Progress 10/01/08 to 09/30/13

Outputs
Target Audience: Scientists (agricultural and human health), poultry industry breeders, students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two PhD students contributed to laboratory work, data analysis and paper writing. How have the results been disseminated to communities of interest? Results were disseminated through the Poultry Workshop at the annual Plant and Animal Genome meeting with presentations and discussions. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Major goals included improved resolution, on the genomic and cytogenetic levels, of the gene and element content of microchromosome 16 as well as improved understanding of the chromosomal architecture and discovery of new regions. The ortholog in turkey was also studied to improve our knowledge of comparative poultry genomes. We also continued to breed our special UCD genetic stocks and make those resources available to other researchers collaborating in NRSP8.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Miller, M.M., C.M. Robinson, J. Abernathy, R.M. Goto, M. Hamilton, H. Zhou, and M.E. Delany. Mapping of genes for olfactory receptors, cysteine-rich-domain containing scavenger receptors and other proteins to GGA 16, the chicken MHC microchromosome. J Heredity.doi:10.1093/jhered/est091 (online 2013; print 2014)
• Type: Book Chapters Status: Published Year Published: 2013 Citation: Delany, M.E. and T.H. O'Hare. 2013. Genetic stocks for immunological research (Appendix I). In Avian Immunology, 2nd Edition (editors: K.A. Schat, B. Kaspers and P. Kaiser). Elsevier: Academic Press, San Diego, CA. ISBN 9780123969651. 456 pp.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Capture array(CA)technology was employed to study specific regions of the chicken genome which had been previously found to be causative for several independently segregating developmental mutations. This was the first time this technology (CA) was employed in poultry. Briefly, we applied the advanced technologies of targeted sequence genomic capture enrichment and next-generation sequencing to discover the causative element for three inherited mutations affecting craniofacial, limb and/or organ development. Since the three mutations (coloboma, diplopodia-1 and wingless-2) were bred into a congenic line series (same genetic background) and previously mapped to different chromosomes, each targeted mutant causative region could be compared to that of the other two congenic partners, thereby providing internal controls on a single array. Of the ~73 million 50-bp sequence reads, ~76% were specific to the enriched targeted regions with an average target coverage of 132-fold. Analysis of the three targeted regions (2.06 Mb combined) identified line-specific single nucleotide polymorphism (SNPs) and micro (1-3 nt) indels. Sequence content for regions indicated as gaps in the reference genome was generated, thus contributing to its refinement. This is the first report of targeted capture array technology in an avian species, the chicken, an important vertebrate model; the work highlights the utility of employing advanced technologies in an organism with only a "draft stage" reference genome sequence. An explanation of the path we used with this unique technology was disseminated via meetings with scientists and members of the commercial poultry breeding companies, by depositing the variants discovered in NCBI GenBank and covering the procedures in a detailed review article published by Gene available as an open access document. In addition, the chicken coloboma mutation was further studied, coloboma exhibits features similar to human congenital developmental malformations such as ocular coloboma, cleft-palate, dwarfism, and polydactyly. Initially, the mutation was linked to a 990 kb region encoding 11 genes; the application of the genetic (fine mapping) and genomic tools (targeted ngs) led to a reduction of the linked region to 176 kb and the elimination of 7 genes. Furthermore, bioinformatics analyses of capture array-next generation sequence data described above identified genetic elements including SNPs, insertions, deletions, gaps, chromosomal rearrangements, and miRNA binding sites within the introgressed causative region relative to the reference genome sequence. Coloboma-specific variants within exons, UTRs, and splice sites were studied for their contribution to the mutant phenotype. Our results suggest three candidate genes for future studies. The three genes, SLC30A5 (a zinc transporter), CENPH (a centromere protein), and CDK7 (a cyclin-dependent kinase), are differentially expressed (compared to normal embryos) at stages and in tissues affected by the coloboma mutation. Of these genes, two (SLC30A5 and CENPH) are considered high-priority candidate based upon studies in other vertebrate model systems. A manuscript is in review on this work. PARTICIPANTS: Portions of this work are collaborative with Dr. Hans Cheng, from the USDA-Avian Disease and Oncology Lab, East Lansing MI. One PhD student was trained and was awarded the PhD degree June 2012: Beth Robb One M.S. student was trained: Ingrid Youngworth TARGET AUDIENCES: Target audiences are other researchers (agricultural and biomedical scientists), poultry breeders and the poultry industry PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The developmental genetic mutations studied segregate within native and commercial poultry stocks and cause syndromes similar to human congenital malformations affecting limb, heart, craniofacial features (e.g., cleft palate). Here we have studied specific genetic lines which serve as important vertebrate models so we can study the genes, their expression patterns and those genetic mechanisms operating during development in vertebrates causing both normal and abnormal syndromes. The work extends the uses of the initial chicken genome sequencing efforts and involves resequencing of whole and specific regions of the chicken genome so to study genes important in growth and development and identify the variation causing the abnormal phenotypes. The variation discovered contributes to refining the draft sequence content. The unique genetic resources under study are valuable models for discovering sequences and pathways important in poultry (chicken and turkey) and vertebrate (i.e., human) development.

Publications

• Robb, E.A., and M.E. Delany. 2012. Case study of sequence capture enrichment technology: Identification of variation underpinning developmental syndromes in an amniote model. Genes. 3:233-247 doi:10.3390/genes3020233 http://www.mdpi.com/2073-4425/3/2/233/
• Robb, E., and M.E. Delany. 2012. Polydactyly in an avian biomedical model: A genetic study of pre-axial variation and genomic maintenance. Cytogenetic and Genome Research 136:50-68.
• Robb, E.A. and M.E. Delany. 2012. Utilization of advanced genomic technologies to identify causative elements of inherited developmental defects in the chicken. International Plant and Animal Genomes XX, January 14-19, 2012, San Diego, CA

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Advances in genomics can be applied to the study of unique phenotypes in biomedical models to generate new knowledge on pathways involved in human congenital malformations. The chicken has long been a premier research model in the field of vertebrate developmental biology due to the advantages of access and ease of manipulation. The power of the chicken as a model organism has been further improved given the release of its genome sequence and progress in creation of tools and reagents (e.g., SNP arrays, ESTs, gene expression and cDNA databases, microarrays) and availability of unique genetic resources and cell lines. Two single gene mutations in the chicken, coloboma (co) and diplopodia-1 (dp-1), were studied for the purpose of identifying the element responsible for each developmental syndrome. These mutant models provide an invaluable means of studying craniofacial morphogenesis as well as limb and organ development. In the recessive condition both co and dp-1 produce syndrome phenotypes shared in common with human congenital malformations including cleft palate, facial-tissue defects, polydactyly, and dwarfism. A 60K SNP array study paired with fine-mapping techniques led to the discovery of a specific chromosomal region associated with each syndrome (co: 240 kb, 5 genes; dp-1: 260 kb, 11 genes). The expression patterns of sixteen genes found within the regions were studied by in situ hybridization during early embryogenesis. Examination of the results allowed further prioritization of candidate genes, suggesting that several genes be emphasized in future studies. In addition, next-generation technologies including a targeted-capture array with deep-sequencing were employed to investigate the regions linked to each mutation. Bioinformatic analyses identified unique genetic features (e.g., SNPs, short INDELs, large deletions) associating with each mutation region. These genetic elements are being evaluated for their contribution to the mutant phenotypes using a variety of genetic and genomic techniques. The results of this study will increase our understanding of the mechanisms underlying similar human disorders and will promote insights into the molecular mechanisms of normal development. Ultimately, this new knowledge could lead to screening tools for preventative medicine applications. PARTICIPANTS: Portions of this work are collaborative with Dr. Hans Cheng, from the USDA-Avian Disease and Oncology Lab, East Lansing MI. One students was trained: Beth Robb (PhD, Genetics). TARGET AUDIENCES: Target audiences are other researchers (agricultural and biomedical scientists), poultry breeders and the poultry industry PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The developmental genetic mutations studied here are common to poultry and a cause of embryo mortality; these are similar to the most common human congenital malformations (affecting limb, heart, craniofacial features). The chicken provides a versatile model to contribute to our understanding of genes, their expression patterns and genetic mechanisms operating during development in vertebrates. The work extends the uses of the poultry genome sequencing efforts and technology development to understand genes important in growth and development. The unique genetic resources under study are valuable models for discovering sequences and pathways important in amniote development.

Publications

• Robb, E.A., C.L. Gitter, H. Cheng and M.E. Delany. 2011. Single nucleotide polymorphism analysis of chicken genetic resources: Variation within and among MHC-congenic lines and mapping of developmental mutations. J. Heredity 102:141-156. (cover art)
• Robb, E.A., and M.E. Delany*. 2011. Chicken developmental mutations that cause embryonic lethal syndromes: The search for causative elements by fine mapping and capture array. International Plant and Animal Genomes XIX, San Diego, CA.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Wingless-2 candidate gene studies: The chicken autosomal recessive and embryonic lethal wingless-2 (wg-2) mutation was first characterized in 1956. The mutant phenotype exhibits complete winglessness, truncated legs, cleft palate, and kidney malformations. During the last year, additional SNP discovery along with fine mapping narrowed the wg-2 mutation to a ~300 kb region encoding four known genes. Our work continues on studying the expression profiles of the genes by qPCR and whole embryo in situ hybridization. We used this mutation for the capture array-NGS effort described below. SNP analyses of developmental mutants: We developed and submitted samples from a number of UCD congenic developmental mutant lines to identify chromosomal regions or improve/narrow the interval for a region of interest for candidate gene studies using the USDA 60K SNP array. A manuscript is in press reporting the results of the study as well as discussion of priority candidate genes (Robb, et al. in press). In brief, the 60K array analyses significantly improved the resolution and number of markers within the trait-associated region for six mutations, co, dp-1, dp-4, Po, ll, and stu as compared to a prior 3K SNP analyses. For example, the 3K array mutant analyses identified one marker for both dp-4 and ll, while the 60K array analyses associated 25 and 42, respectively. Additionally, the 60K enabled the identification of chromosomal regions for two mutations (dp-3 and eu) as no chromosome was identified by the earlier 3K array. During 2010 further fine mapping (breeding/recombination and SNP development/analysis) was conducted to reduce the intervals and two of the mutations were used in the strategy for targeted sequencing of the regions (see below) Capture Array and Next Generation Sequencing:. Three mutations (mapping to three different chromosomes but on the same or related congenic backgrounds): coloboma.003, diplopodia-1.003 and wingless-2.331, were investigated using targeted sequence genomic capture enrichment and next generation sequencing (utilized SeqWright, Inc., service provider) to improve our opportunities to narrow the region and identify the causative element for each mutation. Given the congenic nature of the lines, each targeted mutant region could be compared to the other two congenic partners providing internal control/reference sequences all on one array. Approximately 76.2% (55,749,079) of all 50bp reads aligned to the reference genome with an average target coverage of 132x. Analysis of the three targeted regions (2.05Mb total) indicated 13,265 SNPs and identified 3,903 SNPs unique to the three mutations. The capture array in combination with fine-mapping techniques allowed for the reduction of the total associated regions by 1.3Mb. Verification of the identified SNPs, indels, and gaps in additional individuals is currently underway. To our knowledge this is the first successful employment of targeted capture array technology in chicken. PARTICIPANTS: Portions of this work are collaborative with Dr. Hans Cheng, from the USDA-Avian Disease and Oncology Lab, East Lansing MI. Two students were trained: Andrew Webb (Master's, Genetics), Beth Robb (PhD, Genetics). TARGET AUDIENCES: Target audiences are other researchers and poultry breeders and the poultry industry PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The developmental genetic mutations studied here are common to poultry and a cause of embryo mortality; these are similar to the most common human congenital malformations (affecting limb, heart, craniofacial features). The chicken provides a versatile model to contribute to our understanding of genes, their expression patterns and genetic mechanisms operating during development in vertebrates. The work extends the uses of the poultry genome sequencing efforts and technology development to understand genes important in growth and development. The unique genetic resources under study are valuable models for discovering sequences and pathways important in amniote development.

Publications

• Robb, E.A., H.C. Cheng, and M.E. Delany. 2010. Chromosome regions associated with developmental mutations in congenic inbred lines of chicken. Developmental Biology 344:A103 doi:10.1016/j.ydbio.2010.05.437
• Webb, A.E. 2010. Causative Region Identification by 3K SNP Array and Candidate Gene Analysis of the Wingless-2 Mutation in Chickens. University of California, Davis CA (M.S. thesis).

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Work continued on the wingless-2 mutation. The genetic line was maintained and bred using a SNP genotyping which can assess normal homozygotes, heterozygous carriers and homozygous mutants. This reduces staff personnel time in conducting test matings and decreases the generation time and is more efficient for maintenance of the line (fewer birds are carried). Experiments continues to establish whether TSEN-2 is a strong candidate gene. We sequenced individuals of various genotypes to confirm 7 amino acid differences between wild type and mutants reported earlier. We find the same amino acid differences, however, we find different haplotypes for some of the sequence segregating suggesting that TSEN-2 is not a good candidate. Work is continuing to finalize the results. In addition we began whole embryo in situ hybridization experiments for each of the candidate genes to explore whether the genes are expressed in the right location at the right time to assess their candidacy for causation. We submitted a number of samples from UCD mutant stocks for a 60K SNP analysis and were able to narrow the interval of interest for many of the mutations. Work is continuing to narrow the regions of interest by continued crosses to generate more embryos and recombinants. The borders of the regions of interest were further developed for more SNPs as well. Work continued on the micro-rearrangements between the chicken and turkey genomes. We have assessed 5 different orthologous chromosomes for inversions, chromosome organization (centromeres and telomeres). This research is disseminated through annual meetings, conferences and via invited presentations to scientists and graduate students interested in animal genome organization, structure and function. In addition manuscripts are either in preparation or have been submitted for the scientific community. Several news media interviews were provided on the topics. In addition presentations were made at invited seminars in academic settings. PARTICIPANTS: Portions of this work are collaborative with Dr. Hans Cheng, from the USDA-Avian Disease and Oncology Lab, East Lansing MI (SNP analysis, mutant analysis) and with Dr. Jerry Dodgson, Michigan State University, East Lansing, MI (turkey - chicken analysis). Two students are being trained (mutation analysis study): Andrew Webb (Master's, Genetics), Beth Robb (PhD, Genetics) and one staff researcher works on the turkey project (Andrew Jiang). TARGET AUDIENCES: Target audiences are other researchers and poultry breeders and the poultry industry PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The developmental genetic mutations studied here are common to poultry and a cause of embryo mortality; these are similar to the most common human congenital malformations (affecting limb, heart, craniofacial features). The chicken provides a versatile model to contribute to our understanding of genes, their expression patterns and genetic mechanisms operating during development in vertebrates. The work extends the uses of the poultry genome sequencing efforts and technology development to understand genes important in growth and development. Genome organization at the chromosome level is a critical component of improving our understanding of the connection between genotype and phenotype (of cells, tissues and organisms). The chromosomes are the vehicles for transmission of the sequence information to daughter cells and defining their hierarchical structure expression patterns and also control aspects of genome stability which impact life or death decisions for cells at the molecular level is an important area of study. It contributes to both poultry and vertebrate biological understanding.

Publications

• Webb, A., H. Cheng, and M.E. Delany. 2009. Expression analysis of the candidate gene TSEN-2 in wingless-2 chicken embryos. International Plant and Animal Genomes XVII. San Diego CA. January 10-14, 2009 (poster #508).
• Lee, M.-K., X. Zhang, Y. Zhang, B. Payne, H.J. Park, J.J. Dong, C. Scheuring, M.E. Delany, J. Dodgson, and H.-B. Zhang. 2009. Toward a robust BAC-based physical and comparative map of the turkey genome. International Plant and Animal Genomes XVII. San Diego CA. January 10-14, 2009

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Involvement of a region on GGA12 in the wingless-2 mutation was previously indicated by a SNP study. An interval for the region is from 4.9 - 5.4 Mb. Sequencing determined one gene had seven amino acid predicted differences in mutant versus normal individuals. The TSEN-2 protein is involved in the formation of an endonuclease complex (hetero tetramer) whose function is removal of introns from pre-tRNA's. Last year tissues were collected from mutant and normal individuals during embryonic development for genotyping and RNA analysis. Transcript analysis of TSEN-2 does not indicate a consistent pattern of differences, for tissue-type or embryonic stage, between homozygous normal and mutant embryos from different tissues (representing the primary embryonic layers, endo, meso and ectoderm). In general transcript levels over the period of 4 - 10 days of embryogenesis were either similar or the mutant showed higher levels than the wild type. The ontological analysis, within genotype over time (per tissue), showed that the mutants had a fluctuating pattern whereas levels in the normal genotype were largely uniform. As we know, transcript quantities do not necessarily translate into protein quantities and so levels do not necessarily inform as to protein functionality. We expect to complete the study of TSEN-2 protein levels through Western blot analysis and perhaps immunohistochemistry. Of course, it may well be that mRNA or protein quantities are not the critical issue, but that the amino acid changes impact protein assembly and/or functionality. Or it may also be that TSEN-2 is not the causative gene. Candidate genes analysis for TSEN-2 and other genes in the interval will continue. As reported last year, samples from a group of developmental and congenic UCD genetic lines were used in a SNP analysis employing the original SNPs designed for the USDA-ADOL study of a few years earlier. We examined three developmental mutant lines (limbless, stumpy, talpid-2) to determine a region of interest associated with the mutations as well as several congenic lines to compare variation among their background genotype and as compared to archived samples including the original parent background line (10 or more years prior). It appears that in fact some MHC congenic lines do exhibit background genotype changes. And regions of interest were indicated for some of the mutants (several per mutant). These will be further investigated by a more comprehensive SNP study in 2009. Work commenced on the micro-rearrangements between the chicken and turkey genomes. We have assessed sets of chromosomes for inversions, chromosome organization (centromeres and telomeres). Collaborative work was published on biodiversity of chicken commercial stocks; this analysis was also carried by the non-scientific news media as it is of consequence for conservation and preservation of the genetic reservoir of chickens, for example locally-adapted and native species This research is disseminated through annual meetings, conferences and via invited presentations to scientists and graduate students interested in animal genome organization, structure and function. PARTICIPANTS: Portions of this work are collaborative with Dr. Hans Cheng, from the USDA-Avian Disease and Oncology Lab, East Lansing MI (SNP analysis, mutant analysis) and with Dr. Jerry Dodgson, Michigan State University, East Lansing, MI (turkey - chicken analysis). One student is being trained (mutation analysis study): Andrew Webb (Master's, Genetics) and one staff researcher works on the turkey project (Andrew Jiang). TARGET AUDIENCES: Target audiences are other researchers and poultry breeders and the poultry industry PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The developmental genetic mutations studied here are common to poultry and a cause of embryo mortality, and are similar to common human congenital malformations (affecting limb, heart, craniofacial features). The chicken provides a versatile model to contribute to our understanding of genes, their expression patterns and genetic mechanisms operating during development in vertebrates. Genome organization at the chromosome level is important as a component part of improving our understanding of the connection between genotype and phenotype (of cells, tissues and organisms). The chromosomes are the vehicles for transmission of the sequence information to daughter cells and define through their hierarchical structure expression patterns and also control aspects of genome stability which impact life or death decisions for cells at the molecular level.

Publications

• Muir, W.M., G. K. Wong, Y. Zhang, J. Wang, M.A.M. Groenen, R.P.M.A. Crooijmans, H.-J. Megens, H. Zhang, R. Okimoto, A. Vereijken, A. Jungerius, G.A.A. Albers, C. Taylor Lawley, M. E. Delany, S. MacEachern, and H. H. Cheng. 2008. Genome-wide assessment of worldwide chicken SNP genetic diversity indicates significant absence of rare alleles in commercial breeds. 2008. Proceedings of the National Academy of Sciences (USA). 105:17312-17317.
• Muir, W.M., Wong, G.K., Zhang, Y., Wang, J., Groenen, M.A.M., Crooijmans, R.P.M.A., Megens, H.-J., Zhang, H.M., McKay, J.C., McLeod, S., Okimoto, R., Fulton, J.E., Settar, P., O'Sullivan, N.P., Vereijken, A., Jungerius-Rattink, A., Albers, G.A.A., Taylor Lawley, C., Delany, M.E., and H.H. Cheng. 2008. Review of the initial validation and characterization of a 3K chicken SNP array. World's Poultry Science J. 64:219-225.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: The project emphasized during the prior year developed and continues in collaboration with the USDA-ARS-Avian Disease and Oncology Laboratory. We focused on discovery of new SNPs for GGA 12 to narrow the genetic region of interest for the Wingless-2 (wg-2) mutation. The longterm goal is to find the causative gene. The region was narrowed to about 500 Kb. A number of candidate genes were sequenced in this region and although polymorphisms were evident, only one (TSEN-2) appeared to exhibit variation between the alternate homozygous classes (wild-type versus mutant). Tissue and organ samples from all genotypes (two classes of homozygotes and heterozygous individuals) were collected from embryos, 4 to 10 days of incubation, for RNA and protein level analyses. A second SNP analysis of UC Davis genetic lines was undertaken and made possible due to partial funding from the NRSP-8 Poultry Species Coordinator; the project was organized in collaboration with North Carolina State University. Genome wide SNP analysis was collected for three UCD developmental mutations (limbless (lb), stumpy (st) and talpid (ta)) to determine associations for chromosomal regions of interest ultimately discover the causative gene(s). Also, a number of inbred and congenic lines from current and decade-old samples were include in the genome wide SNP scan analysis to explore mutation rates. Data are to be analyzed during the upcoming year. Whether the samples available are going to be suitable (provide enough power) for such determination remains to be seen. Research continued to determine the extent of mega-telomere variability within and among genetic lines of chicken and cell lines. The number of mega-telomeres within UCD 001 (Red Jungle Fowl inbred line) is highly variable and a mode of 2, 4, or 6 signals in males and 3 or 5 in females has been observed (indicating 1 to 3 loci and involving both sex chromosomes). To date, the chromosomes that carry mega-telomeres in UCD 001 are GGA 9 in all individuals, GGA Z in one male individual, and GGA W in all female individuals; however, unlike UCD 003, GGA 16 and 28 were found to be negative for mega-telomeres. One ADOL individual (progeny from a cross between lines 15I5 x 71) has been studied to date and this female exhibited a mega-telomere on GGA 9 and W, but not GGA 28. Two chicken cell lines are also under analysis, DT40 (ALV transformed B-cell line derived from an Hyline SC female bursal lymphoma) and DF-1 (immortalized CEF line derived from ADOL line 0 embryos, so a mosaic culture system). Both cell lines exhibit a mega-telomere on GGA W but not on GGA 9 and all other mega-telomeres have yet to be analyzed. Interestingly, DF-1 has been reported to be telomerase-negative while DT40 is telomerase-positive, however, DF-1 has a far stronger intensity of telomeric DNA signals (by FISH) compared to DT40 which shows an unusually "dull" profile (yet with unusually distinctive interstitial telomeres on GGA 1). PARTICIPANTS: Lisa Goldberg (research staff, Delany laboratory, UC Davis) Tom O'Hare (PhD graduate student, Genetics Graduate Group, UC Davis) Hans Cheng (USDA-ARS, Avian Disease and Oncology Laboratory, East Lansing, MI) Jerry Dodgson (Poultry Species Coordinator, Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI) Chris Ashwell (Department of Poultry Science, North Carolina State University, Raleigh, NC) TARGET AUDIENCES: Poultry research scientists, biomedical researchers interested in developmental biology, biological research community interested in the relationship between telomere length regulation control and genome stability, lifespan and disease-susceptibility, biotechnology researchers.

Impacts
Understanding the organization and function of the chicken genome is important as it contributes to understanding how to best select genotypes for agriculturally-relevant traits and biotechnology applications. Further, the chicken is an important model for human disease and analysis of the avian genome and its expression during acquired diseases and congenital malformations provides a knowledge base valuable for impacting human health. The genetic mutations studied here are common to poultry and a cause for embryo mortality, and are some of the most common human congenital malformations (affecting limb, heart, craniofacial features). The impact for the telomere length variation analysis research is to improve understanding of genotype-specific telomere length variation and ultimately the molecular and cellular mechanisms that regulate chromosome-specific and genotype-specific variation and impact of such on genome stability and organization. Difference telomere-based thresholds for genome stability may contribute to disease resistance and susceptibility differences among individuals within and among genotypes.

Publications

• Muir William M., Gane Ka-Shu Wong, Yong Zhang, Jun Wang, Martien A.M. Groenen, Richard P.M.A. Crooijmans, Hendrik-Jan Megens, Huanmin M. Zhang, Jim C. McKay, Scott McLeod, Ron Okimoto, Janet E. Fulton, Petek Settar, Neil P. O'Sullivan, Addie Vereijken, Annemieke Rattink, Gerard A.A. Albers, Cindy Taylor Lawley, Mary E. Delany, and Hans H. Cheng. 2007 Chicks and SNPs. 5TH European Poultry Genetic Symposium, Braedstrup, Denmark. 6pp.

Progress 01/01/06 to 12/31/06

Outputs
Work continued related to NRSP-8 Objective 1 on the enhancement and integration of genetic and physical maps of agriculturally important animals for cross-species comparisons and sequence annotation. The project emphasized during the prior year was developed in collaboration with the USDA-ADOL. The objective was to study single nucleotides 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. The specific lines analyzed were wingless-2 (wg-2), coloboma (cm), polydactyly (po), eudiplopodia (eu), diplopodia-1 (dp-1), diplopodia-3 (dp-3), and diplopodia-4 (dp-4). DNA samples from these mutations were included in the SNP analysis of various genetic and commercial lines funded by a USDA-NRI (PD: Hans Cheng, USDA-ADOL). Five of the seven mutations (cm, dp-1, dp-4, po, wg-2) were mapped to a chromosome and in one case (wg-2) a specific region. One candidate gene in the region of wg-2-associated SNPs was studied at a single developmental timepoint post limb outgrowth, however the relative transcript expression between forelimb, hindlimb, and brain among genotypes was similar. All of the developmental mutations with the exception of po are recessive embryonic lethals and thus the lines are maintained by breeding carriers (i.e., heterozygous for the mutation) involving labor- and animal intensive test-crossing. A TaqMan assay was designed and found to be a reliable method for genotyping of the wg-2 line in lieu of test-crossing. Work also continued to explore the genotype variation for the location of mega-telomere loci, previously we mapped loci to specific chromosomes (9, 16, 28, W) in a White Leghorn line (UCD 003). We are now examining loci in the Red Jungle Fowl (UCD 001) and finding variation for some of the positions of the mega telomere arrays, e.g., 16 and 28.

Impacts
Understanding the organization and function of the chicken genome is important as it contributes to understanding how to best select genotypes for agriculturally-relevant traits and biotechnology applications. Further, the chicken is an important model for human disease and analysis of the avian genome and its expression during acquired diseases and congenital malformations provides a knowledge base valuable for impacting human health. The genetic mutations studied here are common to poultry and a cause for embryo mortality, and are some of the most common human congenital malformations (affecting limb, heart, craniofacial features).

Publications

• No publications reported this period

Progress 01/01/05 to 12/31/05

Outputs
Work continued related to NRSP-8 Objective 1 on the enhancement and integration of genetic and physical maps of agriculturally important animals for cross-species comparisons and sequence annotation. Accomplishments included publication of an integrated map of chicken chromosome 17, a microchromosome having homology with human chromosome 9 and containing a Marek's Disease virus QTL. In this work the position of the centromere was refined as was the orientation of the linkage group relative to the p and q arms of the chromosomes. New research was initiated using BACs for fluorescence in situ hybridization analysis to cytogenetically map the ultra-long telomere array sequences in the chicken genome. Another new project developed during the year with the USDA-ADOL to identify single nucleotides polymorphisms in seven UCD developmental mutant stocks to map and identify potential markers for carrier-testing and resolve potential candidate loci.

Impacts
Understanding the organization and function of the chicken genome is important as it contributes to understanding how to best select genotypes for agriculturally-relevant traits and biotechnology applications. Further, the chicken is an important model for human disease and analysis of the avian genome and its expression during acquired diseases and congenital malformations provides a knowledge base valuable for impacting human health.

Publications

• Romanov, M.N., L.M. Daniels, J.B. Dodgson and M.E. Delany. 2005. Integration of the cytogenetic and physical maps of chicken chromosome 17. Chromosome Research 13:215-222.
• Schmid, M., Nanda, I., Hoehn, H., Schartl, M., Haaf, T., Buerstedde, J.-M., Arakawa, H., Caldwell, R.B., Weigend, S., Burt, D.W., Smith, J., Griffin, D.K., Masabanda, J., Groenen, M.A.M., Crooijmans, R.P.M.A., Vignal, A., Fillon, V., Morisson, M., Pitel, F., Vignoles, M., Garrigues, A., Gellin, J., Rodionov, A.V., Galkina, S.A., Lukina, N.A., Ben-Ari, G., Blum, S., Hillel, J., Twito, T., Lavi, U., David, L., Feldman, M.W., Delany, M.E., Conley, C.C., Fowler, V.M., Hedges, S.B., Godbout, R., Katyal, S., Smith, C., Hudson, Q., Sinclair, A., and Mizuno, S. 2005. Second report on chicken genes and chromosomes. Cytogenetic and Genome Research 109:415-479.

Progress 01/01/04 to 12/31/04

Outputs
Work continued related to Objective 1 (Enhance and integrate genetic and physical maps of agriculturally important animals for cross species comparisons and sequence annotation). Accomplishments included (1) first level analysis of the 6.6X draft chicken genome sequence (Nature, 2004), (2) telomere array analysis including mapping of a large ultra-long telomere to the W (manuscript submitted) and identification of telomere repeats in the sequence and (3) improved resolution of chromosome 17 loci relationships and chromosome organization through alignment of the linkage map, sequence map and cytogenetic map (manuscript submitted).

Impacts
The availability of the sequence and the first level analysis of the draft chicken genome change (enhance) the scope of research possible to explore the genetic mechanisms controlling growth, development and disease resistance.

Publications

• International Chicken Genome Sequencing Consortium. 2004. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432:695-716.
• Rodrigue, K.L. 2004. Inheritance of Ultra-Long Chicken Telomere Arrays in a Highly Inbred Chicken Line and Mapping of an Array to the W-Chromosome. M.S. Thesis. University of California, Davis CA.