Brief Summary of Minutes of Annual Meeting

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.

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).