Brief Summary of Minutes of Annual Meeting

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.

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.