Source: UNIVERSITY OF DELAWARE submitted to
ADAPTING CHICKEN PRODUCTION TO CLIMATE CHANGE THROUGH BREEDING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
0224429
Grant No.
2011-67003-30228
Project No.
DEL00692
Proposal No.
2010-04233
Multistate No.
(N/A)
Program Code
A3141
Project Start Date
Apr 1, 2011
Project End Date
Mar 31, 2015
Grant Year
2014
Project Director
Schmidt, C. J.
Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717
Performing Department
Animal And Food Sciences
Non Technical Summary
The rationale for this proposal is that breeding can have a major impact on adapting the chicken and egg producing industries to the climate changes anticipated in the 21st century. One of the most negative impacts of climate change on U.S. chicken production will be increased exposure to heat stress. It is clear genetic variation exists in the chicken's ability to cope with temperatures greater than the thermal comfort zone for maximal productivity. We propose to identify genes that control adaptation to heat stress. Also, the largest single component of the chicken industry's impact on climate change arises from the massive amounts of energy consumed to transport feed. Any increase in nutrient utilization in the chicken would reduce the overall environmental footprint of the industry. Since nutrient utilization is partly genetically controlled, the second major component of this proposal is to identify genes and alleles that regulate nutrient utilization. Our approach will include genetic mapping and extensive gene expression analysis to identify genes in both broilers and egg layers that are important to survival in different climate conditions. The specific aim is to identify genes that control the chickens ability to tolerate heat stress.The single most important impact will likely be on the industry. Once the genes are identified that control heat response and/or nutrient utilization, this knowledge can be used by the chicken breeding industry to implement breeding strategies to improve these traits in production lines. These improvements will reduce mortality and morbidity due to heat and increase nutrient utilization. Because the chicken is likely to remain an important food source throughout the 21st century, the improved genetics of the chicken will play an important role in alleviating hunger.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3153210108060%
3153220108040%
Goals / Objectives
The goals of our research are to better adapt the chicken to production in our changing global climate and to mitigate the environmental footprint of the chicken industry, which this proposal will address through the following objectives: Use genome wide association studies, integrating genome sequencing, SNP analysis, epigenetic analysis and transcriptomics to Identify genes that adapt chickens to the stress of changing climate. Identify genes and alleles to improve nutrient utilization for meat and egg production and mitigate the impact of the chicken industry on the environment. Identify novel alleles in native, backyard African chickens, where the local environment has selected for birds more tolerant of climate variation. Provide this knowledge to breeders assisting their efforts to adapt the chicken to changing climate and mitigate the impact of the industry on earth's climate. EXPECTED OUTCOMES: There will be multiple phases of application of the results of the proposed studies. The initial usage will be within the proposed studies themselves, in which the data of sequencing the African and the commercial chicken lines will inform the selection of specific SNPs as candidates for adaptation to heat stress. This information will guide selection of SNPs to place into the small (384) SNP panels for testing of birds placed into heat-stress trials at ISU. Also, the epigenetic and transcriptome analyses will be mapped to the chicken genome providing a far more detailed gene mapping than currently exists. As our studies map heat sensitive and nutrient utilization traits to the genome, these data will directly affect the gene models within the mapped regions. Two other phases of usage of the results of the proposed work will take place after the results are disseminated. The findings will be of value to the scientific community, as foundational for additional scientific investigations. Perhaps most importantly, the results of the proposed work will be of value to the chicken breeding industry, in providing specific information about SNPs, genes, genetic pathways and biomarkers that can be used in genetic selection for better adaptation of their commercial lines to efficient production under heat-stress conditions. All transcriptome data, including mapped exons, splicing information, alternative initiation and termination sites along with all detected microRNAs will be mapped to the genome and displayed to the scientific community via our Gallus Gbrowse. These data will be invaluable to other studies examining gene expression in the chicken. The data resulting from epigenetic studies will be the first in-depth methylation studies in the chicken, as well as the first tissue specific methylation mapping studies. These data will represent a new class of genomic information for the chicken which has been previously un-characterized and un-tapped for genetic selection. If specific methylation sites can be identified as being important in predisposing an individual bird to thermal conditioning or any other type of conditioning then they can be selected for improvement in the progeny.
Project Methods
Overview of methodologies: Hypothesis driven, candidate gene approaches to identifying genes affecting complex, quantitative traits such as heat stress response or nutrient utilization are unlikely to describe all or even a significant portion of the causative genes. This is because we do not know enough of the underlying biology and pathways to make comprehensive hypotheses that allow us to identify all genes controlling a trait. In contrast, genome-wide association studies (GWAS) are a broader approach, capable of identifying many genomic regions controlling traits, in the absence of preconceived hypotheses about gene identity. Typically GWAS studies focus on associating SNP associations with traits. We propose to extend GWAS to include analysis of SNPs, complete genomic sequences, epigenetic effects and whole transcriptome analysis integrated by statistical and bioinformatic analyses to provide a greater understanding of the relationships between phenotypic traits and the genome. We refer to this approach as a Genome Wide Integrated Study (GWIS). The rationale for this proposal is that breeding can have a major impact on adapting the chicken and egg producing industries to the climate changes anticipated in the 21st century. One of the most negative impacts of climate change on U.S. chicken production will be increased exposure to heat stress. It is clear genetic variation exists in the chicken's ability to cope with temperatures greater than the thermal comfort zone for maximal productivity. We propose to identify genes that control adaptation to heat stress. Also, the largest single component of the chicken industry's impact on climate change arises from the massive amounts of energy consumed to transport feed. Any increase in nutrient utilization in the chicken would reduce the overall environmental footprint of the industry. Since nutrient utilization is partly genetically controlled, the second major component of this proposal is to identify genes and alleles that regulate nutrient utilization. The significance of the understanding gained from this proposal will be multifaceted, including an improved understanding of how genes control the targeted chicken traits. The single most important impact will likely be on the industry. Once the genes are identified that control heat response and/or nutrient utilization, this knowledge can be used by the chicken breeding industry to implement breeding strategies to improve these traits in production lines. These improvements will reduce mortality and morbidity due to heat and increase nutrient utilization. Because the chicken is likely to remain an important food source throughout the 21st century, the improved genetics of the chicken will play an important role in alleviating hunger. All data will be deposited at the NCBI Sequence Read Archive. All gene models, QTL and epigenetic data derived from this work will be displayed in Birdbase Gallus GBrowse, making the information available to the scientific community

Progress 04/01/13 to 03/31/14

Outputs
Target Audience: Graduate students in animal breeding and genetics, commercial breeders of poultry, researchers in animal and poultry breeding and genetics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two Master's students completed their thesis requirements supported by this research grant. An additional eight graduate and six undergraduate students were mentored and trained in conducting research in this project. Summer interns in programs that give research experiences to U.S. students of underrepresented ethnic and racial minority groups were trained in research procedures in the lab and in animal handling. Faculty, graduate students and staff of the project participated in national and international scientific conferences. How have the results been disseminated to communities of interest? Two master's theses have been published and three manuscripts have been submitted for publication in peer review journals. In addition, the results have been disseminated by presentation of research at conferences and in seminars at other universities, nationally and internationally, and direct communications with poultry genetics companies. Seminars on research were presented to first-year graduate students of several different graduate majors. Invited proceedings papers for international conferences have been prepared and submitted for meetings occurring later in 2014. What do you plan to do during the next reporting period to accomplish the goals? With respect to the Ross708 and Legacy heat stress comparison, we currently have an extensive collection of RNA samples from distinct tissues obtained from control and heat stressed birds. We anticipate preparation, sequencing and analysis of libraries prepared from hypothalamus, pituitary, breast muscle, spleen, duodenum, jejunum, ileum and liver during this next reporting period. We anticipate extending our transcriptome studies of probiotic treated birds to include additional libraries from duodenum, ileum and jejunum. In addition, libraries will be prepared from spleen and ceca from control and probiotic treated birds. These studies should provide insight into the improved feed efficiency observed in the probiotic versus control birds. Finally, we will continue to validate the RNAseq experiments using quantitative PCR. Throughout our heat stress trials, we have placed tissue samples in formalin for sectioning. To further validate observations made in the transcriptome studies, we have begun immunohistochemical staining studies of tissues samples using commercially prepared antibodies to specific gene products. Our initial experiments made use of anti-PCNA antibodies to evaluate the cell divisions status of post hatch Ross 708 and Legacy birds. We will extend these staining studies to other gene products as indicated by our transcriptome analyses. This will provide important insight into the relationship between RNA levels and protein in our experiments. We anticipate that manuscripts will be prepared detailing the studies of the hypothalamus, liver, breast muscle and spleen from the heat stress experiments, along with one manuscript from the probiotic experiment. In the next year, we will expand our DNA methylation data collection to additional tissues including spleen, breast muscle and right ventricle. The movement of this effort has allowed for the development of the gene expression data at UDel to identify these tissues as targets for further characterization. Once the DNA methylation data is available we will integrate it with the RNAseq data generated by UDel. The resulting correlation data will provide evidence for regulatory elements (DNA methylation sites) that are involved in the response to heat stress and more importantly the conditioning process. Genome-wide association analysis of the Advanced Intercross line response to heat will be completed and submitted for publication in two or more manuscripts. Global gene expression of immune tissues of birds from two Iowa State University genetic lines under simultaneous heat stress and receiving inflammatory stimulus will be analyzed for statistical significance and then bioinformatically studied to detect pathways involved in heat-stress response. A manuscript will be prepared. Analysis of the 600K SNP data of samples of chickens from Uganda and Rwanda will be completed and a manuscript prepared. Analysis of global gene expression of spleens of birds from three Iowa State University genetic lines under heat stress and receiving inflammatory stimulus done for statistical significance and then bioinformatically studied to detect pathways involved in heat-stress response. Studies of laying hens under heat stress will commence at Virginia Tech University (Michael Persia (co-PI) moved from Iowa State University to VTU). Microbiome samples will be collected from young hens under heat stress, submitted for sequencing, and analysis of heat treatment effects on gut microbial communities will be initiated. In addition, samples will be isolated for both transcriptome and iSTAT analyses. The iSTAT analyses will be used subsequently in a GWAS study to relate allelic variation to blood parameters as a function of heat stress. A panel of prominent national and international speakers, including participants in this project, will be invited to present their research at a workshop at the Plant and Animal Genome Conference (January 2015) on genomics of animal performance under suboptimal conditions, which is being organized by a coPD.

Impacts
What was accomplished under these goals? Over 200 genes responsive to heat stress have been identified in our studies. Recent analyses of our data have indicated that during prolonged heat stress (21 days), gene expression levels may acclimatize and return to baseline levels. Currently, we are developing experiments to investigate much earlier time points during heat stress (1 – 2 hours) along with determining when this hypothesized acclimatization may occur during the three weeks of heat stress. We have continued to explore the transcriptome of additional tissues obtained from heat stress and control birds. In particular we have begun preparing libraries from the hypothalamus and pituitary isolated at various times before and following heat stress. These tissues play an important role in the animal’s response to heat stress and we anticipate this data will provide important insight into metabolic and signaling changes during heat stress. In addition, we have also begun preparing microRNA samples along with microRNA libraries (to date: 24 libraries). These libraries are currently being sequenced. Finally, we have explored the impact of a probiotic (Bacillus subtilis) on the transcriptome and physiology of heat stressed birds. Under control conditions, probiotic treated birds have improved feed efficiency. We have identified a series of genes that appear to be differentially regulated between probiotic treated and control birds. Also preliminary studies show that the probiotic treated birds had a significantly lower body temperature during heat stress than control birds. This difference may have implications for the use of probiotic in ameliorating the impact of heat stress. From the high throughput sequencing data, we have deposited over 100 sequencing libraries with the Roslin Institute Gene Atlas. These data were used to improve the annotation of the chicken genome by improving the exon maps of known genes, along with providing genomic locations of previously unrecognized genes. In addition data from 35 libraries have been deposited with the NCBI archive as supporting data for two submitted manuscripts. One of the most challenging aspects of this study has been data analysis. Our projects are generating large amounts of data that need to be evaluated using multiple analytical techniques. In the initial part of this study, we made use of standard t-tests along with a statistical package, CuffDiff, to identify differentially expressed genes and we had anticipated using these in a computational pipeline to facilitate data analysis by biologists. To reduce the false positive level we observed using those two approaches, we have now developed a pipeline that uses four analytical methods: CuffDiff, Bayseq, DESeq2 and EdgeR. These methods allow the biologist to evaluate differentially expressed data identified by distinct methods and this approach is reducing the number of follow up validation assays that have identified false positives. In the last year, NCSU has refined the approach to evaluating the epigenetic impacts of heat stress and thermal conditioning using whole genome bisulfite sequencing. The reduced cost of sequencing will allow us to perform a more comprehensive level of analysis than previously proposed. In the last year, NCSU has focused on a single tissue (liver) collected from the ISU conditioning/stress trials in order to determine the number of biological replicates necessary to capture the treatment effects on genome-wide DNA methylation patterns. While there is a distinct value in the ability to directly compare DNA methylation patterns with gene expression levels (RNAseq) pooling of samples allows for more coverage depth within a treatment group when sequencing. The current data suggests that the optimal process for evaluating the samples collected will be to perform a low coverage analysis (3-4x) of individual samples using RRBS (Reduced representation bisulfite sequencing) and perform a high coverage analysis (20-25x) of pooled samples within treatment groups using standard bisulfite sequencing. A unique advanced intercross line (F18 and F19 generations) of broiler X Fayoumi chickens was used to initiate a genome-wide association study to identify genomic regions influencing response to heat. In the past year, statistical analysis using the GenSel program identified several genomic regions associated with body temperature, body weight, breast yield, digestibility, and changes of body temperature and weight during heat treatment. Sire heritabilities were estimated for these traits. The results demonstrate the feasibility of genetic selection to improve resistance to the negative impacts of heat exposure in poultry and provide target areas of the genome for future intensive study. Metabolic responses were detected in the liver transcriptome in response to chronic, cyclic heat stress of broiler chickens by using RNA-seq. Two networks were created from the 40 differentially expresses genes: “Cell Signaling, Molecular Transport, Small Molecule Biochemistry” and “Endocrine System Development and Function, Small Molecule Biochemistry Cell Signaling”. Members of the Ras-Raf-MEK-ERK (MAPK) signalling pathway were present in the broiler liver transcriptome in response to both S. Enteritidis infection and heat stress, suggesting that these pathways constitute a common host response to a wide range of stressors. Analysis of the 600K SNP chip data of birds sampled in Africa commenced. This included quality control of the genotypes and preliminary analysis of genetic relatedness of samples collected at distinct geographic regions. Currently we have results for samples from Uganda and Rwanda. Permission was obtained to organize a workshop at the Plant and Animal Genome Conference (January 2015) on genomics of animal performance under suboptimal conditions.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Liang Sun, Susan J Lamont, Amanda M Cooksey, Fiona McCarthy, Catalina O Tudor, K Vijay-Shanker, Rachael M. DeRita, Max Rothschild, Chris Ashwell, Michael E Persia and Carl J Schmidt. Transcriptome response to heat stress in a chicken hepatocellular carcinoma cell line. BMC Genommics, Submitted.
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Richard V. Davis, Susan J.Lamont, Chris M. Ashwell, Max F. Rothschild, Michael E. Persia and Carl J. Schmidt. Transcriptome analysis of post-hatch breast muscle in legacy and modern broiler chickens reveals enrichment of several regulators of myogenic growth. Plos One. Submitted
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Derrick J Coble, Damarius Fleming, Michael E Persia, Chris M Ashwell, Max F Rothschild, Carl J Schmidt and Susan J Lamont. RNA-seq analysis of broiler liver transcriptome reveals novel responses to heat stress. BMC Genomics. Submitted
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Janet deMena, Susan J. Lamont, Max F. Rothschild, Michael Persia, Chris Ashwell, Carl J. Schmidt. Insight into Human Directed Selection through Comparison of Duodenal Gene Expression Patterns Between Modern and Heritage Chicken Lines. Plant and Animal Genomes Meeting 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lakshmi Praveena Kamineni, Susan J. Lamont, Max F. Rothschild, Michael Persia, Iowa State University; Chris Ashwell, Carl J. Schmidt. Comparing Hypothalamic Transcriptomes Between Modern and Legacy Chicken Line. Plant and Animal Genomes Meeting 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Matheus Reis, Antonio Pinto Garcia, Susan J. Lamont, Max F. Rothschild, Michael Persia,Chris Ashwell, Carl J. Schmidt. Comparison of Ileal Gene Expression Between Modern and Legacy Broiler Lines. Plant and Animal Genomes Meeting 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Antonio Pinto Garcia, Matheus Reis, Susan J. Lamont, Max F. Rothschild, Michael Persia,Chris Ashwell, Carl J. Schmidt.Comparison of Jejunal Gene Expression Beteween Modern and Legacy Broiler Lines. Plant and Animal Genomes Meeting 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Richard V. Davis, Susan J. Lamont, Max F. Rothschild, Michael Persia,Chris Ashwell, Carl J. Schmidt. Transcriptome Analysis of Post-Hatch Breast Muscle Development in Legacy and Modern Broiler Chickens. Plant and Animal Genomes Meeting 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Allen Hubbard, Liang Sun, Susan J. Lamont, Max F. Rothschild, Michael Persia,Chris Ashwell, Carl J. Schmidt. Transcriptome Characterization of Heat Shock Response in White Leghorn Chicken Liver Hepatocellular Carcinoma (LMH) Cells. Plant and Animals Genome Meeting, 2014.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2014 Citation: S.J.Lamont, D.J.Coble, A. Bjorkquist, M.F. Rothschild, M.Persia, and C.Schmidt. Genomics of heat stress in chicken. Proceedings, 10th World Congress of Genetics Applied to Livestock Production.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2014 Citation: A. Bjorkquist, C. Ashwell, M. Persia, M. F. Rothschild, C. Schmidt, and S. J. Lamont. QTL for body composition traits during heat stress revealed in an advanced intercross line of chicken.Proceedings, 10th World Congress of Genetics Applied to Livestock Production.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Shurnevia Strickland. Transcriptome and histologic analysis of cardiac differences between modern broiler and heritage chickens. Masters Thesis, University of Delaware, 2014
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Brooke Aldrich Enslen. Characterization of the chicken transcriptome and differential morphometric growth in modern broiler chickens and a heritage line. Masters Thesis, University of Delaware. 2014


Progress 04/01/12 to 03/31/13

Outputs
OUTPUTS: University of Delaware: 1 Extracted RNA and completed sequencing of an additional 119 RNAseq libraries from multiple tissues derived from the Fayoumi, Broiler, Fayoumi x Broiler Advanced intercross lines along with the Heritage and Ross708 lines. 2 Used both Illumina and PacBio sequencing approaches to provide the genomic sequence of the Iowa State Broiler line using DNA provided by Iowa State. Passed data back to Iowa State for analysis and genome building. Preliminary analysis suggests the Illumina method provided 10-fold genome coverage while the PacBio provided 5-fold coverage. 3 Developed a pipeline for depositing RNAseq data with Birdbase and initiated development for automated deposition of data to the Roslin Chicken Gene Atlas project. 4 Initiated development of a pipeline to improve automation of RNAseq data analysis. This project focuses on decreasing the amount of time it takes to identify differentially expressed genes and collect annotation relevant to those genes. This is essential to develop hypotheses regarding the functions of affected genes in the context of heat stress. 5 Completed analysis of RNA libraries derived from LMH cells exposed to heat stress. Manuscript describing this work is in preparation. 6 Completed analysis comparing gene expression patterns in the hearts of non-stressed Ross and Heritage chickens. Manuscript in preparation 7 Completed analysis comparing gene expression patterns in the breast muscle of the non-heat stressed Ross and Heritage chickens. Manuscript in preparation. 8 Completed a differential analysis of genes expressed in different Ross tissues to identify both commonly expressed, and tissues enriched or specific genes. Manuscript in preparation. Iowa State University 1 Reproduced one generation of three ISU chicken genetic lines for testing. 2 Completed live-bird heat-stress trial of generation #2 (about 1,000 birds) of ISU genetic lines, collect physiological parameters on live birds, and harvest tissues for sequence analysis of genome, epigenome and transcriptome. 3 Shipped samples to other two US universities for analyses. 4 Initiated statistical analysis of physiological response to heat-stress, embryonic thermal conditioning, and breed, over two generations. 5 Isolated high-quality genomic DNA from 480 birds for 600K genotyping. 6 Initiated bioinformatic analyses of resequenced pooled samples from inbred Fayoumi and Leghorn chicken lines. 7 Completed arrangements for sampling of 100+ birds in Uganda with a team of students and faculty from the three participating US universities 8 Completed ileal digestibility analysis of samples from generation #1 9 Submitted abstract and delivered research presentation at International Society of Animal Genetics. North Carolina State University 1 Participated in the data collection and sampling at ISU in 2012. 2 Determined the impact of heat stress and conditioning in the ISU experiments on bone mineralization as a measure of skeletal integrity. 3 Purified DNA collected from ISU experiments in preparation for methylation analysis PARTICIPANTS: University of Delaware: All students working on this project participate in the heat stress trials including animal husbandry, necropsies and RNA preparation. Brooke Aldrich: graduate student responsible for determining both tissues specific and commonly expressed genes across all RNAseq libraries. Also conducting RNAseq analysis of brain in the Heritage and Ross708 lines as a function of heat stress. Janet de Mena: graduate student primarily involved in coordinating and analyzing physiological and morphometric data from heat stress trial at UD along with RNAseq analysis of the duodenum. Richard Davis: graduate student conducting RNAseq and micro RNA analysis of control and heat stressed pectoralis muscle in the Ross 708 and Heritage lines. Shurnevia Strickland: graduate student collecting and analyzing of heart RNAseq data in the Ross708 and Heritage lines as a function of heat stress. Liang Sun: graduate student conducting RNAseq analysis of LMH cells along with the liver from the Ross708 and Heritage lines as a function of heat stress. Liang is also responsible for developing the MySQL databases and web interfaces supporting this project. Rachael Derita: undergraduate student who carried out quantitative PCR experiments to validate the liver and LMH RNAseq studies. Rachael also constructed several liver RNAseq libraries under the direction of Liang Sun. Brittany Hazzard: undergraduate student who carried out quantitative PCR experiments to validate the liver and LMH RNAseq studies. Rachael also constructed several liver RNAseq libraries under the direction of Liang Sun Seretha T Suah: undergraduate student from Delaware State University (historically minority institution) who carried out quantitative PCR experiments to validate the liver and LMH RNAseq studies. Rachael also constructed several liver RNAseq libraries under the direction of Liang Sun North Carolina State: Alex Zavelo, graduate student, (primary recruit to perform the epigenetic analyses of project samples), interactions with the Iowa State team during sample collections, collection of samples in Iowa, processing of harvested tissues. Zack Lowman, graduate student, interactions with the Iowa State team during sample collections, collection of samples in Iowa. Mary Pat Bulfin, undergraduate student, processing of harvested tissues. Iowa State University: Erin Sandford, graduate student, preparation of supplies and materials for animal trials, monitoring animals on trial, recording phenotypic responses to heat stress, processing of harvested tissues, development of excel sheets for tracking inventory and phenotypic data, data entry. Derrick Coble, graduate student, preparation of supplies and materials for animal trials, monitoring animals on trial, recording phenotypic responses to heat stress, processing of harvested tissues. TARGET AUDIENCES: The two target audiences for this work include other scientists studying similar phenomena in other species along with poultry producers who may benefit from the genes identified in this study that may contribute to the chickens ability to withstand heat stress. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Response to Heat Stress in the Broiler x Fayoumi and Heritage, Ross708 Trials: The two proposed heat stress trials with the Broiler and Fayoumi intercross lines have been completed, samples shipped to the University of Delaware and North Carolina State for processing, genomic sequencing begun on the Broiler line and more than 100 transcriptome libraries prepared and sequenced from a variety of tissues. Two heat stress trials have been done comparing the Heritage and Ross708 lines at the University of Delaware and the results of these are being analyzed. A third scheduled trial will be completed before the end of year 2 and we anticipate completing the U.D. trials by the end of year 3 (on schedule). African Samples: Dr. Steven Kemp (coPI) has collected genomic samples from chickens in Lamu, Turkana and Western Kenya. The DNA samples have been purified, quality checked and will be routed to the Roslin Institute for SNP analysis. We had originally proposed to sample in Uganda during the first half of the second year of funding. Due to the timing of the funding start date and an outbreak of Ebola, we delayed the Uganda collection trip until the second half of the second year. We will be collecting from October 24 - November 2 in southeastern and southwestern Uganda. Bioinformatics Integration: We have continued to develop the database and web interface for sharing transcriptome data within the group. All gene expression data and associated metadata are now warehoused in a web-accessible MySQL database. This database is providing a resource for supplying our expression data to the Roslin (UK) Chicken Gene Atlas and the Birdbase database. Ultimately, our database will provide a ready conduit for submitting all of this expression data to the NCBI short read archive. 1. Two unanticipated results have been obtained from comparative evaluation of the control (non-heat stressed) Ross and Heritage transcriptomes. The first relates to the post-hatch development of the breast muscle. During the first week post-hatch, the Ross708 pectoralis muscle shows significant enrichment in genes promoting the cell cycle while in the second week it shifts to enrichment for genes involved in cell hypertrophy. The second observation relates to the heart. Modern broilers (Ross708) have an increased incidence of heart failure. During the third week post-hatch, the Ross birds show a significant increase in expression of fibrotic genes (particularly collagen), which may play an important role in heart failure. 2. Through the conduct of the heat-stress trials, a large group of trainees gained knowledge and experience in transdisciplinary studies relevant to climate change. Of these, five are members of non-immigrant underrepresented racial/ethnic groups.

Publications

  • No publications reported this period


Progress 04/01/11 to 03/31/12

Outputs
OUTPUTS: University of Delaware: Conducted heat stress trials using over 600 Ross 708 and Heritage birds. Approximately 200 transcriptome libraries have been prepared and sequenced. We have made the most progress to date evaluating data from the Liver libraries. Conclusions to date include: a.For both control and heat stress samples, significant differences exist in the gene expression patterns when comparing between samples take from the right or left lobe. For example, samples from the right lobe express almost two fold higher levels of Albumin mRNA than left lobe samples. b.Genes responding to heat stress include ones that are well characterized to respond to heat along with previously unrecognized heat responsive genes. 4.We have initiated analysis of all other transcriptome libraries . 5.Trained graduate and undergraduate students in all aspects of the projects. 6.Coordinated with Iowa State University to prepare team accessible, web based database for data collection and analysis. Iowa State University in YEAR 1 was in these main areas: Maintain breeders of three specialized Iowa State University genetic lines of chickens. 2.Hatch Generation #1 chicks for animal trials and identify with individually numbered wing bands. Reserve and raise chicks from Generation #1 from both normal and in ovo incubation treatments, to produce experimental chicks for study in Generation #2. Conduct first heat stress trial with approximately 1,000 chicks. 3. Conduct training of staff, students and postdoctoral trainees in procedures related to the live-bird trial, including collection of phenotypic data and biological samples from live birds and euthanized birds. Initiate cross-institutional, transdisciplinary training with graduate students from NCSU at ISU. 4. Related to all activities, Iowa State was also responsible for accurate record-keeping, adherence to all university and other policies governing research, and timely communications within the project. North Carolina State University in YEAR 1 was in these main areas: 1.Received samples from Iowa State University for subsequent epigenetic analysis. 2.Recruit graduate student to begin experimental analysis of samples from Iowa State University and the University of Delaware during year 2. PARTICIPANTS: Janet De Mena, Graduate Student, provided daily oversight of animal trials and supervised data collection during necropsies, collected and interpreted morphometric data, prepared intestinal transcriptome libraries and attended the Plant and Animal Genome 2012 meeting. Brooke Aldrich, Graduate Student, participated in all necropsies, collected and analyzed morphometric data, prepared brain transcriptome libraries and attended the Plant and Animal Genome 2012 meeting. Liang Sun, Graduate Student, participated in all necropsies, collected and interpreted morphometric data, prepared liver transcriptome libraries, aided in developing web accessible database for sharing data across the team, and presented a poster the Plant and Animal Genome 2012 meeting. Shurnevia Strickland: participated in all necropsies, collected and analyzed morphometric data, prepared heart transcriptome libraries and presented a poster at the Plant and Animal Genome 2012 meeting. Stephanie Shapiro, Undergraduate Student, participated in all necropsies and prepared RNA for library preparation. Monica Sterk, Undergraduate Student, participated in all necropsies and compiled morphometric data. Rachel Derita, Undergraduate Student, participated in all necropsies, developing qPCR approaches to validate transcriptome results. Mel Montagano, Undergraduate Student, participated in all necropsies. Stephanie Chew, Undergraduate Student, participated in all necropsies. Laura Wertman, Undergraduate Student, participated in all necropsies. Iowa State University: Michael Kaiser, Research Associate, provided daily oversight to animal trials including coordinating the ordering and preparation of supplies and materials for animal trials, developing and communicating work schedules, monitoring animals on trial, measuring phenotypic responses to heat stress, euthanizing animals, processing of harvested tissues, maintaining inventory of biological samples, preparing samples for safe shipment to collaborators in project, preparation and Erin Sandford, graduate student, preparation of supplies and materials for animal trials, monitoring animals on trial, recording phenotypic responses to heat stress, processing of harvested tissues, development of excel sheets for tracking inventory and phenotypic data, data entry. Derrick Coble, graduate student, preparation of supplies and materials for animal trials, monitoring animals on trial, recording phenotypic responses to heat stress, processing of harvested tissues. Angelica Bjorkquist, graduate student, preparation of supplies and materials for animal trials, monitoring animals on trial, recording phenotypic responses to heat stress, processing of harvested tissues. Damarius Fleming, graduate student, preparation of supplies and materials for animal trials, monitoring animals on trial, recording phenotypic responses to heat stress, processing of harvested tissues. TARGET AUDIENCES: Researchers in the poultry field. Presentations at Plant and Animal Genome Meeting 2011 PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Through the conduct of the heat-stress trials, a large group of trainees gained knowledge and experience in transdisciplinary studies relevant to climate change. Initiation of the animal experiments in large scale allowed the refinement of protocols and the establishment of a reliable model for heat-stress in poultry. A pipeline is under development to deposit all sequence and experimental metadata with public repositories. Through the mutual scientific networks possessed by the principals, linkages have been formed with the wider agricultural genomics community to address essential issues of the ability to store, transport, and analyze genomic datasets of the scale generated in this project. Data from this project will serve as beta test sets for the development of new pipelines. Through the transdisciplinary team interests and expertise, the important question of "what is the impact of heat stress combined with inflammatory (fever-inducing) stressors on the physiology of poultry" was identified. We have initiated a collaboration with Dr. Peter Kaiser (University of Edinburgh) to share samples and data from birds collected from Kenya.

Publications

  • Sun, L., Ashwell, C., Persia, M.E., Rothschild, M.F, Lamont, S.J., Schmidt, C. J. Transcriptome analysis of broiler chicken liver as a function of heat stress. Plant & Animal Genome XX, January 2012, San Diego, CA
  • Strickland, S. J., Ashwell, C., Persia, M.E., Rothschild, M.F, Lamont, S.J., Schmidt, C.J. Comparative Transcriptome Analysis of Chicken Heart Gene Expression Patterns Between a Modern Broiler and a Heritage Line. Plant & Animal Genome XX, January 2012, San Diego, CA
  • Kaiser, M., Sandford, E.E., Persia, M.E., Rothschild, M.F., Ashwell, C., Schmidt, C.J., and Lamont, S.J. 2012. Physiological differences among chicken breeds in response to embryonic thermal conditioning and post-hatch heat stress. Plant & Animal Genome XX, January 2012, San Diego, CA