres. 28.9% of all markers do not find orthologous sequences in the HGS Build 29 vs 23.6% in HGS Build 21, indicating an increasing number of deletions, particularly of telomeric sequences, in subsequent builds of the human genome
sequence. The human genome sequence assembly currently does not extend beyond the human RH map(s). The 2pt LOD score criterion for creating the 2nd generation porcine EST radiation hybrid map using RHMAP V3 was set at 6. Good statistical support for local gene order was considered to be more valuable than creating large linkage groups and obtaining complete chromosome coverage through a minimum number of linkage groups. This is particularly important in those regions where the human genome sequence is not fully assembled or ambiguous. A total of 588 ESTs (29%) do not identify sequences with significant homology in HGS Build 29. An additionally 4.9% ESTs (100 markers) identify significant matches on human chromosome fragments that do not correspond to the respective porcine chromosome based on bi-directional painting results. We are currently using Carthagene (http://www.inra.fr/bia/T/schiex/Export/WABI.pdf) to calculate an updated version of the 2nd Generation pig human comparative
map (IMpRH7000Rad panel). Markers that introduced new homologies to the comparative map are reevaluated based on their multipoint linkage assignment (using the IMpRH mapping tool), rather than on their 2pt LOD value. These maps will be transferred to the FTP site as they become available. An Excel file (Complete Info for 2035 ESTs.xls) containing all pertinent information (annotation, primers, location in HGS build 29, mapping information) is also available from the FTP site. All vectors generated by our group are public and will continue as such as markers are assigned. Build 30 of the HGS was released June 24th, 2002 and covers > 70% of the human genome in currently 1,736 contigs assembled, covering 2.87 billion base pairs. BLASTN comparisons against the contig assembly and dbEST have been completed. Radiation Hybrid Mapping Project - IMpRH12000rad: A total of 1873 (92% 2035 ESTs on the IMpRH7000rad map) is the current number of framework ESTs currently assigned to all chromosomes
on the IMNpRH12,000-rad panel.. The location of these ESTs is currently being finalized and will be available on our web sites (www.toulouse.inra.fr/lgc/lgc/, http://www.ag.unr.edu/ab/standard.htm), shortly.
Impacts
Nearly 9 million litters of pigs are currently born per year in the commercial swine industry; an industry that continues to grow, in spite of relatively low financial margins. This alone provides an economic incentive to develop genetic reagents that improve gene-mapping data toward practical application and determine how genes and gene clusters, identified through mapping, affect phenotypes and improve efficiency. This in turn will better position the producer to satisfy human food and fiber needs, a stated goal of The USDA's NRI Competitive Grants Program.
Publications
- Milan D, Schiex T, Yerle M, Rink A, Beattie C, Hawken R, Schook L, Yasue H, Fredholm M,Rohrer G. (2002) Integration of Genetic and radiation hybrid maps of the pig: The second generation IMpRH maps. Plant, Animal and Microbe Genomes Conference X, San Diego, CA, January 12-16. W307
- Milan D, Schiex T, Yerle M, Rink A, Beattie C, Alexander L., Hawken R, Schook L, Yasue H,Pomp D., Fredholm M, Rohrer G. (2002) Integration of Genetic and radiation hybrid maps of the pig: The second generation IMpRH maps. International Society Animal Genetics (ISAG) XXVIII D167, 151.
- Anette Rink, Katie Eyer, Ben Roelofs, Craig Beattie. (2002) Current Status of the Physical Map of the Swine Genome. Plant, Animal and Microbe Genomes Conference X, San Diego, CA, January 12-16, W308
- Rink A., Santschi E.M., Vonnahme K.A., Eyer K.A., Roelofs B.M., Sharkey K.J., Sudkamon Lekhong S.,Murphy E.F., Ford S.P., Yerle M., Milan D. and Beattie C.W. (2002) A second generation EST radiation hybrid comparative map of the porcine genome. International Society Animal Genetics (ISAG) XXVIII Gottingen, Germany, August 11-15, A020, p34.
- Rink A., Santschi E.M., Vonnahme K.A., Eyer K.A., Roelofs B.M., Sharkey K.J., Sudkamon L.S.,Murphy E.F., Ford S.P., Yerle M., Milan D. and Beattie C.W. (2003) A secondgeneration EST radiation hybrid comparative map of the porcine genome. Mamm.Genome(In preparation).
- Gadgil C., Rink, A., Beattie, C.W. and Hu W.S. A mathematical model for suppression subtraction hybridization.Comparative and Functional Genomics 3:405-422, 2002.
- Demeure, O. Renard C., Yerle M., Faraut T., Riquet J., Robic A., Schiex T., Rink A. and Milan D. (2003) Rearranged gene order between pig and human in a QTL region on SSC 7. Mamm. Genome (In the Press)
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Progress 01/01/01 to 12/31/01
Outputs
Current Resolution of the IMpRH7000rad panel. The INRA-Minnesota porcine RH (IMpRH) panel Yerle et al. 1998; Hawken et al. 1999) constructed with the PIs collaborators at INRA, Toulouse, France is a web-based ( http://www.toulouse.inra.fr/lgc) public mapping effort. Our initial RH mapping candidates from immune libraries primarily focused on immune response genes, apoptosis, cancer and potential therapeutic targets. We designed and synthesized 4000 primer pairs (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_wwwcgi. of which 79% amplify porcine DNA specifically; 50% ESTs can be mapped. We used the whole-genome RH microsatellite (ms) map of the porcine genome (Yerle et al., 1998; Hawken et al., 1999) to order 1,058 porcine ESTs derived from a panel of normalized cDNA libraries (Rink et al., 2002) to confirm and refine syntenic regions between the human and porcine genomes previously identified by bi-directional painting, identify new syntenic breakpoints and
internal rearrangements and improve overall resolution. We were able to confirm that gene-rich and gene-poor regions of the human genome are conserved in the porcine genome. However, regions of chromosome shuffling cover approximately 25% of the porcine genome. Additionally, we found that 25% of the markers that were mapped on the porcine RH map do not find regions of sequence similarity in the assembled human genome, indicating gaps in all the human chromosomes. Furthermore we mapped 60 ESTs genome wide, which do not confirm previously established synteny. At present, the IMpRH7000rad panel covers 98% of the porcine genome. The sequence of all mapped markers will be released by our colleagues in Toulouse, shortly. Goureau and coworkers (1996) established synteny with human chromosomes for all autosomal pig chromosomes using bi-directional painting. Individual swine chromosomes exhibit synteny with between 1 and 5 human chromosomes with a total of 39 identifiable chromosomal breaks.
Synteny was confirmed for most of these chromosomes. Sequence comparison of our EST markers against V21 of the assembled human genome sequence showed that, on average, three ESTs per swine chromosome found the highest homologies on human chromosomes that are not syntenic based on bi-directional painting results. Bi-directional painting results (Goureau et al., 1996) suggested a total of 39 chromosomal breaks in the porcine genome. We have currently identified a minimum of 60 breaks between chromosomes and 90 internal breaks within regions of synteny. Although our data should be considered preliminary, as the entire comparison is based on V22 of the HGS assembly and small rearrangements currently counted as internal breaks might be due either to mapping errors or to assembly errors in the human genome sequence. As the number of mapped porcine ESTs on the IMpRH7000Rad panel is increased, results will be compared against the freeze, current at the time of completion of the 2nd generation
comparative IMpRH7000Rad map.
Impacts
We believe our approach will rapidly and significantly impact physical map development in swine. The proposals objectives significantly improve the potential to provide long-term genetic improvement of American livestock and the sustainability of this industry as well as a significant impact on NRICGP program 43 "Identifying Animal Genetic Mechanisms and Gene mapping", the special request for proposals on generation, and mapping of genomic reagents and the NAGRP.
Publications
- Rink A, Santschi EM, Eyer KE, Hess M, Karajusuf EK, Sharkey K, Roelofs B, Kabuloglu ZG, Beattie CW (2001). Zooming in on the Porcine-Human Comparative Map. 12th North American Colloquium Animal Cytogenetics and Gene Mapping, July 15-19, Davis, CA.
- Korke R, Lee J, Ming Q, de Leon Gatti M, Rink A, Seow TK, Wong K, Chung M,Beattie CW, Hu W-S (2001). Integrating Genomic, proteomic and metabolic analysis of physiological changes in mammalian cell culture. Fourth conference on Recent Advances in Fermentation Technology (RAFT IV), Nov10-13, 2001, Long Beach, CA.
- Gadgil C, deLeon Gatti M, Korke R, Rink A, Beattie CW, Hu WS (2001). Mathematical Analysis of DNA Hybridization on Microarrays. 220th Natl Mtg ACS, San Diego,CA.
- Korke R, Rink A, Seow TK, Chung M, Beattie CW, Hu WS (2001). Genomic and Proteomic Analysis of Metabolic Shift in Continuous Cell Culture. 220th Natl. Mtg. ACS, San Diego, CA.
- Hu WS, Tzanakakis ES, Abu-Absi SF, Narayanan RA, Jork C, Rink A, Hansen LK,Beattie CW (2001). Genome-Wide Expression Analysis of Hepatocyte Spheroid Formation. NASA CELL SCIENCE CONFERENCE, Houston, TX, March 6-8, 2001
- Santschi EM, Rink A, Beattie CW (2001). Genomic exploration of orthopedic infection using cDNA microarrays. Havemeyer Foundation Workshop on Equine Immunology, Santa Fe, NM, January 24-27, 2001.
- Rink A, Santschi EM, Hess M, Eyer KE, Godfrey ML, Karayusuf EK, Milan D, Yerle M, and Beattie CW (2001). A First Generation EST Radiation Hybrid Map of the Porcine Genome. Plant and Animal Genome IX, San Diego, CA, Jan 13-17, 2001.
- Paszak AA, Wilkie, PJ, Flickenger, GH, Miller LM, Louis CF, Rohrer GA, Alexander LJ, Beattie CW and Schook LB. Interval mapping of carcass and meat quality traits in a divergent swine cross. Animal Biotechnology 12:155-165, 2001.
- Korke R, Rink A, Seow TK, Wong K, Beattie CW, Hu WS Integration of genomic and proteomic approaches to comprehend physiology of cells in bioprocessing. J. Biotechnol. 94:73-92. (March , 2002).
- Milan D, Schiex T, Yerle M, Rink A, Beattie C, Hawken R, Schook L, Yasue H,Fredholm M, Rohrer G. Integration of Genetic and radiation hybrid maps of the pig : The second generation IMpRH maps. Plant, Animal and Microbe Genomes Conference X, San Diego, CA, January 12-16, 2002.
- Rink A, Santschi EM, Eyer KE, Hess M, Roelofs B, Kabuloglu ZG, Karayusuf EK, Sharkey K, Beattie CW (2001). Divergent Molecular and Structural Resolution between the Porcine and Human Genomes. VI Congress of the International Xenotransplantation Association," Meeting the Challenges of the new millennium", September 29th - October 3rd, 2001, Chicago, IL.
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