NC1008: Advanced Technologies for the Genetic Improvement of Poultry (was NC-168)

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[02/26/2004] [09/16/2005] [12/18/2006] [03/12/2007]

Date of Annual Report: 02/26/2004

Report Information

Annual Meeting Dates: 01/10/2004 - 01/11/2004
Period the Report Covers: 01/01/2003 - 12/01/2003

Participants

Okimoto, Ron - University of Arkansas;
Kuenzel, Wayne - University of Arkansas;
Miller, Marcia - City of Hope;
Delany, Mary - University of California, Davis;
Lamont, Susan - Iowa State University;
Dekkers, Jack - Iowa State University;
Muir, William - Purdue University;
Porter, Tom - University of Maryland;
Dodgson, Jerry - Michigan State University;
Foster, Douglas - University of Minnesota;
Ponce de Leon, Abel - University of Minnesota;
Reed, Kent - University of Minnesota;
Petitte, James - North Carolina State University;
Zhu, James - Texas A & M University;
Cheng, Hans - USDA-ARS, ADOL;
Ashwell, Christopher - USDA-ARS, GBL;

Institutions absent;Grossman, Michael - University of Illinois;
Wong, Eric - Virginia Polytechnic Institute & State University;
Smith, Edward - Virginia Polytechnic Institute & State University

Brief Summary of Minutes

The NC-1008 meeting (the meeting formerly known as NC-168) was held in conjunction with the Plant & Animal Genome XII meeting at the Town & Country Resort in San Diego, CA. The meeting was called to order on Saturday at 12:45 by NC-1008 chair, Bill Muir.

Following organization comments by the chair and self-introductions by the attendees, reports were presented as published in the PAG XII program so that others would know when speakers were scheduled. Objective coordinators were the same as last year: Mary Delany ?Objective 1; Jerry Dodgson?Objective 2; and Chris Ashwell?Objective 3. The speakers on Saturday included: Martien Groenen (guest; Netherlands); Jerry Dodgson (MI and NRSP-8 Coordinator); Susan Lamont and Jack Dekker (IA); Hans Cheng (USDA-ARS;ADOL); Mary Delany (CA-UC Davis); Tom Porter (MD); Bill Muir (IN). Presentations continued on Sunday with Larry Cogburn (guest); Marlene Emara (DE); James Zhu (TX); David Burt (guest); Wayne Kuenzel and Ron Okimoto (AR); Tom Wicker (guest); Marcia Miller (CA-City of Hope); Douglas Foster and Kent Reed (MN); Jim Petitte (NC); Urs Kuhnlein (guest) and Chris Ashwell (USDA-ARS;GBL).

The business meeting was called to order by Bill Muir. Sue Lamont moved to approve the minutes (seconded by Hans Cheng) for the 2002 NC-168 meeting. Margaret Dentine, our Administrative Advisor, noted that both NC-1008 and NRSP-8 annual reports should be posted on the NIMSS web site within 60 days after the meeting and reported that NC-1008 has an active program and should continue to emphasize the collaborative nature of our work. She informed members that changes in affiliation, deletions and new membership additions now reside in NIMSS with yearly updates.

Regarding admission of new members, Jerry Dodgson moved (seconded by Mary Delany) that Margaret should act on requests for admission and the committee would be notified of those actions. With urging from others, Marlene Emara agreed to coordinate the writing of the final report for the last five years for NC-168. Muquarrab Qureshi (Animal Genetics National Program Leader) gave updates for the NRICRG funding. Sue Lamont moved (seconded by Jerry Dodgson) that all reports be sent electronically to both NC-1008 and NRSP-8 individuals and administrators by the Wednesday before the next annual meeting, the motion was carried.

The motion was made by Sue Lamont and seconded by Hans Cheng to have the next NC-1008 meeting in conjunction with PAG XIII on January 15-16, 2005, and to maintain Bill Muir?s organizational style (invited speakers, titles organized by topic, and 30 min presentations including questions, unless additional time requested, a maximum of 45 minutes would be allocated to any one station) and was agreed unanimously.

The starting time for next year?s meeting will be 12 noon on Saturday January 15 and the participants agreed to email the chair a month before the meeting the following: 1) title of talk; 2) time requested for talk; and 3) desired time of presentation. The chair would make final adjustments to time and order of presentation as needed.

Kent Reed was nominated and accepted the position as NRSP-8 secretary for next year and Tom Porter was nominated and approved (since he wasn?t at the meeting) as NC-1008 secretary for next year.

Chris Ashwell will be the Chair of NRSP-8 and meeting coordinator for next year and Doug Foster will be Chair NC-1008 next year and coordinate the meeting the following year. It was agreed as a standard operating procedure that the chairs of the NRSP-8 and NC-1008 committees would alternate as coordinators. Only the coordinator would receive funds for travel from the PAG organizers. The organizers offer $100 off the registration cost for up to 6 invited speakers.

To be eligible for the discount the speaker must 1) register through the PAG speaker web site, 2) must register for the entire PAG program, 3) must submit an abstract, and 4) the names must be sent by the coordinator of the Poultry Workshop to the Coordinator of PAG.

It was agreed that these would be offered first to officers of the Poultry Workshop and if declined would be offered to invited speakers. The objective coordinators will remain the same (Objective 1: Mary Delany; Objective 2: Jerry Dodgson; and Objective 3: Chris Ashwell).

The roles of the officers in the reporting process were outlined as follows: the objective coordinators would compile material from individual reports into an objective report and forward to the secretary. The secretary would keep the minutes of the meeting and in addition collect the coordinators reports (prodding as needed) and add publications reported from all stations. The secretary would forward those items to the NC-1008 chair, within 60 days of the meeting, who would review and approve the minutes and report before forwarding to the administrative advisor.

Bill Muir was complimented on his organization of what was the best NC-1008 meeting yet.

The meeting was adjourned almost on time at 5:45.

Minutes Attachment:

Attachment

Accomplishments

ACCOMPLISHMENTS AND IMPACTS: Objective 1. Utilize Modern Molecular and Breeding Technologies to Identify, Locate, Isolate and Characterize Poultry Gene of Economic Importance. Progress was achieved in many areas of poultry (chicken and turkey) genetic and genomic analysis involving the alignment and enhancement of the genetic, physical and cytogenetic maps, in developing the various maps and sequence information for genes/QTLs of economically relevant traits, and in the identification of gene expression profiles. Advances involved substantial collaborative research among technical committee members and included sharing of information, reagents, tools and genetic resources. Results from the previous year included: Feed efficiency studies were conducted by AR using birds having variant forms of the UCP, COII cytb and POMC genes. Segregating mitoD-loop types having different feed efficiencies were found to vary for a substitution in the COII gene; Feather color studies continued for the purpose of developing pooled multiplexed sequencing tests for E locus alleles, recessive white and dominant white. Work progressed on the stereotaxic atlas of the chick brain wherein 50 vector-based plates were completed to date. The plates will allow users to create customized diagramming for research and teaching. CA (Delany) examined profiling of genes regulating telomerase genes and telomere access, which impact genome stability by using quantitative RT-PCR. Several cell systems are under evaluation. An inheritance study was initiated to evaluate telomere recombination using UCD003. CA (Miller) continued work to elucidate the MHC-Rfp-Y gene region. Numerous other genes, including a series of c-lectin-like genes were identified within the various clones. Work was conducted to define break-points within MHC recombinants indicating recombination occurred at various points within a potential hotspot region as well as outside the region. Characterization was initiated by DE for a family of genes known as the F-box protein family; these genes are involved in degradation and control of cell cycle and other regulatory proteins. FBX07 mapped to Gga 1 in the region of DEL001. Sequencing was performed and exon/intron structure was determined. Twenty-one members of the F-box family were identified from chicken ESTs. Studies continued by IA utilizing the Iowa Growth and Composition Resource Population to identify genes associated with skeletal integrity. Candidate genes included BMP6, BMP-receptor 1, Vit D receptor, insulin, among others. BMP6 SNPs were significantly associated with most skeletal traits. Microarray (chicken lymphoid cDNA, 2880 genes) gene expression studies were initiated to assess global gene expression of Leghorn, Fayoumi and broiler lines. No significant variation was identified for the microarray system (activated T cells, mixed lymphoid tissue library) and organ/age sampled (adult spleen). A chicken cDNA microarray (5K array) for the neuroendocrine system was created previously by MD (USDA-IFAFS funds). Initial characterization included gene expression among tissues of the neuroendocrine system (anterior pituitary, pineal gland, and hypothalamus) to assess tissue-specific and shared expression profiles. In all, 426 restricted genes and 4,107 shared genes were found expressed among the three tissues. A developmental profile of gene expression of the neuroendocrine system was examined comparing the late embryonic and neonatal stages indicating activation of such genes as the myelin basic protein, prolactin and decreases in message for type 2 somatostatin. Work continued by MI (Dodgson) on the development and characterization of large insert BAC libraries for the research community; DNA from the female RJF UCD001 chicken used to create the TAMU and CHORI libraries was provided to the Washington U. Genome Center to construct small plasmid libraries for whole genome shotgun sequencing. Overgo hybridization was utilized to assign over 5700 BACs to over 720 markers or genes to integrate the BAC contigs to the existing genetic map. Linkage of the physical map to the genetic marker map is a key component for contig and sequence assembly. The E.Lansing linkage map was expanded to 1249 markers by MI (Cheng); of the 19 new markers, 11 were genes (8 mapped as SNPs) and 8 were SNPs to anonymous sequences. Research by MN emphasized microsatellite marker development and linkage mapping in the turkey. A total of 397 turkey microsatellite markers were developed from small insert libraries. Additionally, to identify suitable markers for mapping, PCR amplification of 772 markers (chicken, quail and turkey) led to the testing of 410 markers for polymorphism in the resource families chosen for linkage mapping. Twenty-six percent were polymorphic and were tested for informativeness and genotyped in the mapping families. All information is available at http://www.tc.umn.edu/~reedx054/Turkeygenome.htm). Approximately 140 RFLP markers from an embryonic cDNA library were mapped in turkey; additional clones were sequenced as a SNP resource. Nucleotide sequencing of the avian pneumovirus (APV) was conducted and indicates substantial nucleotide and amino acid identity between strains isolated from 1996 to 1999. Phylogenetic relationships between European and US strain were established. The sequence of the entire APV/C virus was completed. Work continued to evaluate the host transcriptional response to APV infection indicating upregulation of transcripts involving leukocyte chemoattractants, adhesion molecules and complement as well as genes involved in protein trafficking pathways. RNA expression of novel chicken interleukins, beta-defensins and toll-like receptors was investigated by TX (via real time-PCR) in broilers and layers following LPS stimulation (before/after study). Liver, spleen and lymphocyte samples were studied. Overall the collective data suggest that broilers may have a lowered innate immune response as compared to layers. Objective 2. Develop Methods for Creating New Genetic Variation in Poultry by Gene Transfer and Chromosome Alteration. Reports were provided by MI, MN and NC, with collaboration with CA and USDA-ARS ADOL (ADOL). Viral-based transgenic technologies. MI has begun to test VSV-pseudotyped one-round viral vectors for creating transgenic chickens. MI is also using cloned ALV vectors in combination with cloned MAV for biochemical scale production of proteins in vivo in infected chickens. NC continues characterization and breeding of a transgenic line produced with a replication-defective spleen necrosis virus vector that expresses beta-galactosidase. This line is now at the G3 stage and has shown the expected Mendelian inheritance. Physiological studies on the line have begun. Blastodermal and stem cell culture and analysis of germline commitment. NC continues to examine the expression of DAZL and VASA genes in germline cells. The purpose is to distinguish between an inductive mode or a pre-determined mode of germ cell specification. MI concluded collaborative studies with ADOL on the use of chimeric chicken techniques and blastodermal cell culturing to generate germline transgenic birds. The effects of growth factor extracts on cell growth and telomere maintenance were performed in collaboration with ADOL and CA. MN has recently isolated chicken stem cell-like cells from 6 day-old SPAFAS chicken embryos and is currently investigating what growth factors and media conditions will maintain a viable stem cell population without the use of heterologous feeder layers. Sperm-mediated gene transfer (SMGT). BioAgri Corp. reported successful SMGT with several agricultural animal species including chickens. MI and ADOL obtained reagents from BioAgri in an attempt to duplicate their results. Rigorous tests of SMGT using DNA that encodes the RCASBP(A) replication competent avian leukosis virus (ALV) retroviral vector were performed. Despite every attempt to reproduce the BioAgri conditions, SMGT has not been successful in their hands. Studies of immortal avian cell lines. MN has succeeded in developing the world?s first and only non-virally immortalized continuously growing turkey cell line (TT-1). MN has ascertained that this new turkey-specific cell line supports the propagation of avian pneumovirus (APV). Objective 3. Develop, Compare and Integrate Emerging Technologies with Classical Quantitative Genetics for Improvement of Economic Traits in Poultry. IN used compute simulations to examine Genome wide MAS. For all combinations of parameters examined, a small population size was better than large. For a poultry breeder this does not mean that inbreeding will be a problem, rather the breeding structure must change to utilize this method. IN showed that a subdivide merge scheme of breeding can be utilized. With this method the population is divided into many small population, selection occurs within line and periodically the lines are crossed and re-subdivided. The rate of inbreeding is the same as for one large population of similar cumulative size. ADOL and IA continued previous work to identify QTL conferring resistance to Marek?s disease in commercial layers was performed by both. Previously, using the Marek?s disease (MD) resistant inbred Line 6 and the MD susceptible Line 7 to generate a F2 progeny resource population, 14 QTL were identified conferring susceptibility to Marek?s disease. To verify if the QTL that was identified in experimental crosses existed in commercial chickens, and to identify new QTL, two resource populations were developed in collaboration with Hy-Line. For genotyping, ~120 informative microsatellite markers have been scored on the parents, F1 sires, and selective DNA pools (within MHC haplotype and cross) for all the BC progeny. Statistical analyses identified many markers that show suggestive association with disease resistance traits in one or more analyses. ADOL also continued efforts to identify candidates for genetic resistance to Marek?s disease (MD) using a comprehensive two-hybrid screen was conducted using genes unique to virulent Marek?s disease virus (MDV) strains. ADOL also validated a SNP protocol for assessment of chicken genotypes conferring susceptibility or resistance to infection by subgroup E and subgroup B avian leukosis viruses (ALV). A comprehensive scan of the chicken genome using microsatellite markers was carried out on a meat-type x egg-type resource population (wide cross) by ADOL. USDA-ARS,GBL (GBL), in collaboration with IA, has completed genotyping the F2 generation of the Iowa Growth and Composition Resource Population (IGCRP). A genome scan was conducted and included 269 microsatellite markers. Initial analysis of data from GGA1 by interval mapping suggests the presence of QTL for age dependent growth related traits. IA and GBL also jointly collected data for nutrient absorption/retention phenotypes for the IGCRP advanced intercross which indicated that significant differences between the founder lines and resulting crosses exist for absorption of specific, environmentally important, nutrients. GBL has continued work to identify candidate genes to incorporate into QTL studies by identifying several genes involved in phosphorus absorption in the small intestine by using microarrays consisting of 3,072 unique cDNAs specific for the small intestine. IA has developed A three-generation resource population to detect quantitative trait loci (QTL) affecting egg production and egg quality traits in layers,. AR has developed a subpopulation of their F2 broiler cross developed to study abdominal pigmentation that has been typed for both their alleles at the dominant white (I) locus and the extended black (E) locus. Please see the NC1008 homepage for the full accomplishments report.

Publications

ARKANSAS, UNIVERSITY OF ARKANSAS, Fayetteville, AR Cisar C.R., J.M. Balog, R. Okimoto, N.B. Anthony, and A.M. Donoghue. 2003. The chicken bone morphogenetic protein receptor type II (BMPR2) gene maps to chromosome 7. Animal Genetics. 34: 465-476. Cornett, L.E., J.D. Kirby, J.A. Vizcarra, J.C. Ellison, J. Thrash, P. R. Mayeux, M.D. Crew, S.M. Jones, N. Ali and D.A. Baeyens. 2003. Regulatory Peptides 110:231-239. Jurkevich, A., L.R. Berghman, L.E. Cornett and W.J. Kuenzel. 2003.Immunohistochemical visualization of the vasotocin VT2 receptor in the pituitary gland of the domestic chicken. Poultry Sci. 82 (Suppl.): 81. Jurkevich, A., L.R. Berghman, L.E. Cornett and W.J. Kuenzel. 2003. Immunohistochemical characterization of chicken pituitary cells containing the vasotocin VT2 receptor. Soc. Neurosci. Abstr. 33: #710.10. Kuenzel, W.J. 2003. Neurobiology of molt in avian species. Poultry Science 82:981-991. Kuenzel, W.J., M.M. Abdel-Maksoud, T. Elsasser and J.A Proudman. 2003. Sulfamethazine advances puberty in male chicks by effecting a rapid increase in gonadotropins. Comp. Biochem. Physiol. A (in press) Kuenzel, W.J., M.B. Ferrari, T. Rathinam and H. Li. 2003. Cerebrospinal fluid- contacting neurons in the lateral septal organ (LSO) appear to function as encephalic photoreceptors. Soc. Neurosci. Abstr. 33: #827.9. Li, H., M. Ferrari and W. Kuenzel. 2003. Light-induced reduction of cytoplasmic free calcium in neurons proposed to be encephalic photoreceptors (EPRs) in chick brain. Poultry Sci. 82 (Suppl. 1): 45-46. Ling, M.K., M.C. Lagerstrom, R. Fredriksson, R. Okimoto, N.I. Mundy, S. Takeuchi, H.B. Schioth. 2003. Association of feather colour with constituatively active melanocortin 1 receptors in chicken. Eur. J. Biochem. 270: 1441-1449. Reiner, A., D.J. Perkel, L.L. Bruce, A.B. Butler, A.Csillag, W. Kuenzel, L. Medina, G.Paxinos, T. Shimizu, G. Striedter, M. Wild, G.F. Ball, S. Durand, O. Güntürkün, D.W. Lee, C.V. Mello, A. Powers, S.A. White, G. Hough, L. Kubikova, T.V. Smulders, K. Wada, J. Dugas-Ford, S. Husband, K. Yamamoto, J. Yu, C. Siang, and E.D. Jarvis. 2004. Revised nomenclature for avian telencephalon and some related brainstem nuclei. J. Comp. Neurol. (in press). Reiner, A., D.J. Perkel, L.L. Bruce, A.B. Butler, A. Csillag, W. Kuenzel, L.Medina, G.Paxinos,T. Shimizu, G. Striedter, M. Wild, G.F. Ball, S. Durand, O. Güntürkün, D.W. Lee, C.V. Mello, A. Powers, S.A. White, G. Hough, L. Kubikova, T.V. Smulders, K. Wada, J. Dugas-Ford, S. Husband, K. Yamamoto, J. Yu, C. Siang, and E.D. Jarvis. 2004. The Avian Brain Nomenclature Forum: Terminology for a new century in comparative neuroanatomy. J. Comp. Neurol. (in press). Scarbrough, K., J.D. Kirby, L.E. Cornett and R. Okimoto. 2003. Chromosomal assignment and mapping of the vasotocin receptor 1, homologue to the mammalian V1a and Vasotocin receptor 2 homogogue to the mammalian V1b receptors in the domestic fowls. Anim. Genetics 34:384-397. Sharma, P.M., D.W. Salter, W.S. Payne, and R. Okimoto. 2003. Chicken melanosomal matrix protein is a candidate gene for the dominant white phenotype. Plant, Animal, and Microbe, Genome XI. Abstract guide. Sharma, P.M., W.G. Bottje, and R. Okimoto. 2003. Association of variant alleles of the uncoupling protein with feed efficiency in broiler chickens. Poultry Sci. 82 (suppl. 1) page 23. Takeuchi, S., S. Takahashi, R. Okimoto, H. B. Schiöth, T. Boswell. 2002. Avian melanocortin system: alpha MSH may be acting as an autocrine/paracrine hormone. 5th International Melanocortin meeting. Sunriver Oregon. Published in Ann NY Acad Sci. 2003, 994: 366-372. Thilakar, R. and W. Kuenzel. 2003. Effect of electrolytic lesions of the lateral septal organ on gonadal development in male broiler chicks (Gallus domesticus). Poultry Sci. 82 (Suppl. 1): 44. Ugrankar, R.B., K. Cheng, and R. Okimoto. 2003. DNA sequence of melanocortin 1-receptor gene in Coturnix japonica: correlation with three E locus alleles, E, e+, and erh. Discovery. 4: 83-88. CALIFORNIA, CITY OF HOPE, Duarte, CA Goto, R.M., Afanassieff, M., Ha, J., Iglesias, G.M., Ewald, S.J., Briles, W.E., and Miller, M.M. 2002. Single strand conformational polymorphism (SSCP) assays for major histocompatibility complex B genotyping in chickens. Poultry Science 81:1832-1841. Iglesias, G.M., Soria, L.A., Goto, R.M., Jar, A., Miquel, M.C., Lopez, O.J., and Miller, M.M. 2003. Genotypic variability at the major histocompatibility complex (B and Rfp-Y) in Camperos broiler chickens. Animal Genetics 34:88-95. Liu, W. Miller, M.M. and Lamont, S.J. 2002. Association of MHC class I and class II gene polymorphisms with vaccine or challenge response to Salmonella enteritidis in young chicks. Immunogenetics 54: 582-590. Thoraval, P., Afanassieff, M., Bouret, D., Luneau, G., Esnault, E., Goto, R.M., Chausse, A.-M., Zoorob, R., Soubieux, D., Miller, M.M., and Dambrine, G. 2003. Role of nonclassical class I genes of the chicken major histocompatibility complex Rfp-Y locus in transplantation immunity. Immunogenetics. 55:647-651. CALIFORNIA, UNIVERSITY OF CALIFORNIA, DAVIS, Davis CA Daniels, L.M. and M.E. Delany. 2003. Molecular and cytogenetic organization of the 5S ribosomal DNA in chicken (Gallus gallus). Chromosome Research 11:305-317. Delany, M.E.. 2003. Genetic diversity and conservation of poultry. In Poultry Genetics, Breeding and Biotechnology (editors: W.E. Muir and S.E. Aggrey). Chapter 15, pp. 257-281. CABI Publishing, Wallingford, UK. Delany, M.E. 2003. Organization and dynamics of chicken telomeres. In Proceedings of the 52nd Annual National Breeders Roundtable, St. Louis MO. Delany, M.E., L.M. Daniels, S.E. Swanberg, and H.A. Taylor. 2003. Telomeres in the chicken: Chromosome ends and genome stability. Poultry Science 82:917-926. Delany, M.E., and L.M. Daniels. In press. The chicken telomerase RNA gene: Conservation of sequence, regulatory elements and synteny among viral, avian and mammalian genomes. Cytogenetic and Genome Research. Fulton, J.E. and M.E. Delany. 2003. Diminishing poultry genetic research resources: Rescue needed. Science 300:1667-1668. Swanberg, S.E., and M.E. Delany. In press. Dynamics of telomere erosion in transformed and non-transformed avian cells in vitro. Cytogenetic and Genome Research. DELAWARE, UNIVERSITY OF DELAWARE, Newark,DE Bliss, T. W., M. G. Emara, and C. L. Keeler, Jr. 2003. Construction and Characterization of EST libraries From Differentially Stimulated Avian Macrophage Cells. 103rd American Society for Microbiology general meeting, Washington, DC, May 18-22. Emara, M. G., and H. Kim. 2003. Genetic markers and their application in poultry breeding. Poultry Sci. 82:952-957. Kim, H., and M. G. Emara. 2003. Characterization of the F-box Protein Family in Chickens. J. Heredity, submitted. Kim, H., C. J. Schmidt, K. S. Decker, and M. G. Emara. 2003. A double-screening method to identify reliable candidate non-synonymous SNPs from chicken EST data. Animal Genetics 34:249-254. INDIANA, PURDUE UNIVERSITY, West Lafayette, IN Cheng, H.W., P. Singleton and W.M. Muir. 2003. Social stress differentially regulates neuroendocrine responses in laying hens: I. Genetic basis of dopamine responses under three different social conditions. Psychoneuroendocrinology 28:597-611. Cheng, H.W., P. Singleton and W.M. Muir. 2003. Social stress in laying hens: Differential effect of stress on plasma dopamine concentrations and adrenal function in genetically selected chickens. Poult. Sci. 82:192-198. Muir, W.M.. 2003. Indirect Selection for Improvement of Animal Well-Being. Chapter 14, p247-256. In Poultry Breeding and Biotechnology Eds. WM Muir and S Aggrey. CABI Press Cambridge MA. Muir, W.M.. 2003. Incorporating Molecular Information in Breeding Programs, Applications and Limitations. Chapter 28 p549-562. In Poultry Breeding and Biotechnology Eds. WM Muir and S Aggrey. CABI Press Cambridge MA. IOWA, IOWA STATE UNIVERSITY, Ames, IA Deeb, N. and Lamont, S. J. 2003. Use of a novel outbred by inbred F1 cross to detect genetic markers for growth. Anim. Genet. 34:205-212. Goodenbuhr, J.M., Kaiser, M. G., and Lamont, S.J. 2004. Linkage mapping of Inhibitor of apoptosis protein-1 (IAP1) to chicken chromosome 1. Anim. Genet. (in press). Kaiser, M. G., Lakshmanan, N., Arthur, J. A., O?Sullivan, P., Kuhn, M., and Lamont, S. J. 2003. Experimental population design for estimation of dominant molecular marker effect on egg-production traits. Anim. Genet. 34:334-338. Kaiser, M. G., Lanning, J. D., Ingleby, L. J., and Lamont, S. J. 2003. Interval mapping of QTL for salmonella colonization in F1 crosses if outbred by inbred chickens. In: Proc. Plant and Animal Genome XI, San Diego, CA, January 11-15. Kramer, J., Malek, M., and Lamont, S. J. 2003. Association of twelve candiate gene polymorphisms and response to challenge with Salmonella enteritidis in poultry. Anim. Genet. 34 339-348. Kramer, J., Malek, M., and Lamont, S. J. 2003. Candidate gene analysis to salmonella resistance in meat-type chickens. In: Proc. Plant and Animal Genome XI, San Diego, CA, January 11-15. Lamont, S. J. 2003. Unique population designs used to address molecular genetics questions in poultry. Poultry Sci. 82:882-884. Lamont, S. J., Pinard-van der Laan, M.-H., Cahaner, A., van der Poel, J. J., and Parmentier, H. K. 2003. Selection for disease resistance: direct selection on the immune response. Pp. 399-418. In: Poultry Genetics, Breeding and Biotechnology. Muir, W. M. and Aggrey, S. E., eds., CAB International. Li, H., Deeb, N., Zhou, H., Mitchell, A.D., Ashwell, C.M., and Lamont, S.J. 2003. Chicken quantitative trait loci for growth and body composition associated with transforming growth factor-b genes. Poultry Sci. 82:347-356. Liu, W. and Lamont, S.J. 2003. Candidate gene approach: potential association of Caspase-1, Inhibitor of Apoptosis Protein-1, and Prosaposin gene polymorphisms with response to Salmonella enteritidis challenge or vaccination in young chicks. Anim. Biotech. 14:61-76. Liu, W., Kaiser, M.G., and Lamont, S.J. 2003. Natural Resistance-Associated Macrophage Protein 1 Gene Polymorphisms and Response to Vaccine against or Challenge with Salmonella enteritidis in Young Chicks. Poultry Sci. 82:259-266. Lonergan, S. M., Deeb, N., Fedler, C. A., and Lamont, S. J. 2003. Breast meat quality and composition in unique chicken populations. Poultry Sci. 82:1990-1994. Lonergan, S. M., Deeb, N., Fedler, C. A., and Lamont, S. J. 2003. Breast meat quality and composition in unique chicken populations. Poultry Sci. 82(Suppl. 1):48. Malek, M. and Lamont, S. J. 2003. Association of INOS, TRAIL, TGFb2, TGFb3, and IgL genes with response to Salmonella enteritidis in poultry. Genet. Sel. Evol. 35 (Suppl. 1):S99-S111. Malek, M., and Lamont, S. J. 2003. Response to Salmonella enteritidis in chickens associated with INOS, TRAIL, TGFB2, TGFB3, and IGL genes. In: Proc. Plant and Animal Genome XI, San Diego, CA, January 11-15. Malek, M., Hasenstein, J., and Lamont, S. J. 2004. Analysis of chicken TLR4, CD28, MIF, MD-2, and LITAF genes in a Salmonella enteritidis resource population. Poultry Sci. (in press). McElroy, J. P., Cheng, H. H., Fulton, J., Soller, M., Lipkin, E., Dekkers, J. C. M., and Lamont, S. J. 2003. Markers associated with Marek?s disease survival in commercial layers. Poultry Sci. 82(Suppl. 1):24. Wang, J. 2003. Interval mapping of QTL with selective DNA pooling data. Ph.D. Dissertation, Iowa State University. Wang, J., Fulton, J., and Dekkers, J. 2003. Mapping quantitative trait loci affecting economic traits in layers. Poultry Sci. (Suppl. 1):49. Wang, J., Fulton, J., and Dekkers, J. 2003. Accuracy of detecting quantitative trait loci by selective DNA pooling. J. Anim. Sci. 81 (Suppl. 2): Wang, J., Koehler, K., Soller, M., and J.C.M. Dekkers, J.C.M., 2003. Least squares interval mapping to detect QTL with selective DNA pooling. Plant and Animal Genome XI: 69. Zhou, H., Deeb, N., Mitchell, A., Ashwell, C., and Lamont, S. J. 2003. Associations of BMP genes with skeletal integrity traits in chickens. Poultry. Sci. 82(Suppl. 1):23. Zhou, H. and Lamont, S. J. 2003. Associations of six candidate genes with antibody response kinetics in hens. Poultry Sci. 82:1118-1126. Zhou, H. and Lamont, S.J. 2003. Association of transforming growth factor b genes with quantitative trait loci for antibody response kinetics in hens. Anim. Genet. 34:275-282. Zhou, H. and Lamont, S. J. 2003. Chicken MHC class I and II gene effects on antibody response kinetics in adult chickens. Immunogenet. 55:133-140. Zhou, H. and Lamont, S. J. 2003. Network interactions among fifteen candidate genes for immunity hens. In: Proc. Plant and Animal Genome XI, San Diego, CA, January 11-15. Zhou, H., Li, H. and Lamont, S. J. 2003. Genetic markers associated with antibody response kinetics in adult chickens. Poultry Sci. 82:699-708. MARYLAND, UNIVERSITY OF MARYLAND, College Park, MD Cogburn LA, Wang X, Carre W, Rejto L, Porter TE, Aggrey SE, Simon J (2003) Systems-wide chicken DNA microarrays, gene expression profiling, and discovery of functional genes. Poult Sci. 2003 Jun;82(6):939-51. MICHIGAN, MICHIGAN STATE UNIVERSITY, East Lansing, MI Dodgson, J.B. 2003. The future of molecular genetics in poultry breeding. In W. M. Muir and S.E. Aggrey (eds.), Poultry genetics, breeding and biotechnology. CABI Publishing, New York, NY, pp. 685-695. Dodgson, J.B. 2003. Chicken genome sequence: a centennial gift to poultry genetics. Cytogenetics and Genome Research, in press. Dodgson, J.B. and Romanov, M.N. 2004. Use of chicken models for the analysis of human disease. In N.C. Dracopoli, J.L. Haines, B.R. Korf, D.T. Moir, C.C. Morton, C.E. Seidman, J.G. Seidman, and D.R. Smith (eds.) Current Protocols in Human Genetics. John Wiley & Sons, Hoboken, NJ, in press. Lee, M.-K., C.W. Ren, B. Yan, B. Cox, H.-B. Zhang, M.N. Romanov, F.G. Sizemore, S.P. Suchyta, E. Peters, and J.B. Dodgson 2003. Construction and characterization of three BAC libraries for analysis of the chicken genome, Animal Genetics 34:151-152. Ren C., M.K. Lee, B. Yan, K. Ding, B. Cox, M.N. Romanov, J.A. Price, J.B. Dodgson and H.B. Zhang. 2003. A BAC-based physical map of the chicken genome. Genome Research 13:2754-2758. Romanov M.N., J.A. Price, and J.B. Dodgson. 2003. Integration of animal linkage and BAC contig maps using overgo hybridization. Cytogenetics and Genome Research, in press. MINNESOTA, UNIVERSITY OF MINNESOTA, St. Paul, MN Alvarez, R., H.C.Lwamba, D.R. Kapczynski, M.K. Njenga, B.S. Seal. 2003. Nucleotide and predicted amino acid sequence-based analysis of the avian metapneumovirus type C cell attachment glycoprotein gene: Phylogenetic analysis and molecular epidemiology of U.S. Pneumoviruses. J. Clin. Microbiol. 41:1730-1735. Chaves, L.D., B.J. Ostroski, and K.M. Reed. 2004. Myosin light chain genes in the turkey (Meleagris gallopavo). Cytogenet Genome Res. In press. Christman S.A., B.-W. Kong, M.M. Landry, H. Kim, and D.N. Foster. 2003 Fluctuations in the p53 and Rb/p16INK4a pathways contribute to the spontaneous immortalization of a chicken embryo fibroblast cell line. Molec. Biol Cell. (suppl)14: 165a-166a. Dranchak, P, L.D. Chaves, J.A. Rowe, and K.M. Reed. 2003. Turkey microsatellite loci from an embryonic cDNA library. Poultry Sci. 82:526-531. Harry, D.E., P.J. Marini, D. Zaitlin, and K.M. Reed. 2003. A first generation map of the turkey genome. Genome. 46:914-924. Kong, B-W., H. Kim, and D. N. Foster. 2003. Expression analysis and mitochondrial targeting properties of the chicken manganese-containing superoxide dismutase. Biochem. Biophys. Acta. 1625:98-108. Kong, B-W., H. Kim, and D. N. Foster. 2003. Cloning and expression analysis of chicken phospholipid-hydroperoxide glutathione peroxidase. Animal Biotechnology. 14:19-29. Lwamba H.C., D.A. Halvorson, K.V. Nagaraja, E.A. Turpin, D. Swayne, B.S. Seal, M.K. Njenga. 2002. Antigenic cross-reactivity among avian pneumoviruses of subgroups A, B, and C at the matrix but not nucleocapsid proteins. Avian Dis 46:725-729. Lwamba H.C., R.S. Bennett, D.C. Lauer, D.A. Halvorson, M.K. Njenga. 2002. Characterization of avian metapneumoviruses isolated in the USA. Anim. Health Res Rev. 3:107-117. Munir, S. and V. Kapur. 2003. Regulation of hohst cell transcriptional physiology by the avian pneumovirus provides key insights into host-pathogen interactions. J. Virol. 4899-4910. Njenga, M. K., H.C.M. Lwamba, B.S. Seal. 2003. 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Identification of chicken stem lymphocyte antigen 6 complex, locus E (LY6E, alias SCA2) as a putative Marek?s disease resistance gene via a virus-host protein interaction screen. Cytogen. Genome Res., in press. Niikura, M., Liu, H.-C., Dodgson, J.B., and Cheng, H.H. A comprehensive screen for chicken proteins that interact with proteins unique to virulent strains of Marek?s disease virus. Submitted. Okumura, F., Shimogiri, T., Shinbo, Y., Yoshizawa, K., Kawabe, K., Mannen, H., Okamoto, S., Cheng, H.H., and Maeda, Y. Linkage mapping of four chicken calpain genes. Submitted. Zhu, J.J., Lillehoj, H.S., Allen, P.C., Van Tassell, C.P., Sonstegard, T.S., Cheng, H.H., Pollock, D., Sadjadi, M., Min, W., and Emara, M.G. 2003. Mapping quantitative trait loci associated with resistance to coccidiosis and growth. Poultry Science 82:9-16. USDA, USDA-ARS; GROWTH BIOLOGY LABORATORY, Beltsville, MD Li, H., Deeb, N., Zhou, H.t Mitchell, AD., Ashwell, C.M., and Lamont, S.J. 2003. 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Impact Statements

Date of Annual Report: 09/16/2005

Report Information

Annual Meeting Dates: 01/15/2005 - 01/16/2005
Period the Report Covers: 01/01/2005 - 12/01/2005

Participants

Ashwell, Christopher - North Carolina State University;
Cheng, Hans - USDA-ARS, ADOL;
Delany, Mary - University of California, Davis;
Dodgson, Jerry - Michigan State University;
Foster, Douglas - University of Minnesota;
Kuenzel, Wayne - University of Arkansas;
Lamont, Susan - Iowa State University;
Miller, Marcia - City of Hope National Medical Center;
Muir, William - Purdue University;
Petitte, James - North Carolina State University;
Ponce de Leon, Abel - University of Minnesota;
Porter, Tom - University of Maryland;
Reed, Kent - University of Minnesota;
Wong, Eric - Virginia Polytechnic Institute & State University;
Zhang, Huaimin - USDA-ARS, ADOL;
Zhu, James - Texas A & M University

Brief Summary of Minutes

The NC-1008 meeting was held in conjunction with the Plant & Animal Genome XIII meeting in San Diego, on January 15 and 16, 2005. The business meeting was held on Sunday, January 16. The business meeting was called to order on January 16th at 3:00 by NRSP-8 chair, Chris Ashwell.

Following organizational comments by the chair and self-introductions by the attendees, reports were presented as published in the PAG XIII program.

The speakers who presented reports/papers to the group included: Tom Porter (MD); James Zhu (TX), Bill Muir (IN), Doug Foster (MN), Kent Reed (MN), Julie Long, (Guest, USDA-ARS-Beltsville), Eric Wong (VA), Mary Delany (CA), Wayne Kuenzel (AR), Doug Rhoads (AR), Sue Lamont (IA), Marcia Miller (COH), Paul Siegel (Guest, Va Tech), Chris Ashwell (NC), Jim Petitte (NC), Jerry Dodgson (MI and NRSP-8 Coordinator), Parker Antin (Guest, University of Arizona College of Medicine), Hans Cheng (USDA-ARS-ADOL), Huanmin Zhang (USDA-ARS-ADOL), Joan Burnside (Guest, University of Delaware), Alice Kuo (Guest, USDA-ARS-GBL), Pete Kiaser (Guest, Institute for Animal Health-UK), Fred Leung (Guest, University of Hong Kong), Martin Groenen (Guest, Wageningen University).

The first order of business was to approve the minutes of the previous year's meeting. Sue Lamont offered a correction that "Dekkers" was misspelled in last year's minutes and Doug Foster added the correction that Marlene Emara at Delaware had been omitted from the list of meeting attendees. With those corrections, the minutes were approved unanimously.

There was no outstanding old business to discuss.

Reports were then presented by:

Administrative advisor Margaret Dentine gave her report to the group and noted that NC1008 is exactly what a multistate project is supposed to be. The midterm review is coming up next year. This is a very productive committee. Examples of productivity should be easy. We have a great story to tell genome websites, etc. Use NIMSS for communication. Distribute minutes through NIMSS please. NIMSS is updated.

Species coordinator report was presented by Jerry Dodgson Highlights for the year included the following: Genome sequence paper, Physical map paper, the 2.8 million SNPs paper. Companion reports will appear in Genome Research (1/05). He is still distributing resources. Microsatellite Kit #7 covers genome. Affymetrix chip for the chicken is now available. The chicken is now #7 among all species in ESTs submitted. Radiation hybrid map is available through Alain Vignal. As coordinator, Jerry will argue for post-sequence tools program for USDA-NRI. Jerry commented that this year's was a "well organized, well run meeting.

CSREES advisor Muquarrab Qureshi distributed his report (CSREES - Genetics and Genomics Portfolio) to members. He then posed an important question to determine if the chicken that was sequenced had been named. He summarized what the status of operations in the agency. Performance-based budgeting has arrived at CSREES. A major goal that is near completion is sequencing of the bovine genome at 6X coverage. The USDA has committed 10M$ for sequencing of the swine genome. Input was requested for the RFA2006 for the NRI competitive grants program. The molecular tools program is not off the books, just paused. Muquarrab also encouraged the group members to tell their respective experiment station directors about the success of out regional project(s).

New business:

No officers are turning over in the next year.

For the annual report the following individuals agreed to summarize progress for each of the objectives: Mary Delaney objective 1, Jim Pettitte objective 2, Sue Lamont objective 3. It was also agreed that everyone would summarize their contributions by objective and send them to the appropriate individual so that organization would be smoother.

A discussion took place on recruiting new members. Abel Ponce de Leon noted that the officers should search for new members at meetings. Administrative advisor Margaret Dentine remarked that the room has quite a few senior members and that new faculty hires should be encouraged to join the group.

There was discussion regarding the invitation of guest speakers that resolved in agreement that outside speaker should continue to be invited.

Discussion with regard to changes in the organization for future meetings was initiated by Chris Ashwell who noted that when meeting in conjunction with PAG and other regional projects the group is trapped by the master schedule. Chris also suggested that we begin the meeting earlier on Saturday in order to accommodate the number of speakers and provide additional time. Abel Ponce de Leon suggested that each station should report every other year and more time should be made for topic discussion. Administrative advisor Margaret Dentine noted that shorter station reports would allow for more discussion. Bill Muir and Sue Lamont agreed that everyone could and should talk less. Jerry Dodgson noted that the current years schedule was appropriate to allocate time by station with additional time to be given to the larger stations. Tom Porter moved that time be allocated by the individual presenter. The motion was seconded by Jim Petitte. The motion passed unanimously. As for the scheduling of the 2006 meeting Chris Ashwells suggestion to start the meeting at 9:00am was unanimously agreed upon.

Jerry Dodgson moved that the meeting be adjourned.

Sue Lamont made a special announcement prior to adjournment noting that Dr. Robert Shoffner should be acknowledged for his many contributions to the field of poultry genetics upon his passing. This acknowledgement was affirmed by voice call.

The meeting was adjourned at 4:00 in time for the following NRSP-8 business meeting.

Accomplishments

Objective 1. Utilize Modern Molecular and Breeding Technologies to Identify, Locate, Isolate and Characterize Poultry Gene of Economic Importance. Significant progress was made in poultry (chicken and turkey) genetic and genomic analysis. Research focused on the continued development and alignment of genetic, physical and cytogenetic maps. Advances were achieved in the sequence status for genes/QTLs of economically relevant traits, and also in the identification and elaboration of gene expression profiles associated with growth, development, nutrient utilization and disease. Particularly noteworthy are the release 6.6X draft chicken genome (March 2004) and the publications in Nature of the first level genome (Hillier et al., 432:695) and SNP (Wong et al., 432:171) analyses involving international consortia of scientists, including NC1008 scientists and their collaborators. Advances in Objective 1 involved substantial collaborative research among technical committee members and included sharing of information, reagents, tools and genetic resources including cell lines and specialized stocks of chicken and turkey. Extensive individual station annual reports were prepared and distributed by the members in advance of and during the meeting; the following review provides only a very brief outline of the highlights of a very productive year of research on Objective 1 for the contributing stations. SDD (spermatozoa dysfunction degeneration) is a dominant mutation resulting in production of degenerate sperm and low fertility. The AR station identified males with an SDD phenotype from several broiler breeder lines and showed that the defect is inherited as a single dominant gene in 2 successive generations. The SDD mutation is tentatively mapped to GGA7. New microsatellites and SNPs are being developed in this region to confirm and refine this map position. The follicle stimulating hormone, beta chain (FSHb) promoter is being examined with a concentration on the response elements since the pulsatile release of FSH is much different than LH. A brain structure called the lateral septal organ (LSO) contains specialized neurons that respond to direct photostimulation. When the LSO is removed surgically, development of reproductive system following exposure to a long photoperiod is significantly attenuated. It is suggested that the LSO contains encephalic photoreceptors responsible for detecting long-day photoperiods and bringing birds into sexual maturation. Research at the UC Davis CA station (in vitro and in vivo) explored the molecular organization and expression of genes involved in the telomere-telomerase pathway controlling genome stability. The chicken TERT gene was cloned and compared to available vertebrate TERTs; new regions of vertebrate domain homology were identified. Both 5' and 3' genomic regions were analyzed to establish elements relevant for gene regulation; the chicken TERT gene as found for other vertebrates, contains a c-my E-box along with a series of conserved regulatory element motifs. Gene profiling was conducted for telomerase RNA and reverse transcriptase, telomere binding proteins and c-myc in DT40, chicken embryo fibroblasts, gastrula stage embryos and chicken embryonic stem cells showing abnormal levels of transcripts in DT40 relative to the other cell types. A key finding was that TR rather than TERT may be the rate limiting transcript controlling telomerase activity. At the COH the Ca station continues to assemble BAC clones into a map for the chicken MHC B and Y gene regions. Two BAC clones forming a single contig for the B region are fully sequenced. Currently two contigs containing seven BAC clones represent the Y region. Two Y BACs are fully sequenced. The IA station investigated bone morphogenetic protein (BMP) receptor 2 using primers published by Cisar et al., 2003, for associations with skeletal integrity traits in two F2 resource populations. Sequence polymorphisms between founder lines (broiler, Leghorn and Fayoumi) were detected, and PCR-RFLP methods were developed to type the F2 males. A BMPRII SNP showed significant (P < 0.05), or approaching significant (0.05 < P < 0.10), associations with percent shank weight and height/weight ratio in the broiler-Leghorn cross, and with the same two traits plus bone mineral content and density, tibia length and shank percent length in the broiler-Fayoumi cross. The MD station has produced cDNA microarrays containing more than five thousand genes expressed in the chicken neuroendocrine system, validated an RNA amplification protocol for use with samples from individual embryonic pituitary glands, and used these tools to study changes in gene expression profiles within the hypothalamus and anterior pituitary gland of chickens during late embryonic and early post-hatch development. Genes were identified whose expression levels changed in accordance with hatching and the differentiation of several anterior pituitary cell types. They have confirmed expression profiles of these by quantitative Real Time RT-PCR. The MI station contributed to the assembly of the physical BAC contig map of the chicken and its alignment to both the chicken sequence and linkage map via overgo hybridization techniques. Efforts also began to extend this approach to a comparative chicken-turkey physical map. The USDA-ARS-ADOL continues to coordinate the East Lansing genetic map. Currently, the map includes 1276 markers. Efforts are underway to enhance the integration with the draft genome sequence, especially the genetic mapping of unassigned BAC contigs and the development of SNPs. Research by the MN station emphasized microsatellite marker development and linkage mapping in the turkey. Genotyping of current markers was completed during the Summer/Fall of 2004 and genetic map construction is now complete. The current linkage map includes 314 loci arranged in 29 linkage groups with a total length of 2011 cM. An additional 38 markers show significant 2-point linkages but could not be ordered and 17 markers remain unlinked at this time. The linkage map has been aligned with the chicken genome sequence by in silico mapping and application of the INRA chicken RH-panel. A 461-marker comparative map was constructed. Work is underway to integrate the previously constructed maps of the turkey genome with the new comparative map. In addition a BAC clone containing the MHC B-locus has been isolated from the CHORI-260 turkey BAC library and is being sequenced. The NC station has been evaluating a small intestine-derived Na/Pi IIB cotransporter for cDNA structure and predicted protein organization. Expression level is highest in the duodenum. The mRNA is found specifically within the vertical cryptvillus axis of the small intestine. The gene maps to GGA 4 where a QTL for bone, egg shell, and egg production traits was identified suggesting this genes is a positional candidate for dietary phosphorus retention. A microsatellite marker is found within intron 5 and specific alleles were associated with phosphorus retention in commercial broilers. Nutrient retention traits are key components for minimizing environmental impacts of animal agriculture. The TX station has been studying chemokines and the receptors roles in host defense, organogenesis, hematopoiesis, and neuronal communication. Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. These genes were annotated and named according to the nomenclature of the mammalian genes based on phylogenetic and syntenic analysis, which can be used for the functional inference of these genes in chickens The VA station has been studying the transport of nutrients (amino acids, peptides, and sugars) across the intestinal epithelia, mediated by membrane bound transporter proteins. The expression of a number of these transporters was shown to be developmentally regulated. For example, the peptide transporter PepT1 and the sugar transporters SGLT1 and GLUT2 are induced prior to hatch, which indicates that these transporters show temporal specific expression prior to the ingestion of feed. Objective 2. Develop Methods for Creating New Genetic Variation in Poultry by Gene Transfer and Chromosome Alteration. The MN station reported on the status of the SC-1 spontaneously immortalized chicken embryo fibroblast (CEF) cell line whose origin has been reported previously. Although this cell line had been in culture for almost four years, its growth rate has remained lower than that of primary CEF cells and the morphology was not as uniform as observed in primary cells. The SC-1 cell line was treated with chicken embryo extract (CEE) to determine whether growth rates could be increased and cell morphology enhanced. The CEE also was tested on primary CEF cells, another spontaneously immortalized CEF cell line (DF-1), and on two other non-virally and non-chemically immortalized CEF cell lines (BCEFi and HCEFi). Concentrations of CEE e 100 µg/ml inhibited growth of all cells tested. However, addition of 50 µg/ml CEE enhanced the growth rate and improved the morphology of the SC-1 cells. The increased growth rate and improved morphology of the SC-1 cells achieved with CEE treatment was retained following removal of CEE, and these improvements should aid in increasing the utility of the SC-1 cell line as a cellular/molecular reagent. The MN station also reported that the spontaneously immortalized TT-1 cell line that was specifically developed for the propagation of avian metapneumovirus (aMPV) is now at passage 145. Cell cycle analysis of TT-1 cell showed population doubling levels of 1.2-1.4. FACS analysis showed a higher proportion of S phase and G2/M phase cells compared to primary turkey turbinate cells. Finally, there were differences between TT-1 and Vero cell propagated aMPV plaque formation. Considerably more and larger areas of syncytial plaque formation were observed using Vero host cells compared to the more punctuate, non-syncytial areas seen with TT-1 cells suggesting that the homologous turkey cell substrate displays a different infectious nature than the heterologous monkey Vero cell substrate. The MN station has continued attempts at growing long-term cultures of chicken stem cell-like cells from 7 day-old SPAFAS chicken embryos. As a novel approach to this challenge MN has utilized the recent identification of the chicken LIF (cLIF) sequence (Horiuchi et al., JBC, 279:24514-24520, 2004) to clone an open reading frame for the 211 amino acid protein which was stably transfected the recombinant cLIF expression vector construct into DF-1 cells that will serve as a non-inactivated feeder layer in transwell plates for the propagation of putative chicken stem cells with or without the addition of 50 ug/ml chicken embryo extract. In collaboration with the MD staion , MN has explored the possibilities of either life span extending or immortalizing chicken embryonic somatotrophic pituitary cells isolated from 12-14 d old chicken embryos. Cells were able to be cultured but finally senesced after about 3 months, reaching passage 12. Given previous attempts at immortalizing pituitary cells, life span extension was quite probably achieved, although the ultimate goal of immortalization was not realized. Further attempts with additional cell cycle regulatory constructs are planned. Progress at the MI station focused on maintenance and development of test lines including albino and feather pigmentation lines described previously, whose endogenous viral genes are being eliminated or minimized by breeding. MI is also testing VSV-pseudotyped one-round viral vectors for creating transgenic chickens. One-round vectors get around some of the negative effects of traditional retroviral vectors but are inefficient at generating a suitable number of transgenic progeny. VSV-pseudotyping allows for concentrating and stabilizing the viruses used, and this method has been reported to be highly successful in generating quail transgenics (Mizaurai et al., Biochem. Biophys. Res. Commun. 286:456-463, 2001). To date, MI has been unable to generate adequate titers of VSV-pseudotyped BBAN virus to use in birds. New host cell lines are being developed and transfection protocols being modified. The MI station has also employed avian myeloblastosis virus (AMV) as a potential cost-effective source of membrane proteins, in particular, viral env proteins, for structural analysis. In association with its normal helper virus (typically a mixture of myeloblastosis-associated virus, MAV, 1 and 2), AMV generates extensive leukemia and very high virus titers in birds. To insure consistency, MI has co-transfected cloned AMV and MAV-1 recombinant DNAs into cells, followed by passage of the virus in birds. Optimal times of infection (3-5 days) have been established. The viral preps show increased virulence during the first few passages in birds. We have also shown that MAV-1 can easily be replaced by the widely used cloning vector RCAS(BP). This system has the potential to generate other membrane proteins that could be cloned into RCAS. After passage in birds, clonal cell lines can be established that appear to express GFP at varying levels. In previous years, the MI station has demonstrated that retroviral vectors can be introduced into birds that induce resistance to challenge viruses (of subgroups other than the introduced vector) by expression of soluble viral receptor (e.g., tva, the receptor for subgroup A avian leukosis-sarcoma viruses (ASLV)) or soluble viral envelope protein (SU fragment of env). A new project has commenced at MI to use this system as a test of the use of RNAi expression against exogenous viruses in adult birds. Initial RNAi targets for both tvb and env have been designed, cloned into the entry vectors and are now being verified by sequence analysis. The various RNAi retroviral vectors will first be tested for generation of viral resistance in infected cell lines. If successful, the system will be tested in somatic transgenic birds in collaboration with Henry Hunt and Huanmin Zhang at USDA-ARS-ADOL. The efficient production of germ line chimeras using PGC transfer into early embryos has been hampered by the limited availability of PGCs obtained from blood and the early germinal ridge and the preparation of suitable recipient embryos. Presently, it has been difficult to efficiently fractionate the PGC's from the total somatic cell population. The NC station has developed a Fluorescence Activated Cell Sorting (FACS) technique to isolate PGCs, whose identity was confirmed using Periodic Acid Schiff (PAS) staining or anti-Embryonic Mouse Antigen (EMA)-1 staining followed by microscopic evaluation. Less than 0.1% of the blood cell population was collected as SSEA-1 positive cells. Similarly, approximately 2% of the gonadal cell population was collected as SSEA-1 positive cells. Therefore, a small number (~1000-9000) of PGCs are able to be recovered from each isolate. The sorted SSEA-1 positive cells were maintained in vitro for two days to demonstrate their viability. In addition, sorted cells have been used at the NC station to produce germline chimeras. Compared to density gradient centrifugation and magnetic particle sorting, FACS has proved to be the best means of obtaining a pure population of PGCs. The NC station also used busulfan (1,4-butanediol dimethanesulfonate) to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in more a consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a 3-fold increase of PGCs in stage 27 embryos. Subsequently, germline chimeras were produced at the NC station by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras demonstrated an increase of 5-fold when compared to untreated recipients and the number of donor-derived offspring from the germline chimeras also increased 8-fold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production. Transgenic birds developed at the NC station carrying the lacZ gene were produced by infecting early embryos with a replication defective spleen necrosis retroviral vector encoding the lacZ gene. To further characterize this line, the 5' end of the proviral insert with flanking regions of genomic DNA was cloned and sequenced using inverse-PCR indicating that the insertion site is located on GGA11. The location was confirmed by using down-stream sequence information from the avian genome database, PCR primers were designed to develop a procedure for genotyping wild-type, hemizygous and homozygous birds. As expected for SNV, sequence analysis indicated that the process of integration of the proviral DNA resulted in the generation of a 5bp virus-specific repeat at the site of integration of the viral DNA. An examination of 10kb genomic sequence up-stream and down stream of the insertion site indicated that the proviral DNA lies within intron 1 of a 3 exon predicted gene, which appears to be neurotactin. This observation is consistent with the preference of genomic repeat structure as a preferred site of integration for retroviral genomes. Objective 3. Develop, Compare and Integrate Emerging Technologies with Classical Quantitative Genetics for Improvement of Economic Traits in Poultry. The USDA-ARS-ADOL station continues to work on genetic resistance to Marek's disease (MD). Collaborative efforts with Janet Fulton (Hy-Line) and Sue Lamont and Dr. Jack Dekkers (Iowa State U.) had led to the identification of numerous QTL in BC and F6 resource populations. Studies are underway to fine map each QTL. Evaluation of the candidate genes CPPP and OPN suggested that CPPP is significantly associated with MD resistance while OPN is only suggestive at best and may be complicated (e.g., epistasis or haplotype). A PCR-based assay has been developed to identify chickens genetically resistant to ALV subgroup B, D, and E infections. Finally, the mini l has been applied to efficiently generate specific point mutations in an infectious Marek's disease virus BAC clone (Cheng, ADOL). QTLs for traits of low heritability are difficult or impossible to locate. A method to overcome these limitations using a BLUP model based on a dense map a SNP's across the genomes was examined (GMAS). A gene level simulation was used to examine these issues. Results showed that traits of high heritability (.5), the accuracy of selection with GMAS reached about 88%, whereas with traditional BLUP the accuracy was 82%. However, the accuracy of BLUP rapidly dropped off rapidly in generations where the genotype is predicted based only on ancestors information whereas GMAS continued at a higher accuracy. For traits with a low heritability of .1, the accuracy of selection was 70% with GMAS and 3 generations of training, while BLUP only approached 60%. This is the first report of marker assisted selection where the response to selection exceeds BLUP where the QTL had to be found from the data. This method will allow the actual implementation of MAS in animal breeding on a wide scale for any trait, and simultaneously for all traits, the only real issue is if it can be cost effective (Muir, IN). It is well recognized that commercial broilers are more susceptible to infectious diseases than other slow growing chickens. The gene expression profiling of immune effectors and regulators indicates that broilers have a weaker innate immune response than layers. The results suggest that therapeutic intervention and genetic selection to enhance the innate immunity are needed to improve the health of commercial broilers. A chicken genome-wide microarray and bioinformatic tools were developed to facilitate research in chickens (Zhu, TX). In the past year, we completed the validation in backcrosses and F2 populations of candidate DNA markers for immune response previously identified in selected chicken lines. The newly released chicken genomic DNA sequence was also used to further test flanking markers (SNP-based) at 10-kb intervals from the putative loci. We also showed in the past year that a Gallus gallus nuclear gene possessed sufficiently strong phylogenetic signal to replicate mitochondria DNA classifications of Galliformes. In our turkey work, we used markers previously described to show the genetic relatedness among non-commercial and commercial domestic turkeys, information that may be useful for introgression of genetic backgrounds (Smith,VA). Two related, informative chicken F2 populations were used for genome-wide linkage and QTL analysis. All 742 F2 birds were phenotyped for 42 traits related to growth (8), body composition (12), skeletal integrity (12), and metabolic factors (10), and genotyped for 269 microsatellite markers. Given 1008 tests conducted for all marker-trait associations, 98 QTLs were significant at the 1% chromosome-wise level in the broiler-Leghorn cross; and 89 QTLs in the broiler-Fayoumi cross (Lamont, IA). Our objective was to identify appropriate statistical models for analysis of marker associations in selectively genotyped survival data. Survival data were simulated with a Kaplan-Meier function estimated from a real data set. Linear regression was found robust to deviations from normality inherent to survival data and selective genotyping, and may be preferred over survival analysis models for detection of marker associations because of ease of implementation and interpretation (Dekkers & Lamont, IA). This study characterized a method to simultaneously identify SNPs and estimate SNP allele frequencies from DNA pools, using relative peak heights of nucleotides in sequencing traces (ABI Prism 377). The correlation between estimated frequencies (from the DNA pools) and observed frequencies was 0.89 and the slope was 0.99. Therefore, estimation of SNP allele frequencies from DNA pools using relative peak heights of nucleotides in sequencing traces is accurate and feasible and can increase the efficiency of SNP studies by providing accurate, rapid and economical profiles of SNP allele frequencies of populations (Lamont & Dekkers IA). The potential to use population-wide linkage disequilibrium (LD) in commercial chicken breeding populations to detect QTL for economic traits was explored by examining the extent of marker to marker LD in commercial chicken breeding lines, under the assumption that it is predictive of the extent of marker-QTL LD. The extent of LD did not differ between chromosomes and lines, but did differ significantly (P<0.0001) between regions of d10 cM within lines. These results indicate that marker-QTL LD may be a useful route to MAS in these lines (Dekkers, IA).

Publications

Publications Attachment:

Attachment

Impact Statements

See Accomplishments Section

Date of Annual Report: 12/18/2006

Report Information

Annual Meeting Dates: 01/14/2006 - 01/15/2006
Period the Report Covers: 10/01/2005 - 10/01/2006

Participants

AR-University of Arkansas-W, Kuenzel, D. Rhoads, J.Kirby;
CA-City of Hope-M. Miller;
CA-University of California, Davis-M. E. Delany;
DE-University of Delaware-M. Emara;
GA-University of Georgia-S. Aggrey;
IA-Iowa State University-S. J. Lamont, J. Dekkers;
IN-Purdue University-W. Muir;
MI-Michigan State University-J.B. Dodgson;
MN-University of Minnesota-D.N. Foster, K. Reed, A. Ponce de Leon;
NC-North Carolina State University-C. Ashwell, J. Petitte ;
TX-Texas A & M University-J. Zhu;
USDA-USDA-ARS-ADOL-H. Cheng, H. Zhang;
VA-Virginia Polytechnic Institute & State University-E.Wong, E. Smith ;

Institutions absent-University of Maryland-T. Porter

Brief Summary of Minutes

Members present: Ashwell, Cheng, Delany, Dodgson, Emara, Foster, Kuenzel, Lamont, Miller, Muir, Pettite, Reed, Rhoads, Wong, Zhang, Zhu, Fulton, Qureshi, Saif

Members absent: Dekkers, Porter, Smith

Visitors: Burgess (MS), Song, and others

The meeting was called to order at 3:40 p.m. by NC1008 Chair Doug Foster. Minutes from the 2004 meeting were approved.

Old Business: There was no additional old business to discuss.
New Business: Shane Burgesss (MS) approval is in the works and should be official this year. James Zhu (TX) offered that Huaijun Zhou, a new faculty member at TAMU, will be a potential NC1008 station member.

Mo Saif was introduced as the new administrative advisor. He congratulated the committee members for sending their progress reports electronically before the meeting. The midpoint report for the project was submitted and received a complimentary review. The NC1008 committee has a very good reputation.

Muquarrab Qureshi also welcomed Mo Saif as the new administrative advisor. He commended the committee for putting on another excellent program. 135-140 publications were listed in the annual reports. The committee should focus on impacts. Muquarrab presented results for Hatch funding for FY06, $240M and for NRI $181M, which represents flat funding. He announced that there are new openings for a National Program Leader in Agrosecurity and as an Associate Administrator of CSREES. It is a great time for Animal Genomics with the announcement of the swine genome and additional funding to complete the chicken genome. There will be an Animal Production Stakeholder Workshop April 10-12, 2006 at the Marriot Hotel in Kansas City.

The recent review of NRSP8 was very complimentary. NRSP8 was considered to be one of the best projects and it was recommended that it maintain current funding levels. The newsletter was thought to be very helpful.

A summary of the NRI Program titled National Research Initiative Animal Genome Programs written by Peter Burfening was distributed. A key point was that since 1999, 26 grants had been awarded in poultry from NRI a success rate of 24.5%. More funds need to be put into the program to ensure that grants rated as outstanding are funded.

The next meeting will be prior to PAG XIV in San Diego, Jan 13-14, 2007. New officers for 2005 were elected. For NRSP-8, Kent Reed (MN) becomes Chair; Huanmin Zhang (USDA-ADOL) was elected secretary. For NC1008, Tom Porter (MD) becomes Chair; Eric Wong (VA) was elected secretary. New objective coordinators will be: Objective 1: Chris Ashwell (NCSU); Objective 2: Jerry Dodgson (MI); Objective 3: James Zhu (TX). Requests for bioinformatics resources to be developed for avian work need to be brought to the attention of James Reecy. Swine and aquaculture have utilized the resources, poultry has not. Mary Delany (CA) was nominated and elected secretary for the overall NRSP8 species coordinators group. Jerry Dodgson (MI) requested suggestions from committee members on how best to spend coordinators funds. The meeting was adjourned at 4:05 pm.

Accomplishments

Objective 1. Develop High Resolution Integrated Maps to Facilitate the Identification of Poultry Genes and Other DNA Sequences of Economic Importance. Progress was made in poultry (chicken and turkey) genetic and genomic analysis as well as in tool/resource development. A clearly noticeable trend was the utilization of the available chicken tools, the sequence and ESTs, to develop resources for turkey and for usage in poultry biological research to understand gene and sequence organization as well as function. Research also continued on the development and alignment of genetic, physical and cytogenetic maps. Advances in Objective 1 have involved considerable collaborative research among technical committee members and included sharing of information, reagents, tools and genetic resources including cell lines and specialized stocks of chicken. Kuenzel (AR). Scientists from four universities, UA Fayetteville (UAF), UA for Medical Sciences (UAMS), UA Little Rock (UALR), and Texas A&M Univ., collaborated to determine the functional role of the vasotocin II receptor (VT2R) that was recently cloned and sequenced in the chicken. Over the past two years the receptor was mapped to GGA 26 and its sequence was shown to be homologous to the vasopressin 1b (V1b) receptor in mammals. A polyclonal antibody was made against VT2R. Using immunocytochemistry (ICC) the receptor was found predominantly in the cephalic region of the anterior pituitary and associated with cell membranes of pituitary cells. A series of experiments was completed where antibodies to all six types of pituitary cells were utilized to determine which cell type contained the VT2R. Using double ICC, it was determined that the most abundant cell type containing the receptor was corticotrophs (ACTH containing cells). Similarly, melanotrophs (MSH containing cells) displayed a high number of cells with VT2Rs. Lactotrophs (prolactin containing cells) had 10-15% showing the receptor while thyrotrophs (thyroid stimulating hormone), somatotrophs (growth hormone) and gonadotrophs (luteinizing hormone containing cells) showed no co-localization of the VT2R. Due to the high association with corticotrophs, a study was completed with awake birds to examine the neuroendocrine stress response in such chickens. A cannula was implanted in a ventricle of the brain and a catheter implanted in the jugular vein of male broilers. Equimolar concentrations of corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT) were administered into the brain and corticosterone was assayed in the plasma. Results have shown that CRH or AVT were effective in releasing significantly higher levels of CORT compared to saline injected controls and that CRH was more efficacious in releasing the hormone. Rhoads (AR), with the collaboration of Kirby and Froman have generated four generations of pedigreed families from four different broiler breeder males from two commercial grandparent breeding lines. Males were evaluated for sperm viability (SDD) and mobility. The SDD trait was mapped in one family to a 3 Mb region of GGA 7, work in another family indicates a tentative location to a central region of GGA 1 (one family). They are working to refine the map position and develop a genetic test for SDD. Male fertility continues to be a major problem in advanced broiler breeding lines. Two major determinants of male infertility are sperm degeneration and sperm mobility. Research has been aimed at identifying the underlying genetic and physiological causes of sperm degeneration. Our goal is to understand the origins of the defect and develop genetic tests that can be used to increase male fertility. Delany (CA-UC Davis). Research continued on telomere organization and function as well as on telomerase gene expression. A developmental study provided evidence that the telomerase RNA gene is down-regulated as tissues lose telomerase activity and this is the first report of such for any vertebrate organism. Analysis of TERT splice variant expression was conduced in cell systems known to be positive or negative for telomerase activity. Nineteen splice variants were found among the samples, with several held in common, but also some exhibiting a unique profile. Variants were generated by intron insertion, exon skipping, and alternate usage of splice donor and acceptor sites. Most of the variants were predicted to produce premature termination codons, but several had potential for translation to a modified TERT protein. Work continued to map the ultra long (aka mega-) telomeres and to date four loci have been verified. Interestingly the main in common feature of the involved chromosomes is that they also encode other highly repetitive tandem arrays, coding or non-coding. The prior study provided a series of cytogenetically-verified BACs which can be used to identify the majority of microchromosomes; further, the work contributes to the alignment of the sequence and cytogenetic maps. UCD participate in the ADOL SNP project to establish SNPs associated with a series of developmental mutations. Miller (CA- City of Hope Medical Center). Work to define the identity and function of genes within the major histocompatibility complex (MHC) in the chicken continues. Progress has been made on three fronts. 1) CD1 genes (one active and one likely a pseudogene), encoding MHCI molecules that present lipid-type antigens to T cells, were mapped to the MHC region (Miller et al. 2005). While this is an interesting finding vis-à-vis MHC evolution the finding also identifies one more means by which the chicken MHC might affect disease incidence should the genes differ in expression among different genetic lines. 2) Y MHCI molecules were shown to be alloimmunogenic and well-expressed on blood cells providing data supporting the likelihood for a role of Y MHCI in immunity (Hunt et al. in press). 3) BG1 has been identified as a candidate gene influencing the incidence of Mareks disease in chickens (Miller et al. submitted abstract). Work continues to focus on how this occurs. Emara (DE). Control of the cell cycle has important implications for a variety of biological processes. For instance, the oncoprotein, Meq expressed by Mareks disease virus (MDV) affects the c-jun pathway and results in transformation of T lymphocytes. DE has initiated a project to characterize additional cell cycle regulators that are involved in the jun pathway, including cyclin E and the F box proteins, Fbxw7 and Skp2. Over expression of cyclin E or Skp2 (oncogenes) and loss of Fbxw7 (tumor suppressor) expression can lead to cell transformation. The chicken cyclin E1 gene maps to GGA 11; alternative transcripts for cyclin E1 have been identified; and the E1 gene encodes a conserved product of 407 amino acids. The chicken Fbxw7 gene maps to GGA 4, near a Mareks disease QTL and the product is conserved with an F box motif and seven WD domains. The chicken Skp2 gene maps to GGA Z and it encodes a protein of 443 amino acids, with an F box motif and a leucine-rich region. Two SNPs were identified in the chicken Skp2 gene. Serum starvation experiments also confirm that chicken Skp2 can function as an oncoprotein. The role of these genes in MDV-induced lymphomagenesis remains to be determined. Lamont (IA). Iowa State University maintains many unique chicken lines (highly inbred; MHC- congenic; closed populations; advanced intercross lines) for research. These lines serve as valuable resources for identifying genes of economic importance. Semen from all of the highly inbred and partially inbred lines was collected at ISU, and the staff of the National Animal Germplasm program cryopreserved the samples on site and then transported them to Ft. Collins for long-term storage. Straws were frozen from 151 males in 21 genetic lines. The need for multiple sites for storage of valuable genetic material was clearly emphasized by the passage of an F1 tornado through the ISU campus during this collection procedure. Dodgson (MI). MI has been generating avian BAC contig maps and integrating them with the respective linkage maps primarily by screening the libraries for genetic markers using overgo hybridization. These efforts generated over 7800 BAC assignments to over 900 distinct chicken markers or genes. Recently, similar efforts applied to the turkey CHORI-260 library have generated 3772 BAC assignments for 336 markers/genes. To date, only cross-species (chicken and zebrafinch) overgos have been employed, but we are beginning to use turkey marker overgos. Efforts have also recently begun to develop a BAC contig map for the turkey, along with a comparative turkey-chicken map, based on overgo mapping, BAC fingerprints and limited BAC end sequencing. We also tested the utility of overgo mapping to generate avian comparative maps outside the Phaseanids, in particular, focused on generating a map, especially of the Z chromosome, of the zebrafinch. Recently, NHGRI accepted proposals to generate a BAC contig map and a draft sequence of the zebrafinch genome, and our data will contribute to that effort. Cheng (USDA-ADOL). ADOL coordinated a large genotyping project that screened 3072 SNPs on 2580 experimental and commercial birds. As a result, we developed 2733 (89%) working assays on the Illumina platform, 2551 validated SNPs (93%), and 1980 new genetic markers. Consequently, we provided a high confirmation rate of the ~3 million in silico chicken SNPs, generated a much higher density genetic map that has enhanced the second genome sequence assembly, and demonstrated that a high density SNP can identify tightly linked markers for simple and complex traits. Furthermore, this is the first public genotyping project that surveys almost an entire agricultural industry. With our rich dataset, we are able to address whether directional and destabilizing selection occurs, examine the impacts of industrialization on the loss of diversity, etc. Reed (MN). The turkey genetic mapping continues to expand. During the past year nearly 300 microsatellite (ms) markers (provided by D. Burt, Roslin Institute) were screened on the UMN/NTBF and 65 new ms markers developed. As a result 438 markers are now genetically linked in the UMN/NTBF families and all of the linkage groups on the Roslin map have been integrated with the UMN comparative map. This represents a 39% increase in marker number and significantly increases marker density. Marker interval on the turkey genetic linkage map is estimated at 5 cM. Analysis of genotypes for a second population (NTE) will add approximately 60 ms and 140 RFLP markers to the genetic map during the coming year resulting in a genetic linkage map of approximately 600 markers. Over 1700 turkey DNA sequences have now been assigned positions in the chicken genome sequence. Ashwell (NC). A project undertaken at the NC station is focused on evaluating the expression of genes, in broilers fed diets formulated with varying dietary crude protein levels, but including all essential amino acids with the goal of reducing nitrogen composition in the waste. Breast muscle samples from standard and low crude protein dietary treatment groups were subjected to microarray analysis in order to identify differences in gene expression. This analysis indicated significant effects on the expression of genes in several classes including, energy metabolism, differentiation, growth, and apoptosis. These differences in gene expression in the low crude protein diet treatment group in relation to the standard group, may in part, explain the equivalent performance associated with lower dietary CP levels, which reduces the nitrogen load in animal waste. Zhu (TX). In mammals, natural killer (NK) cell C-type lectin receptors are encoded in a gene cluster called nature killer receptor gene complex (NKC). Two chicken C-type lectin-like receptors were identified in a region on Chromosome 1 that is syntenic to the mammalian NKC region. The region containing NK C-type lectin like receptors in GGA 1 has been previously identified to be associated with disease resistance in chickens. Identification of the NK receptors can be used to study the association of the genes with disease resistance. Toll-like receptors (TLR) are conserved components of the innate immune system that recognize specific microbial components. A novel chicken TLR containing 21 leucine-rich repeat (LRR) domains, a transmembrane domain, and a Toll/Interleukin-1 receptor domain was identified. Sequence analyses show that the identified chicken TLR has 38% homology to human TLR9 and contains a DNA binding motif, which indicates that unmethylated DNA is the ligand of this receptor. Wong VA. The VA station has been studying the transport of nutrients (amino acids, peptides and sugars) across the intestinal epithelia, which is mediated by membrane bound transporter proteins. The abundance of mRNA for these transporters was determined by real time PCR and was found to vary depending upon the intestinal segment and the time of development (embryonic day 20 to 7 days post hatch). For example, the abundance of PepT1 mRNA is induced prior to hatch and peaks at day 3; in contrast expression of selected amino acid transporters increased, decreased or stayed constant from E20 to D7. Furthermore, PepT1 is expressed in all segments (duodenum, jejunum, and ileum) of the small intestine, whereas the amino acid transporters are expressed mainly in the distal half of the small intestine. Thus, nutrient transporters are differentially expressed in not only a temporal but also a spatial manner. Objective 2. Develop Methods for Creating New Genetic Variation in Poultry by Gene Transfer and Chromosome Alteration Rhoads and Kirby (AR) have completed initial experiments with liposome delivery for sperm mediated gene transfer. Results suggest low hatch rate and transient persistence of the transgene in chicks. Plans are to investigate methods to improve hatch rate and further examine the presence of the transgene in newborn chicks. Dodgson (MI) has employed avian myeloblastosis virus (AMV) as a potential cost-effective source of membrane proteins, in particular, viral env proteins, for structural analysis. This system was used to generate large quantities of ALV viral envelope glycoproteins and are now applying it to other viral glycoproteins. Previously, it was demonstrated that retroviral vectors can be introduced into birds that induce resistance to challenge viruses. Work has begun on a project to use this system as a test of the use of RNAi expression against exogenous viruses in adult birds. We have replaced mammalian pol III promoters in our entry vectors with homologous chicken promoters (chicken U6-1 to -4, chicken H1) and have begun to test these in both DF1 and OU2 cells. Very recently, reports suggest that larger portions of a miRNA gene driven by a pol II (or pol III) promoter are more succesful in mammals. This approach will be adapted for chickens. Foster (MN) has established an immortal turkey turbinate cell line (TT-1) as a homologous cell substrate for the propagation of avian pneumovirus (AMPV). Kinetic analysis suggests that AMPV can infect and replicate more rapidly and generate larger more infectious plaques in TT-1 compared to Vero cells. The differential infectious phenotype observed in TT-1 cells might be the result of the differences in the efficiency of enzymatic activities of the viral fusion (F) protein. The highly variable AMPV G protein is a major determinant for distinguishing virus subtypes and sequence analysis revealed that the complete 1.8kb G gene was found when AMPV was propagated in TT-1 cells, but was essentially deleted when grown in Vero cells, resulting in no G gene mRNA expression. To determine if the deletion occurred during the process of attenuation, the TT/AMPV and Vero/AMPV G gene products were analyzed. In TT-1 cells, there were no changes in the AMPV G gene from passages 11 to 28. However, passage 11 Vero cells contained both the full-length (1.8kb) and the spliced G gene, while only the spliced G gene was detected in passage 28. Chicken primordial-like germ cells from 6.5 day-old East Lansing Line 0 (ev-0) embryos have been in culture for 6 months (26 passages) without the use of feeder layers. Petitte and Mozdziak (NC) have characterized the proviral insertion in the transgenic line NCSU-Blue1. The proviral DNA lies within intron 1 of a 3 exon predicted gene, which appears to be fractalkine/neurotactin. The provirus appears to be inserted in reverse orientation. Fractalkine transcripts were detected in brain RNA from wild type and hemizygous chickens, but were not detected in brain RNA from homozygous birds. This suggests that homozygous birds from this line represent an insertional knock-out for fractalkine. The efficient production of germ line chimeras using PGC transfer into early embryos has been hampered by the limited availability of PGCs obtained from blood and the early germinal ridge. Germ line chimeras were produced using sorted gonadal germ cells when injected into stage X, germinal crescent and stage 17. This observation taken together with the scientific literature indicates that germline chimeras can be made using any source of PGCs up to stage 27-28 as the donor cells and any stage of embryo development up to stage 17 can be used as a recipient. Petitte (NC) in collaboration with NIEHS characterized an enzyme in chickens that is responsible for the endogenous phytase activity previously observed in the chicken small intestine. Multiple Inositol Polyphophate Phosphatase (MIPP) or HiPer1 normally remains locked in the endoplasmic reticulum of the cell and is poorly secreted. The enzyme is active across a wide range of pH, does not require glycosylation for activity and is 100 times more active than the same enzyme in rats and humans. The chicken MIPP enzyme is a highly efficient phytase and can digest phytate (myo-inositol hexakisphosphate) to inositol monophosphate. The amino acid sequence responsible for retention of MIPP in the endoplasmic reticulum was identified, and its function verified in cell culture. Future work will entail the development a line of transgenic chicken that expresses a secreted form of the chicken MIPP gene in the intestine. Such an animal should show increased P utilization from plant phytate. Objective 3. Develop, Compare and Integrate Emerging Technologies with Classical Quantitative Genetics for Improvement of Economic Traits in Poultry. ADOL continues to work on genetic resistance to Mareks disease (MD). Reassessment of the ADOL 6 x 7 F2 MD resource population had confirmed at least 5 of the 15 previously identified QTL, revealed 2 new QTL, and identified 2-way epistatic interactions that shared a QTL on chr1. In addition, all 19 6C.7 RCS strains were fingerprinted in the SNP project described in objective 1. Efforts are underway to identify optimal MD virus challenge conditions to characterize the lines. Collaborative efforts with Janet Fulton (Hy-Line) and Sue Lamont and Jack Dekkers (IA) had led to the identification of numerous QTL in BC and F6 resource populations. Polymorphisms in avian leucosis virus (ALV) receptor genes tva and tvb that confer genetic resistance were confirmed, and Pyrosequencing assays developed. In cooperation between IA and Hy-Line, candidate gene and genome scan analyses were conducted in an advanced (F6) intercross between two partially inbred commercial Leghorn lines that differed in resistance to Marek's disease to detect QTL for survival following challenge with highly virulent Marek's disease virus. A polymorphism in the Rh-associated glycoprotein gene was found to be associated with survival and a total of 11 putative QTL were identified at a 20% proportion of false positives significance threshold. Eight of these represent QTL that had strong main effects that came to consistent expression in the different families and blood types. The feasibility of detection and mapping QTL in breeding populations with a high-density marker map using population-wide linkage disequilibrium was investigated by simulation at IA. Designs that allow adequate power and mapping precision were developed. Designs and statistical models for global gene expression analysis using micro-arrays based on pools of mRNA samples from multiple individuals were investigated at IA. For a given total number of individuals and arrays, simulated pooling resulted in loss of power compared to arraying all samples individually but under some conditions, loss of power was small and it was possible to find a low-cost pooling design with power close to that of an individual design. IA, in cooperation with Aviagen, studied 12 immune-related genes for associations with general mortality and other performance traits in three elite commercial broiler chicken lines raised in high and low hygiene environments. From a total of 56 identified single-nucleotide polymorphisms (SNPs) in 12 genes, 14 SNPs that had moderate allelic frequencies in at least two of the lines were typed in 100 progeny-tested sires from each of the three elite lines using RFLP techniques. These data were then used in association analysis. The traits measured on the progeny (total progeny number = 145,467) were broiler-type traits related to growth, yield, support or mortality. Association analyses were conducted with sire allele and/or haplotype substitution effect models using progeny mean data. Ten of the 12 immune-related genes had SNP associations with at least one trait. Most detected effects were with mortality and growth traits. The myostatin gene was also studied. Five SNPs and 8 haplotypes were identified. Myostatin SNPs had associations with growth, mortality, eviscerated carcass weight, blood oxygen, and vaccine response. Most gene SNP-trait associations varied by genetic line or with environment. These results indicate that variation in candidate genes with important broiler traits can be identified in multiple environments, and may facilitate marker-assisted genetic selection to improve traits in commercial environments. NC has continued to develop new microsatellite markers for use in fine-mapping QTL regions. This data indicates that there is at least one usable microsatellite marker every 40,000 bp and at least one polymorphic microsatellite marker every 115,000 bp in commercial populations. Using the chicken genome sequence to increase marker density can significantly decrease the time to develop new informative microsatellites and allows for ease in targeting specific chromosomal regions. NC has further characterized the MHC B locus at the sequence level in numerous layer lines from around the world to provide a reproducible means to determine B haplotypes. The microsatellite marker LEI0258 is known to be physically located within the MHC. DNA from 51 distinct serologically defined MHC types was sequenced and organized into distinct sequence based haplotypes. This information will be a useful tool to identify new MHC haplotypes in outbred populations of chickens particularly for the initial development of serological reagents. NC is also developing a new resource population focusing on the detection of quantitative trait loci (QTL) involved in the regulation of disease resistance and immune response. This resource population consists of reciprocal crosses, F1,and F2 of the two lines of White Leghorns that have undergone long-term selection for high or low antibody response to sheep red blood cell antigen. Screening of the parents of the F1 generation for 260 microsatellite markers and SNP typing (Illumina) have identified a significant number of polymorphic markers for future use. DE has genotyped broiler chickens for the microsatellites, LEI0258 and MCW0371 to determine their use as markers for the major histocompatibility complex (MHC). At least 9 LEI0258 alleles, ranging in size from 195 to 397 bases were identified in the two F2 resource populations that were phenotyped for either coccidiosis or Mareks disease resistance. Genotyping of the MCW0371 microsatellite and determination of MHC allelic associations with disease resistance in broilers are ongoing. DE further mapped the chromosome 1 QTL that affected oocyst shedding during coccidiosis infection of broiler chickens to the microsatellite marker, MCW0318. An interesting candidate gene near MCW0318 is lymphocyte activation gene 3 (LAG-3; CD223). The LAG-3 gene was simultaneously identified in DNA microarray studies to be reduced in expression after Eimeria infection of chickens. The LAG-3 molecule has an important role in maintaining T cell homeostasis during antigen-driven immune responses. The chicken LAG-3 transcript is 1515 bp with 8 exons (similar to human LAG-3) and it encodes a protein with 504 amino acids. Very few ESTs have been identified for LAG-3 and they are mainly expressed in lymphoid tissues. We have confirmed the lympocyte-specific expression of LAG-3 by RT-PCR and demonstrated a rapid down-regulation of LAG-3 with coccidial infection. MD has performed additional transcriptional profiling screens using their cDNA microarrays. Genes were identified that respond directly to glucocorticoids in cultures of anterior pituitary cells. Six of these were confirmed by quantitative RT-PCR. These genes are candidates for mediating the effects of glucocorticoids on expression of genes. Gene expression profiles in the anterior pituitary and hypothalamus were compared between lines of chickens genetically selected to have high or low abdominal fat. Genes were identified (and confirmed by quantitative RT-PCR) that were differentially expressed in the neuroendocrine system before differences in body fat. MD is currently sequencing the 5'-flanking region of these candidate genes in a Fat X Lean reference population in an attempt to identify genetic markers associated with differences in body fat.

Publications

Publications Attachment:

Attachment

Impact Statements

see above

Date of Annual Report: 03/12/2007

Report Information

Annual Meeting Dates: 01/13/2007 - 01/14/2007
Period the Report Covers: 10/01/2005 - 09/01/2006

Participants

AR University of Arkansas W. Kuenzel; D. Rhoads
CA City of Hope M. Miller
CA University of California, Davis M. E. Delany
GA University of Georgia S. Aggrey
IA Iowa State University S. J. Lamont, J. Dekkers
IN Purdue University W. Muir
MD University of Maryland T.E. Porter, J. Song
MI Michigan State University J.B. Dodgson
MN University of Minnesota D.N. Foster, K. Reed
NC North Carolina State University C. Ashwell; J. Petitte
MS Mississippi State university S. Burgess; F. McCarthy
TX Texas A & M University H. Zhou
USDA USDA-ARS; ADOL H. Cheng; H. Zhang
VA Virginia Polytechnic Institute & State University E. Wong

Institutions absent
DE University of Delaware M. Emara
WI University of Wisconsin J. Bitgood
IL University of Illinois M. Grossman

Brief Summary of Minutes

Members present: C. Ashwell, S. Burgess, H. Cheng, M. Delany, J. Dodgson, D. Foster, W. Kuenzel, S. Lamont, M. Miller, W. Muir, J. Petitte, T. Porter, M. Qureshi, K. Reed, D. Rhoads, M. Saif, J. Song, E. Wong, E. Young, H. Zhang, H. Zhou

Visitors: J. Fulton, A. Paszek, Carl Schmidt

The annual meeting was held in conjunction with the Plant and Animal Genome Conference and consisted of the Poultry Genome Conference followed by the Business Meeting.

Meeting Schedule
Poultry Genome Workshop
Supported by a Grant from the USDA-CSREES (to T.E. Porter)
SATURDAY
9:00 AM Welcome
9:10 AM Station report, USDA Avian Disease and Oncology Laboratory; Hans Cheng and Huanmin Zhang --"Genetic characterization of Marek's disease resistance"
9:40 AM Station report, University of Arkansas; Douglas Rhoads -- "Genetic analysis and mapping of sperm degeneration in broiler breeders" and Wayne Kuenzel -- "Potential genes involved in activation of gonadal development in male chicks"
10:30 AM Station report, University of California-Davis; Mary Delany -- "Cytogenetic analysis of telomeres in normal and transformed chicken cells"
11:00 AM Station report, City of Hope, National Medical Center; Marcia Miller -- "Potential Disease Resistance Genes in the Chicken MHC-B Extended Class II Region"
11:30 AM Station report, Mississippi State University; Shane Burgess -- "AgBase: Facilitating Analysis of Chicken Functional Genomics Datasets"
11:50 AM Station report, University of Georgia; Sammy Aggrey -- "Molecular basis for feed efficiency"
1:30 PM Station report, Purdue University; William Muir -- "Innate Genetic Differences In Birds Differing In Aggressive Behavior As Determined By Affymetrix Genechip Chicken Genome Array"
2:00 PM Station report, Texas A&M University; Huaijun Zhou -- "Development of new chicken genome array and its application"
2:30 PM Station report, Virginia Tech; Ed Smith -- "Dissection of genetic factors for immune response and round heart heart disease in birds" and Eric Wong -- "Differential expression of intestinal nutrient transporters in posthatch broilers"
3:30 PM Station report, North Carolina State University; Jim Petitte -- "Avian Primordial Germ Cells" and Chris Ashwell -- "Genomic Approaches To Identifying The Underlying Cause Of Traits Of Economic Importance In Poultry"
4:00 PM Station report, Michicgan State University; Jerry Dodgson --"Progress towards a Turkey Genome Physical Map and Antiviral RNAi in Chicken Cells"
4:30 PM Station report, Iowa State University; Susan Lamont and Jack Dekkers -- Application of high-density SNPs in chickens: linkage disequilibrium and trait-association results

SUNDAY
8:30 AM Station report, University of Minnesota; Kent Reed -- "Refinement and application of genetic maps in the turkey" and Douglas Foster -- "Gene expression in a fully immortalized CEF cell line"
9:00 AM Station report, University of Maryland; Jiuzhou Song and Tom Porter -- "Characterization of genetic polymorphisms and gene expression related to body fat"
9:30 AM Roger Coulombe, Utah State University -- "Characterizing Genes Associated with Aflatoxin B1 Hypersensitivity in Turkeys"
10:30 AM Michael Romanov, Zoological Society of San Diego -- "Genomic resources and tools to investigate factors associated with chondrodystrophy in California condors"
11:15 AM Elisabeth Lebihan-Duval, Institut National de la Recherche Agronomique, France -- "Recent Results On The Genetic Variation Of Chicken Technological Meat Quality"
1:15 PM Fred Leung, University of Hong Kong -- "A Chicken Full-Length cDNA Database And Its Applications"
2:00 PM Helen Sang, Roslin Institute, Scotland -- "Applications Of Lentiviral Vector Transgenesis In The Chicken"

The Business Meeting was called to order at 2:55 pm by Tom Porter, Chair of NC1008.

T. Porter thanked the CSREES for funding to support the travel expenses of the invited speakers at the Workshop (Coulombe, Romanov, Lebihan-Duval, Leung, and Sang).

J. Dodgson presented the NRSP8 Coordinators Report. The Second Build for the chicken genome was released May 2006. The coordinates form the First Build and the Second Build are different and some of the annotations have not been transferred to the Second Build. There are still some resources available to committee members such as BAC library filters and DNA microarryas (FHCC 13k, Agilent and Nimblegen arrays). The NRSP8 rewrite is due in 2007.

Minutes from the 2005 Annual Report were approved.

M. Saif, the administrative advisor reported that the NC1008 project terminates in Sept 2008 and that the rewrite for the NC1008 project is due by the end of 2007. The committee that reviews these projects will meet in January 2008. He congratulated the committee for an excellent workshop and for sending the reports electronically prior to the meeting. He reminded committee members to make sure that progress reports contain impact statements. Each subject should be accompanied by a 2-3 sentence impact statement, which can be easily extracted when needed. M. Saif also indicated that NC1008 was the only committee that held its Business Meeting at the end of the workshop. There is an advantage to conducting the Business Meeting prior to the workshop, especially during years when the rewrite is due, to allow sufficient time to discuss important issues. He is very supportive of the productivity and excellence of this committee and does not feel that there will be a problem with renewal.

M. Qureshi introduced Eric Young, the new NRSP8 administrator for Poultry. He thanked T. Porter and K. Reed for organizing the workshop and conducting one of the best workshops, due to the number of invited speakers. He indicated that it was a challenge to provide the funds for the outside speakers during this year of federal budget uncertainty. He distributed a copy of his report, which provided personnel updates at CSREES and provided statistics for the USDA NRI competitive grants programs. There will be one vacant position open as National Program leader for Animal Science. All grants for 2007 must be submitted electronically via Grants.gov in pdf format. Committee members should review the instructions carefully and work with their institutional grants personnel early to ensure that they are prepared to process grants submitted electronically. M. Qureshi reported that 27% of the proposals in the Animal Genomics Program were funded with an average award size of approximately $500,000. The USDA is now tracking the number of graduate students (22) and postdocs (9) trained with CSREES grants. He encouraged members to keep including graduate students and postdocs in their grants. If Congress does not pass a budget by Feb 2007, the government will operate under continuing resolution for the rest of the year, which will likely result in some financial problems.

The issue was raised from last years discussion about including Industry members. This was considered to be important for the committee and Janet Fulton and Albert Paszek were nominated and approved as members. Both agreed to be members.

Site of the next meeting was not decided, but an email poll will be conducted to see if it should be held prior to PAG XV in San Diego Jan. 2008.

T. Porter volunteered to write the 2006 annual report.

The following volunteered to lead the rewrite of specific objectives of the Project.

Objective 1: Chris Ashwell
Objective 2: Jim Petitte
Objective 3: Sue Lamont

Chris Ashwell volunteered to assemble the rewritten project.

The meeting was adjourned at 3:30 pm.

Although significant concerns were raised in the ensuing email poll and the site for the next meeting, sufficient support did not exist for changing the venue at this time. Next year's meeting will be held again in conjunction with the Plant and Animal Genome conference. The meeting will be organized by Kent Reed and Tom Porter.

Accomplishments

Objective 1. Develop High Resolution Integrated Maps to Facilitate the Identification of Poultry Genes and Other DNA Sequences of Economic Importance. ADOL continues to curate the East Lansing genetic map. In the past year, 710 SNPs were added, which brings the total number of genetic markers to 3209. The East Lansing and Wageningen maps were combined into a consensus map that contained 3850 genetic markers, which was used in the second genome sequence assembly. Of the 3072 SNPs screened in our Illumina panel generated in 2005, 233 changed chromosomal positions from the first to the second genome assemblies. At AR, the promoter regions from the chicken DAZL gene, a PGC specific promoter, has been cloned. The promoter is being fused to a reporter gene to allow us to determine time of induction and extent of expression (collaborator: NCSU). To protect scientifically valuable research genetic lines of highly inbred chickens, semen samples were collected again this year from all adult males of the 21 highly and partially inbred lines held at Iowa State University (IA). Semen samples were placed into the long-term cryopreservation bank in the National Animal Germplasm program in Ft. Collins, CO. At CA (City of Hope) work continues on construction of a gene map for the chicken MHC B and Y regions. The B map for Red Jungle Fowl now spans 242 kb and encompasses 46 genes including TRIM, C-type lectin and Ig superfamily type genes. At CA (UC Davis), research continued to explore variation for telomere length within genomes (among chromosomes) and among different genotypes. Differences among genotypes are evident as telomere profiles differ (slightly) between inbred lines, although locations for some mega-telomere loci are held in common. Work was also initiated to examine integration of MDV into the chicken genome using MDV-BACs (collaborator: H. Cheng). We are studying the hypothesis that there is targeted integration into the mega-telomeres. This work will assist in determining clonality of tumors within individuals and will establish commonalities (and/or preferences) of integration sites among individuals. A project emphasized in the lab during the prior two years developed in collaboration with the USDA-ADOL. Our objective was to study single nucleotide polymorphisms (SNPs) in seven UCD developmental mutant stocks so to map and identify potential markers for carrier-testing and begin to resolve potential candidate loci. Five of seven mutations (cm, dp-1, dp-4, po, wg-2) were mapped to a chromosome and in one case (wg-2) a defined region of several Mb. One candidate gene (Sef; similar expression to fibroblast growth factor) in the region of wg-2-associated SNPs was studied at a single developmental time point post limb outgrowth (Sef expression has previously been found to be in the right place at the right time in limb outgrowth, suggesting this is a reasonable candidate despite a location). However, the relative transcript expression of forelimbs and hindlimbs among the genotypes (+/+, +/wg, wg/wg) was similar although the ratios relative to brain (highest expressing tissue) within each genotype showed a slight genotype-effect. Efforts are underway at MI to generate a turkey BAC-contig map that is aligned with the chicken genome sequence. To date, we've completed over 40,000 turkey BAC fingerprints. Generation of ~20,000 turkey BAC end sequences nearly done. Additional shotgun sequencing of the turkey genome is planned. We've made nearly 8000 turkey BAC-marker assignments. This includes most of the existing STS genetic linkage map markers available in turkey, as well as numerous chicken markers. Over 700 genetic markers (primarily microsatellites) have been genotyped by researchers at MN on the UMN/NTBF and Nte mapping families. Combined analysis of both mapping families found 684 markers to be significantly linked to at least one other marker in the UMN database; 41 linkage groups have been identified. Through the use of common marker sets, in silico mapping and RH mapping, 35 of the 41 linkage groups are aligned with the chicken genome. Over 1700 turkey DNA sequences have been compared and assigned to positions with the chicken genome sequence. Two MHC-B BAC clones were identified and sequenced. Gene annotation indicates three class IIb genes in the sequenced turkey haplotype, one more than in the sequenced chicken haplotype. DNA sequence polymorphisms (SNPs) identified in the turkey MHC were used to develop genotyping assays for genetic mapping in the UMN/NTBF mapping families. Segregation analysis found two turkey MHC-B SNPs (BTN2 and C4) were genetically linked. Genetic linkage was not observed between the MHC-B and MHC-Y SNPs. Gene expression profiles of turkey skeletal muscle between and within growth-selected and control turkey lines at three developmental stages were compared using three turkey skeletal muscle cDNA libraries. To date, each non-subtracted library has been extensively sequenced (15,648 reads) with 6624, 5088 and 3936 reads from the 18-day embryo, 1-day post-hatch poult, and 16-wk post-hatch turkey, respectively. NC has been using microarray analysis to identify differential chicken gene expression in response to dietary nutrient restriction. Adaptation to P and Ca restricted diets has been previously reported in chickens. Animals respond to nutrient restriction by increasing absorption rates and utilization efficiency, which decreases excretion of the restricted nutrients. NC investigated if birds had the capacity to adapt similarly and used microarray analysis to identify genes whose expression was significantly altered in response to dietary P restriction. Previous experiments with regard to market weight have suggested that broiler chickens fed a restricted phosphorus diet from hatch to 90 hours of age were better suited to a restricted diet fed from 22 to 38 days of age than those fed a control diet in the first 90 hours post hatch. Microarray analysis was used to identify additional genes whose expression was significantly altered in response to this dietary P restriction. Interestingly the list of differentially expressed genes includes the intestinal Na/Pi cotransporter type IIb previously shown to be induced by dietary P restriction (Yan et al, 2007). Within this list there are genes that are involved in cell signaling, transport, and proliferation. The studies conducted at NC are some of the first to focus on nutritional and thus environmental impact of poultry production from a genetic point of view. This information obtained in the studies of nutrition related genes can then be used to implement marker assisted selection practices to identify and select for superior individuals in breeding programs that can reduce the environmental impact of nutrients in the excreta. The outcome of this research will provide a means to improve the innate ability of poultry to utilize environmentally important nutrients such as N and P, therefore reducing their excretion, and therefore greatly aid in reducing the cost of poultry litter disposal and in maintaining the productivity of poultry industry. At TX, a new chicken 44K Agilent whole genome array was developed. To validate this new developed chicken 44K array, 4 major tissue samples (liver (L), spleen (S), cecal tonsil (CT), and ileum (IL)) from the six two weeks old commercial broiler chickens were collected. There were four biological replicates for every two tissues comparison. The results demonstrated that this new developed chicken oligonucleotide array is very informative and tissue-specific. Objective 2. Develop Methods for Creating New Genetic Variation in Poultry by Gene Transfer and Chromosome Alteration CA completed and published research in collaboration with Origen Therapeutics (Burlingame, CA) on long term culture procedures of stem cells (embryonic stem cells, primordial germs cells) and their utilization/utility for making transgenics (Origen) and analysis of features related to genome stability and differentiation status of these cell systems (UCD). Differentiation status was examined by profiling telomerase activity, a hallmark feature of cells with high proliferative potential (toti-, multi-potent cells as well as renewable stem cells). Both the long term embryonic stem cells (cultured from Stage X embryos) pre- and post manipulation and primordial germ cells (cultured for hundreds of days) were found to be telomerase positive. Long term cultures were typically found to be normal in terms of karyotype to the level of analysis conducted, although occasionally cell lines were found with macrochromosome aberrations (in one case involving a deletion of a large portion of GGA 2) and of course these would not be suitable for use. Thus, the analysis of cells to be used for transgenic purposes as to their chromosomal status is an important parameter which needs consideration. MI generated a Gateway-compatible entry plasmid containing the micro RNA (miRNA) sequence of chicken miR-30a that allows RNAi cassettes to be inserted into an ALV subgroup(A) retroviral destination vector. RNAi targeted either against the viral envB gene or host receptor tvb gene has been shown to be an effective antiviral strategy against subgroup(B) ALV. Similar reductions in plaque number and size of Marek's disease virus (serotype III) have been achieved by constructs that target the essential gB glycoprotein gene. Work conducted at MN indicated that the phenotype of the spontaneously immortalized chicken cell line SC-2, changed dramatically at about passage 80, appearing smaller and more compact than at earlier passages. Passage 43 SC-2 cells expressed undetectable levels of p53 mRNA, but the elevated levels detected by passage 95 did not correlated to functional protein activity. The altered expression of genes involved in the p53 and Rb pathways, specifically, p53 and p21WAF1, may have contributed to the immortalization of the SC-2 CEF cell line. The regulation of chicken p15INK4b was shown to increase substantially at senescence and was transcriptionally silenced in two immortalized chicken cell lines. Short-hairpin RNA (shRNA)-mediated knockdown of chicken INK4b provided only modest lifespan extension, suggesting that other factors contribute to senescence in CEFs. The transgenic chicken line developed in NC, now designated NCSU-Blue1, is a useful tool for several areas of research, and they have been used for studies of early embryonic development (Stem Cells Dev. (2006) 15:17-28). Functional beta-galactosidase was expressed in all tissues of the digestive tract, particularly in the small intestine. This should give the birds the ability to hydrolyze lactose, which normally cannot be utilized as a source of energy in birds. NC examined this possibility through a feeding trial in which isocaloric diets containing 0, 5, 7.5 and 10% dietary lactose were fed to wild type and transgenic birds from 10-24 days of age. Transgenic birds were observed to have a greater ability to digest lactose through the hydrolysis of lactose to galactose and glucose than those of nontransgenic wild-type chicks at least by 10%. The greater lactose digestibility of the transgenic birds does not result in better growth performance. These studies point to the fact that the nutritional requirements of transgenic poultry can be significantly altered and the nutritional requirements may need to be re-evaluated in birds with high expressing transgenes. The culture of PGCs from male and female embryos at the NC station will have significant applications in reproductive biology, developmental biology and transgenics. Work with the NCSU-Blue1 line of transgenic chickens suggests that the nutritional requirements of transgenic poultry may need to be evaluated, particularly in high expressing lines Objective 3. Develop, Compare and Integrate Emerging Technologies with Classical Quantitative Genetics for Improvement of Economic Traits in Poultry. ADOL continues to work on genetic resistance to Mareks disease. Evaluation of the line 6 x 7 F2 resource population for two-epistatic interactions identified a large number (239) of highly significant interactions involving loci located throughout the genome that account for MDV viremia titers in infected birds. Recombinant congenic strains (RCS) developed from lines 6 (background) and 7 (donor) were evaluated and shown that the 19 RCS generated are both genetically and phenotypically variable from one another, thus, serve as unique genetic resources for identifying and characterizing QTLs or candidate genes conferring genetic resistance to Mareks disease as well as other traits of interest. Based on prior two-hybrid results, the MDV protein R-LORF10 may be the responsible protein in the novel up-regulation of MHC class II cell surface expression based upon defined MDV recombinants via MDV-BAC clones. In addition, a worldwide and genome-wide assessment was made for commercial poultry by genotyping 2551 informative SNPs spaced throughout the chicken genome on 2580 unique individuals including 1440 commercial birds. Results from several analytical methods combined with theory indicate that individual commercial breeding stocks have lost 70% or more genetic diversity of which no more than 10% can be recovered by combining all breeds from commercial poultry. These results emphasize a need for concerted national and international efforts to preserve chicken biodiversity. AR initiated a SNPlotype mapping project for Sperm Degeneration and Sperm Mobility, with partial support from Director funds. A detailed full-length cDNA sequencing project to characterize over 450 novel transcripts expressed in the chicken reproductive tract was begun. In work conducted at IA, Fatness QTL were mapped in two F2 resource populations that were established by crossing one broiler sire with dams from two unrelated highly inbred lines (Fayoumi and Leghorn). Thirty-three markers in 8 regions on chromosomes 1 to 4 showed significant association (1% FDR) with AF. We evaluated the efficiency of the pQTL transcriptome mapping approach, which combines the mRNA pooling with eQTL transcriptome mapping. Through simulation studies, we found that pQTL transcriptome mapping using the standard regression method achieved statistical power comparable to the power that can be achieved by eQTL transcriptome mapping. We evaluated the influence of heritability used in analysis of SNP data on the significance and magnitude of SNP effects and their standard errors (SE) and developed an approximation that would allow results for alternate levels of heritability to be obtained without reanalysis, using actual data from a broiler breeder population. Use of SNP-trait associations detected in one population for use in other populations requires LD between loci to be consistent across populations. We used genotype data for over 100 SNPs on two chromosomes from 10 broiler breeder lines to evaluate similarities in LD across the lines and compared this to phylogenetic relationships among lines estimated using differences in SNP allele frequencies. The correlation between LD measured by r2 between lines for SNP at short distances is a good predictor of line relationships, although somewhat less so than the typical allele frequency-based distance. At MD, analysis of global gene expression in the neuroendocrine system of chickens genetically selected for high and low body weight or body fat was accomplished using custom cDNA microarrays produced in collaboration with DE. Hundreds of genes were identified that are expressed at different levels in the pituitary gland or hypothalamus in either fat versus lean chickens or high growth versus low growth chickens. These are excellent candidate genes for controlling body fat and body growth in chickens. Single nucleotide polymorphisms (SNPs) have been identified in many of these genes and a study has been initiated at MD to assess these SNPs for their utility as genetic markers in marker assisted selection programs. mRNA splicing variants have been identified in BDNF, which is known to control body fat accumulation. In collaboration with ADOL, it was determined that these BDNF splice variants are associated with chicken lines which accumulate different amounts of body fat. In other work at MD, novel computer algorithms are being developed for analysis of microarray results from time-series experiments. This work has the potential to identify numerous genetic markers for use in marker assisted selection programs aimed at improving growth performance and reducing body fat in broiler chickens. MS has developed and demonstrated computational tools, as well as proteomics techniques, to improve the structural annotation of the chicken genome. This is especially important for identifying those genes unique to birds for which obvious mammalian homologs do not exist. Resistance to MD is the result of complex interactions between chicken and MDV genes and current research is aimed at defining one molecular genetic mechanism that may be a critical determinant of this host-pathogen relationship. Results demonstrate the applicability of high throughput proteomics followed by computational modeling to understand biological function in the chicken. Databases are being maintained and computational tools developed that facilitate chicken researchers ability to derive biological meaning from their functional genomics datasets. This is broadly applicable throughout the chicken research community regardless of the field of study. NC has focused on the identification of QTL for immune response and disease resistance in lines differentially selected for antibody production. NC has developed a new resource population which consists of reciprocal crosses of lines divergently selected for antibody response to sheep red blood cells. Selection for immune response parameters may lead to improved general disease resistance in part due to they are difficult to measure and have low to moderate heritability. To determine if SNP allele frequencies were altered as a result of selection for SRBC response in the HAS and LAS lines a representative sample of unrelated individuals were genotyped using the Illumina GoldenGate Assay system. Illumina evaluated 3072 SNP loci dispersed across the genome. No loci were fixed within either line. 30 genome regions consisting of one or more SNPs showed significant differences in allele frequency between the HAS and LAS lines. These loci are located on 19 chromosomes with specific regions of Gga1, Gga2, Gga5, Gga6, Gga18, and Gga28 that correspond to regions previously associated with disease resistance or antibody response QTL. The nature of selection appears to favor the contribution of a large number of loci with relatively small effects as opposed to single loci with large effects. Further characterization of these selected lines and their intercross population will provide additional information on the complexity of antibody response in the chicken as well as the genetic basis of selection in general. A second round of SNP genotyping by Illumina also supported in part by the NRSP-8 species coordinator with cooperation from 5 institutions is currently underway and includes samples to selectively genotype the F2 generation of this population. In work at TX, the chicken 44K Agilent array was used to analyze RNA of heterophils from SE-resistant (line A) and SEsusceptible chickens (line B) with SE (I) or without SE infection (N). The results indicated that: for the comparisons of SE infection with non-infection, 3096 genes in line A and 3312 genes in line B were differentially expressed (P<0.05). In the comparison of linage (line A and line B) difference, 4377 genes in the non-infected and 4333 genes in the infected groups have shown differential expression (P<0.05). The results discovered in the present study have laid a solid foundation to elucidate cellular and molecular mechanisms of SE infection in chickens. RNA interference was used to specifically inhibit expression of NF- B and to elucidate the role of NF- B in the signal transduction pathway of Salmonella infection in chicken HD11 cell line. After a 24-hour transfection, the treated cells were followed by Salmonella enteritidis infection (MOI=100) or non-infected for 1 and 4 hours. Four candidate genes (interleukin (IL)-1², IL-6, Toll like receptor (TLR)-4 and TLR-15) were selected to examine the effect of NF-kB inhibition on gene expressions with SE infection by the real-time quantitative PCR. The results showed that, with 1.6-fold of inhibition of NF- B gene expression, the gene expression of IL-6 was consistently and significantly increased at both 1 hour and 4 hours with Salmonella challenge; whereas the gene expression of IL-1² and TLR-15 were increased at 4 hours only. The results of the current study have laid foundation for uncovering the gene networks of innate immune system in chickens. VA has examined the spatial and temporal expression of nutrient (amino acid, peptide, and monosaccharide) transporters in the small intestine of late embryonic and early posthatch chicks. Expression of these transporters showed different developmental profiles and different levels in the intestinal segments. The peptide transporter PepT1 is expressed highest in the duodenum, the monosaccharide transporters are expressed highest in the jejunum and the amino acid transporters are expressed predominantly in the ileum. Because early nutrition plays an important role in overall growth performance, optimizing diets to match the absorptive capacity of the posthatch chick may result in increased growth performance.

Publications

ARKANSAS, UNIVERSITY OF ARKANSAS, Fayetteville, AR D.P. Froman, J.D. Kirby, and D.D. Rhoads. (2006) An Expressed Sequence Tag Analysis of the Chicken Reproductive Tract Transcriptome. Poultry Science 85:1438-1441. Klein S., Jurkevich A., Grossmann R. (2006). Sexually dimorphic immunoreactivity of galanin and colocalization with arginine vasotocin in the chicken brain (Gallus gallus domesticus). J Comp Neurol 499:828-839. Kuenzel, W.J. and C.D. Golden. 2006. Distribution and change in number of Gonadotropinreleasing hormone-1 neurons following activation of the photoneuroendocrine the chick, Gallus gallus. Cell Tissue Res. 325(3):501-512. Kuenzel, W.J., A.M. Rowland, P.B. Pillai, T.I. OConnor-Dennie, J.L. Emmert and Wideman. 2006. The use of vitamin A-deficient diets and jugular vein ligation intracranial pressure in chickens, Gallus gallus. Poultry Sci. 85:537-545. CALIFORNIA, CITY OF HOPE, Duarte, CA Miller, MM. 2006. Why do we need to conserve what we have? A post-genome sequencing perspective on existing chicken strains. Poult Sci. 85:243-245 Hunt, H.D., Goto, R.M., Foster, D.N., Bacon, L.D., and Miller, M.M. 2006. At least one YMHCI molecule in the chicken is alloimmunogenic and dynamically expressed on spleen cells during development. Immunogenetics, 58:297-307. CALIFORNIA, UNIVERSITY OF CALIFORNIA, DAVIS, Davis, CA Chang, H., and M.E. Delany. 2006. Complicated RNA splicing of chicken telomerase reverse transcriptase revealed by profiling cells both positive and negative for telomerase activity. Gene 379:33-39. van de Lavoir M.-C., Diamond, J.H., Leighton, P.A., Mather-Love, C., Heyer, B.S., Bradshaw, R., Kerchner, A., Hooi, L.T., Gessaro, T., Swanberg, S.E., Delany, M.E., and Etches, R.J. 2006. Germline transmission of genetically modified primordial germ cells. Nature 441: 766-769. van de Lavoir, M.-C., Mather-Love, C., Leighton, P., Diamond, J.H., Heyer, B.S., Roberts, R., Zhu, L., Winters-Digiacinto, P., Kerchner, A., Gessaro, T., Swanberg, S., Delany, M.E., and R.J. Etches. 2006. High-grade transgenic somatic chimeras from chicken embryonic stem cells. Mechanisms of Development 123:31-41. Delany, M.E. 2006.Avian genetic stocks: the high and low points from an academia researcher. Poultry Science 85:223-226. OHare, T.H., and M.E. Delany. 2005. Telomerase gene expression in the chicken: telomerase RNA (TR) and reverse transcriptase (TERT) transcript profiles are tissue-specific and correlated with telomerase activity. AGE 27:257-266. Swanberg, S.E. and M.E. Delany. 2006. Telomeres in aging: Birds. In Handbook of Models for Human Aging (editor: M. Conn), Chapter 29, p. 339-349. Academic Press (Elsevier, Inc.). Burlington MA (USA). GEORGIA, UNIVERSITY OF GEORGIA, Athens, GA Lagarrigue S., Pitel F., Carré, W, Abasht B., Le Roy P., Neau A., Amigues Y. Sourdioux M., Simon, J., Cogburn L., Aggrey S., Leclercq B., Vignal A., and Douaire M., 2006. Mapping quantitative trait loci affecting fatness and breast muscle weight in experimental meat type chicken lines divergently selected on fatness. Genetics Selection Evolution. 38:8597. Nahashon, S.N., S.E. Aggrey, N.A. Adefope, A. Amenyenu, and D. Wright, 2006. Growth characteristics of pearl gray Guinea Fowl as predicted by the Richards, Gompertz, and Logistic models. Poultry Science 85:359363. Abasht B. Pitel F., Lagarrigue, S.,BihanDuval, E., Pascal, P., Olivier, D., Simon J., Cogburn L., Aggrey S., Vignal A., and Douaire M., 2006. Fatness QTL on chicken chromosome 5 and interaction with sex. Genetics Selection Evolution 38: 297311. Nahashon, S.N., S.E. Aggrey, N.A. Adefope, A. Amenyenu, and D. Wright, 2006. Modeling growth characteristics of meat type Guinea Fowl. Poultry Science 85: 943946. Carre, W., X. Wang, T. E. Porter, Y Nys, J. Tang, E. Bernberg, R. Morgan, J. Burnside, S. E. Aggrey, J. Simon and L. A. Cogburn, 2006. Chicken genomic resources: sequencing and annotation of 37,557 ESTs from single and multiple Tissue cDNA libraries and CAP3 assembly of a chicken gene index. Physiological Genomics 25:514524. INDIANA, PURDUE UNIVERSITY, West Lafayette, IN Devlin, RH Sundström, LF and WM Muir. 2006. Interface of biotechnology and ecology for environmental risk assessments of transgenic fish. Trends in Biotechnology 24:89-97. Sun,W., V. M. Margam, L. Sun, G. Buczkowski, G. W. Bennett, B. Schemerhorn, W. M. Muir and B. R. Pittendrigh 2006. Genome-wide analysis of henobarbitalinducible genes in Drosophila melanogaster Insect Molecular Biology. Insect Molecular Biology 15: 455464 W.M. Muir, J. Romero-Severson, S.D. Rider Jr., A. Simons, and J. Ogas. 2006. Application of One Sided t-tests and a Generalized Experiment Wise Error Rate to High-Density Oligonucleotide Microarray Experiments: An Example Using Arabidopsis. J. Data Science 4, 323-341. Muir, W.M. and P. Bijma. 2006. Incorporation of competitive effects in breeding programs for improved performance and animal well-being. WCGALP 17:806-812 Bijma, P. and W. M. Muir 2006. Genetic analysis and improvement of traits affected by interaction among individuals WCGALP 17:974-980 IOWA, IOWA STATE UNIVERSITY, Ames, IA Abasht, B. Dekkers, J.C.M, and Lamont, S.J. 2006. Review of quantitative trait loci Identified in the chicken. Poultry Sci. 85:2079-2096. Cheeseman, J.H., Kaiser, M.G., Ciraci, C., Kaiser, P. and Lamont, S.J. 2006. Breed effect on early cytokine mRNA expression in spleen and cecum of chickens with and without Salmonella enteritidis infection. Devel. Comp. Immunol. 31: 52-60. Grapes, L., M. Z. Firat, J.C.M. Dekkers, M.F. Rothschild, and R.L. Fernando. 2006. Optimal haplotype structure for linkage disequilibrium-based fine mapping of quantitative trait loci using identity-by-descent. Genetics 172: 1955-1965. Hangalapura, B. N., Kaiser, M. G., van der Poel, J.J., Parmentier, H. K., and Lamont, S. J. 2006. Cold stress equally enhances in vivo pro-inflammatory cytokine gene expression in chicken lines divergently selected for antibody responses. Develop. Comp. Immunol. 30:503-511. Hasenstein, J.R., Zhang, G., and Lamont, S.J. 2006. Analyses of five gallinacin genes and the Salmonella enterica serovar enteritidis response in poultry. Infect. Immun. 74:3375-3380. Kaiser, M.G., J.H. Cheeseman, Kaiser, P., and Lamont, S.J. 2006. Cytokine expression in chicken peripheral blood mononuclear cells after in vitro exposure to Salmonella enterica serovar Enteritidis. Poultry Sci 85:1907-1911. Lamont, S.J. 2006. Perspectives in chicken genetics and genomics. Poultry Sci. 85:2048-2049. McElroy, J.P., Kim, J.J., Harry, D.E., Brown, S.R., Dekkers, J.C., and Lamont, S.J. 2006. Identification of trait loci affecting white meat percentage and other growth and carcass traits in commercial broiler chickens. Poultry Sci. 85:593-605. McElroy, J.P., Zhang, W., Koehler, K.J., Lamont, S.J., and Dekkers, J.C.M. 2006. Comparison of methods for analysis of selective genotyping survival data. Genet. Selec. Evol. 38:637-655. Soller, M., Weigend, S., Romanov, M.N., Dekkers, J.C.M., and Lamont, S.J. 2006. Strategies to assess structural variation in the chicken genome and its associations with biodiversity and biological performance. Poultry Sci. 85:2061-2078. Tuggle, C.K., J.C.M. Dekkers, and J.M. Reecy. 2006. Integration of structural and functional genomics. Anim. Genet. 37 S1:1-6. Ye, X., Avendano, S., Dekkers, J.C.M. and Lamont, S.J. 2006. Association of twelve immune-telated genes with performance of three broiler lines in two different hygiene environments. 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