Duration: 10/01/2002 to 09/30/2007

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The tremendous efforts of animal genomics researchers involved in the ongoing National Research Support Project-8 (NRSP-8) and past Multistate Research Projects (NC-209, NC-210/220, NC 168, NE-60) have resulted in the extremely successful species genome projects. These projects have provided chromosomal sequence data, physical and linkage maps, and molecular genetic markers of production, health, and product quality traits in cattle (dairy and beef), sheep, pigs, poultry, and more recently horses. However, the full value and applications of the species genome projects will be realized only when the actual genes and gene products (proteins) that coordinate and regulate important animal traits are known and understood. The time has come to harvest the value of species genome projects by linking chromosomal DNA information to expression profiles, phenotypes, and functions of specific genes and proteins in relevant models of animal husbandry. In cattle, for example, knowledge of the genes involved in nutrient partitioning, mammary development, and muscle growth will enable researchers to develop accurate genetic selection strategies using biologically relevant molecular markers for milk and meat production/quality. In addition, knowledge of the genes involved in digestion and nutrient absorption will allow researchers to develop optimal nutritional regimes for cattle of particular genetic backgrounds reared in a variety of environments. Similarly, a better understanding of the genes that regulate female reproduction will enable researchers to find real solutions to the problem of poor conception rates in high producing dairy cows. Finally, knowing what genes come into play when cattle are stressed by the environment, or challenged by infectious pathogens and the continued metabolic demands required to support high production, will enable researchers to develop novel management and breeding strategies and therapeutic drugs that promote animal health, well being, and food safety.

A major unifying feature of health, reproduction, production, and product quality traits that are important to the sustainability of US dairy and beef industries is their complex physiology, which involves intricate coordination of multiple gene expression events in a large variety of tissues at different stages of an animals productive lifetime. Traits such as these have mostly proven refractory to in-depth molecular analysis by traditional methodologies, but are excellent candidates for functional genomics and proteomics studies. These complex traits also form a unifying backbone of the research proposed under this new Multistate Research Project. Participants of the Project will use a variety of genomics and proteomics techniques to determine the location, structure, function, effect, and expression of genes affecting health, reproduction, production, and product quality in dairy and beef cattle. Identified genes and proteins will be shared amongst participants for contribution to ongoing SNP (single nucleotide polymorphism) analyses, mapping efforts, and for analyses of statistical associations with the economically important health, reproduction, production, and product quality traits recorded in the numerous resource populations contributed to the Project by various participants (see below).

Importantly, recent development of mixed and tissue-specific bovine complementary DNA (cDNA) libraries have allowed bovine genome researchers to obtain large numbers of expressed gene sequences (known as expressed sequence tags, or ESTs), which can be used to link chromosomal DNA sequences to specific cellular functions and phenotypes (Beckmann et al., 1997; Duggan et al., 1999). Thus, ESTs are critical tools for bovine functional genomics efforts and the ultimate development of transcript maps that are helping researchers decipher the independent and interconnected functions and variations of cellular proteins that result in particular phenotypes. Bovine ESTs that are sequenced, mapped, and compared to known genes and full-length cDNAs recorded in public databases are already being used by Project participants to develop bovine-specific microarray interrogation systems capable of monitoring thousands of gene expression changes in a variety of comparative experiments (e.g., Yao et al., 2001; Burton et al., 2001). Therefore, when combined with cDNA microarray technologies, existing bovine EST collections are powerful tools for discovering genes in livestock that associate with outcomes of various husbandry practices, genetic selection schemes, environmental extremes, nutritional regimes, reproductive status, and infection scenarios. These EST libraries and cDNA microarray technologies will be available to the proposed Project through its participants (see below).

However, if the activity or yield of gene transcription is not proportional to the functional activity of the encoded proteins, then knowledge about gene transcription events gained through functional genomics studies can be limited with regard to which proteins are actually responsible for altering a given trait (Matthews et al., 1998; 1999). This limitation is compounded if a given functional activity results from and (or) is regulated by multiple gene products (Lin et al., 2001; Jackson et al., 2001). Another limitation of stand-alone functional genomics approaches is that relatively little information is gathered about whether expressed proteins undergo post-translational modifications or where functional activity is localized. These limitations can be especially critical if achievement or alteration of a given trait results from expression of multiple proteins. Consequently, to fully understand the metabolic and regulatory status of many cells and tissues resulting in particular phenotypes, the ability to simultaneously identify and analyze the expression patterns of multiple proteins is required (Jensen et al., 1999). Current methods for separation and identification of proteins by 2-dimensional (2-D) electrophoresis, paralleled by development of mass spectrometric techniques sensitive enough to be applied to biological systems, have given rise to protein-based gene expression analysis (proteomics). As antibodies to these proteins are developed, microarray technology can be used for even more rapid screening of expressed proteins. When coupled with information from functional genomics analyses, proteomics contributes to a very comprehensive understanding of how and why particular phenotypes occur. Proteomics approaches to identifying and studying the structure, function, and expression of candidate genes affecting health, reproduction, production, and product quality in cattle will be used in the proposed Project (see below). Where appropriate, gene function studies will be supported using transgenesis and cloning technologies already in use by Project participants (see for example, Kerr et al., 2001).

Because of the far-reaching nature of the proposed gene discovery efforts that link actual genes to relevant chromosomal locations and protein variants that determine the health, reproduction, production, and product quality phenotypes of interest (Objectives 1 and 2 below), the proposed Project requires connected efforts of researchers from multiple disciplines and geographical locations. Multistate Research Projects are the perfect and necessary forums to foster such research efforts. The current proposal was developed in collaboration with ongoing efforts of participants from NC-209, NE-112, NCR-199, NRSP-8, and several members of the National Bovine Functional Genomics Consortium (see below), as well as several new investigators from AZ, KY, IL, MI, PA, and VT with keen interest in joining the Project (refer to Appendix E). Therefore, this proposal was developed from multistate and multidisciplinary perspectives and will require the support and connections that are fostered through Multistate Research Project activities. As with any large genomics/proteomics efforts, it will be increasingly important to communicate new information from this type of work to other interested animal researchers, industry representatives, government agencies, and the public at large. Objective 3 (see below) of the proposed Project will ensure that this is accomplished.

Related, Current and Previous Work

One approach to identifying, locating, cloning, and characterizing the huge number of genes that regulate economically important traits of farm animals is embodied in the mapping efforts of NRSP-8 and related Multistate Research Projects. A different, but complementary approach is to use the tools and techniques of functional genomics and proteomics to rapidly identify and characterize the tens or hundreds of genes and gene products that regulate economically important traits in agricultural animals. This can be accomplished by high throughput monitoring of tissue-specific messenger RNA (mRNA) and protein expression changes that occur in tissues under relevant production scenarios. These genomic, functional genomics, and proteomics approaches to gene identification and characterization in cattle are the ones proposed in the current Multistate Research Project proposal. Primary experimental models for mRNA and protein expression profiling by participants of the new Project (see below) represent some of the most pressing husbandry problems and opportunities for dairy and beef producers across the US. To achieve the unprecedented generation of cattle functional genomics and proteomics knowledge proposed in the current application, Multistate researchers participating in this Project must share resources, expertise, and outreach efforts. This resource sharing has already begun to take shape through highly successful collaborative research activities that developed out of the current NC-209 Multistate Research Project (10-1-96 to 9-30-02). Concrete examples of available shared resources, expertise, and outreach activities available for the newly proposed Multistate Research Project through participating Stations include the following.

Resources of The National Bovine Functional Genomics Consortium: Recently, multiple NC-209 researchers (AZ, CA, MI, USDA-BARC) and collaborating colleagues (Cornell, Idaho, Missouri, USDA-MARC) formed a National Consortium (the National Bovine Functional Genomics Consortium, or NBFGC) to initiate the development and application of comprehensive bovine functional genomics tools for the study of genes involved in milk quality and well being of dairy cows. USDA-IFAFS funds are being used by the NBFGC to complete five major outcomes that will directly benefit the research objectives of the currently proposed Multistate Research Project. These include:

(1) identification and clustering of existing bovine ESTs (~180,000) into ~18,000 unique genes;

(2) expansion of an existing bovine EST database to include these unique gene clusters (http://gowhite.ans.msu.edu);

(3) development and testing of a gene discovery cDNA microarray set made up of the ~18,000 unique genes, as well as of smaller sub-arrays composed of ~6,000 genes identified from the gene discovery work as important in the regulation of milk quality and well being;

(4) expansion of the bovine EST database (http://gowhite.ans.msu.edu) to accommodate gene expression annotations resulting from microarray experiments.

(5) development and maintenance of a web site (hosted by Michigan State University, Department of Animal Science) for dissemination of bovine genomics information resulting from NBFGC research efforts to the general public, to educate extension personnel, producers, consumers, university students, and school children in K-12.

The EST collections, cDNA microarrays, microarray equipment and expertise, and supporting bioinformatics resources developed by NBFGC will be available to the currently proposed Multistate Research Project through its participants.

Bovine Genetic Mapping Resources: Transcript maps of EST sequences and identification of SNPs within genomic counterparts of EST transcripts have been powerful methods for positional cloning and linkage mapping of genes. Current NC-209 and NBFGC researchers and their collaborators have played an integral part in developing and using these techniques. Participants of the currently proposed Multistate Research Project bring with them extraordinary bovine mapping resources in the forms of dairy and beef cattle resource populations (OH, MI, MN, USDA-BARC) and ordered BAC libraries (USDA-BARC) that help speed the positioning of EST-based markers on the bovine physical map. For example, a unique tool for investigating the regulation of expression of genes controlling growth in cattle has been developed at the Ohio Station. Lines of Angus cattle have been established by divergent selection for serum IGF-I concentration. Serum IGF-I concentrations differ significantly between the high and low lines such that calves with lower IGF-I exhibit more rapid postweaning weight gains. Currently, the high line averages 90 ng/mL more serum IGF-I than the low line (P < 0.01) in the spring replicate and 122 ng/mL more serum IGF-I than the low line (P <0.01) in the fall replicate of the selection experiment. Also, Michigan State University is currently developing an F2 intercross beef cattle resource population utilizing foundation animals from the Angus and Limousin breeds for identifying genomic regions and ultimately genes controlling economically important traits including growth and carcass merit. The Angus and Limousin breeds not only exhibit wide variation in muscling and carcass traits, but both breeds are also used extensively in commercial beef cattle populations, thus allowing QTL results from the experimental population to be applied in commercial populations. Current plans include production of 500 F2 offspring and collection of growth, carcass merit and meat quality phenotypic data. Tissues from a subset of F2 animals will also be collected for gene expression analyses. Furthermore, the University of Minnesota is in the process of constructing a F2 Holstein population based on crossing an unselected (since 1964) control line of Holstein cows with nationally selected sires. This F2 Holstein population will be used in the proposed project to identify QTL associated with health and production traits and to conduct functional genomics studies on these traits. The University of Minnesota is also developing a BTAY contig map for the euchromatic region, a BTAY-EST map (based on comparative human and mouse information), and is studying Y chromosome-specific expressed sequences. Finally, USDA-BARC will provide access to a unique resource in the form of the Cooperative Dairy DNA Repository (CDDR) and together with the University of Illinois, the Dairy Bull DNA Repository (DBDR), for fine mapping of genes. Each of these genetic mapping resources will be available to the participants of the proposed Project

Proteomics Expertise: Identification and understanding the expression and activities of protein products of genes in relevant tissues and isolated cells will be the ultimate foundation for understanding traits of interest to the US cattle industry. Participants of the proposed Multistate Research Project offer expertise in numerous proteomics technologies. For example, the University of Kentucky (a new member to this Project) will share equipment for sample concentration, 2-D electrophoresis, image capture and analysis, mass spectrometry, and associated data base-searching software for identification of proteins in the model systems for study during this Project (see below). In addition, Michigan State University offers fluorescence activated flow cytometry and cell sorting expertise and equipment, and has plans to develop limited in-house monoclonal and polyclonal antibody production capabilities. The cDNA microarray equipment offered through the Michigan Station is capable of expansion to protein spotting should funds become available to support such large-scale proteomics work during the proposed Project.

Model Systems of Cattle Health, Reproduction, Production, and Product Quality:

(1)Health: Three participating Stations of the proposed project (AZ, MI, VT) are studying genetics of cattle health using well defined in vivo models and in vitro cell culture systems. The University of Arizona is using NBFGC-developed cDNA microarrays and real time RT-PCR to study effects of heat stress on genes regulating immunity and mastitis susceptibility in heat-stressed dairy cows. The Arizona Station offers an additional unique resource in the form of a controlled environment complex capable of simulating conditions of desert regions, including temperature, humidity and solar radiation, and to simulate daily and seasonal cycles. Michigan State University is using a variety of techniques, including NBFGC-developed cDNA microarrays, DDRT-PCR, real time RT-PCR, gel shift assays, nuclear run on assays, DNA foot-printing assays, and flow cytometry to study genetic- and stress-induced susceptibility to three serious diseases in cattle: mastitis, bovine respiratory disease complex (BRDC), and Johnes disease. The animal model used to study mastitis susceptibility is the periparturient dairy cow. BRDC susceptibility is being studied using a well-characterized truck transport model of husbandry stress. Susceptibility to and pathogenesis of Johnes disease is being investigated using three separate models: experimental disease challenges of young dairy calves with known genotypes at potential candidate gene loci, natural disease outbreaks in chronically infected adult dairy cows, and cultured macrophage cells exposed to M. paratuberculosis. Participants from the University of Vermont have successfully employed transgene technologies (mouse and cattle) to study mastitis resistance genes in dairy cows. Together with participants from the Michigan Station, the Vermont Station will use transgenesis and cloning technologies during the proposed Project to continue studying the function of genes involved in mastitis resistance and susceptibility.

(2) Reproduction: Participants from the Michigan Station have developed a normalized bovine oocyte cDNA library that is ready for spotting on microarrays. These microarrays will be used by participants from the Michigan Station to study the functional genomics of fertility in dairy cows.

(3) Production & Product Quality: The University of Kentucky has a well developed model of metabolic capacity in the life cycle of forage-fed cattle, which focuses on identifying proteins that regulate glutamate transporter expression by intestinal epithelia and hepatic cells. The Kentucky Station also has developed a primary monolayer hepatocyte culture system, and characterized amino acid transport activity of a bovine immortalized cell line, to study the expression of bovine glutamate and glutamine transporters. Participants from the Arizona and California Stations have well-developed in vivo and in vitro model systems to study mammary development and milk quality in dairy cows, and participants from the Michigan and Ohio Stations have developing models and resource populations to study skeletal muscle development and growth in beef cattle. Therefore, ample expertise and tissue collection possibilities for multiple model systems of interest to the functional genomics and proteomics work of the proposed Project will be available through its participants.

Novel Study Designs: The Pennsylvania State University is developing new study designs to explore genetic variability for susceptibility to complex diseases in farm animals. The Pennsylvania Station plans to develop a dairy cattle genetic resource that has potential to provide basic information on the genetics of susceptibility to mastitis, metabolic diseases and reproductive disorders. Twenty full-sib (FS) families of elite Holstein dairy cattle with 30 daughters per family (total of 600 daughters) will be developed using multiple ovulation and embryo transfer technology. Simulation studies indicate that this FS design provides a statistical power >0.80 for detecting linkage to a disease locus that accounts for ~10% of the disease trait variation (i.e., QTL effect size ~0.45 s_p). At the QTL-fine mapping stage, these large families will also allow efficient testing for linkage disequilibrium (LD) or association between disease trait and marker loci using transmission disequilibrium test (TDT) and variance component approaches. This FS design offers several advantages in comparison to designs currently used to map genes for complex diseases in dairy cattle. Half-sib (HS) family designs such as the daughter and granddaughter designs require genotype and phenotype data in thousands of animals. F₂ intercross and backcross (BC) designs derived from crossing outbred lines are also powerful designs. However, these designs have two main limitations: (i) the QTL identified in the experimental population may have limited application in commercial populations; and (ii) the F₂ and BC designs are suited for studying only one trait (or group of related traits) per study design. The proposed FS design can be utilized in the study of several disease traits that may be segregating in this contemporary resource population and the results will be amenable for commercial application. Furthermore, these large FS families can be analyzed using powerful identical-by-descent (IBD) based methods of multipoint linkage/association analyses already available for biomedical researchers.

The proposed Project does not overlap with other related National or Multistate Research Projects, though it does complement several of these Projects. NRSP-8, for example, is a multi-species project devoted to the development and dissemination of genome research tools for species genome map development, comparative genome map development, and maintenance of the animal genome databases. NRSP-8 was not designed to study, apply, or educate the public about bovine genomics/proteomics research. Thus, while the efforts of NRSP-8 are critical for the successful development of genome mapping technology in cattle, and the genome mapping efforts of the NRSP-8 Bovine Technical Committee are complementary to the currently proposed project, there is no overlap between the objectives of NRSP-8 and the proposed Project. Indeed, the proposed Project adds significant value to the NRSP-8 Project, providing interpretation, application, and dissemination of bovine genome information through targeted model systems that are relevant to cattle production in the US. Several current NC-209 committee members that will participate in the new project are also members of NRSP-8, thus ensuring excellent exchange of results between the two Projects. Similarly, the new NC-210/220 Project complements but does not overlap with the currently proposed Project because it exclusively focuses on the genetics and functional genomics of pigs. The same is true of NE-60 and NC-168, which focus on the genetics and genomics of poultry. Other cattle Projects which complement but do not overlap with the currently proposed Project are S-284 (with an emphasis on health and fitness of cattle with no genomics or proteomics approaches being used), NE-112 (with an emphasis on mastitis resistance to enhance food safety with no genomics or proteomics approaches being used), and NCR-199 (with an emphasis on national cattle evaluation).

Objectives

1. Determine the location, structure, function and expression of genes affecting health, reproduction, production, and product quality in cattle.
   
2. Interpret and apply genomics and proteomics information by developing statistical/bioinformatics methods and utilizing molecular tools in cattle.
   
3. Develop and deliver educational materials about bovine genomics research to consumers and stakeholders.
   
4. 
   

Methods

Objective 1: Determine the location, structure, function and expression of genes affecting health, reproduction, production, and product quality in cattle.

While researchers are beginning to gain insight into the chromosomal locations that regulate important traits in cattle, much discovery work is left regarding the generation of practical genomics information that can be applied to genetic improvement (and other manipulations) of health, reproduction, production, and product quality traits in cattle. This work will involve finer mapping of the bovine genome, association studies to identify candidate genes, and aggressive exploration of the structure, function, and expression of relevant candidate genes. In collaboration with Project participants working under Objective 2 (below), a number of Stations will work together to determine the location, structure, function and expression of genes affecting health, reproduction, production, and product quality in cattle. For example, participants from USDA-BARC, IL, MI, MN, and PA will use current and expanding marker information from the CDDR, DBDR, and developing dairy and beef cattle resource populations to identify, map, and study associations of new genes with traits for mastitis, metabolic diseases, reproductive disorders, milk production, growth, and meat quality. Marker information will also be used for simulation studies and to fine map chromosomal regions already shown to be involved in health, reproduction, production, and product quality in cattle. Candidate genes identified in this Project through the functional genomics and proteomics efforts (see below) will also be contributed to these mapping efforts. Participants from AZ, CA, IA, MI, OH, PA, USDA-BARC, and VT continue to study the structure of numerous candidate genes of skeletal muscle growth, carcass and meat quality, milk production, milking rate, milk quality, digestion/nutrient utilization, female reproduction, stress susceptibility/adaptation, and susceptibility to three important bovine infectious diseases (mastitis, respiratory disease complex, and Johnes disease). Participants from many of these Stations are members of the NBFGC and have developed a wide variety of tissue-specific bovine EST libraries (skeletal muscle, embryo, digestive tract, mammary gland, oocyte, leukocytes), clustered EST collections representing > 18,000 unique bovine genes, resource populations, and livestock transgenic/cloning technologies to identify and study additional candidate genes. For example, at least 10 candidate genes already under study at these Stations have been subjected to extensive SNP analyses and are currently being assessed for associations with numerous phenotypes related to health, reproduction, production, and product quality traits in cattle (through the current NC-209 project and NBFGC efforts). These molecular genetic efforts will continue in the newly proposed Project, and will be substantially expanded to include new candidate genes provided through the mapping, cDNA microarray, and proteomics efforts. Existing and newly identified candidate genes also will be characterized in terms of intron/exon boundaries, possible occurrence of alternative splice variants, and the structure of key regulatory elements to provide valuable new information about how relevant allelic variants (or mutated sites) likely interplay to alter the expression of complex phenotypes under study. This information will contribute significantly to ongoing annotations of bovine genome maps and EST databases, including the NBFGC database housed and maintained at Michigan State University (http://gowhite.ans.msu.edu). Improving knowledge about the chromosomal location and molecular genetic structure of candidate genes of health, reproduction, production, and product quality in cattle accomplishes half the battle of providing practical information that can be applied to dairy and beef production on the farm. However, the other half of the battle, which is still in its infancy, is to know how the relevant genes actually function in various tissues that express and utilize the protein products of the genes. Participants from AZ, CA, IA, KY, MI, and VT have identified and will continue to study the functions and tissue expression of multiple candidate genes of stress and disease susceptibility, pathogenesis, immunity, reproduction, skeletal muscle and adipose tissue development, meat quality, gut-liver-kidney function, whole animal nitrogen metabolism, mammary tissue differentiation and growth, and mammary fatty acid metabolism. Facilitating this work is the ongoing development of cDNA microarrays by participants of the currently proposed Project. Microarray technology is an exploratory method for understanding biological pathways important in physiological processes. Multiple participants (AZ, CA, IA, MI, USDA-BARC) will collaborate on the continuing development and extensive utilization of gene discovery cDNA microarrays to generate significant preliminary gene expression data that will benefit all future gene expression profiling efforts in cattle. Each collaborating Station will also contribute RNA from bovine tissues collected from the animal models listed above to study health, reproduction, production, and product quality traits in cattle. In addition, several participants (KY, MI, VT) have begun to use proteomics and transgenesis/cloning technologies to explore the expression and biological function of several candidate genes of gut function, inflammation, and mammary health. These efforts will continue as a vital and new area of candidate gene exploration in the proposed Multistate Research Project. The results will indicate how the candidate genes actually function in various tissues that express and utilize the protein products of the genes. These outcomes complement the improved knowledge about the chromosomal location and molecular genetic structure of candidate genes of health, reproduction, production, and product quality in cattle and provide practical information that can be applied to dairy and beef production on the farm. Objective 2: Interpret and apply genomics and proteomics information by developing statistical/bioinformatics methods and utilizing molecular tools in cattle.

Statistical methods, experimental designs, and bioinformatics tools are critical to map quantitative trait loci (QTL), to interpret results from gene expression and proteomics studies, and to apply results of genome research in cattle breeding to optimize food production and resistance to complex production diseases. Statistical analysis of QTL mapping data not only is necessary to identify and narrow down the chromosomal regions that may contain genes or QTL, but also will affect the future research direction and costs in pinpointing the genes pursued in Objective 1. Since any small genetic distance may correspond to a large physical distance, any improvement in statistical accuracy for mapping analysis may result in significant savings in laboratory costs and research time for finding the relevant genes. Sound experimental designs are critical for structural and functional genomics studies in Objective 1 to ensure research effectiveness and gathering of genetics/genomics data of biological relevance. Likewise, studies on structure and function of genes in Objective 1 will allow the development of statistical methods and utilization of molecular tools in genetic improvement of cattle, as well as real or empirical data for developing sound experimental designs and methods of statistical analysis. To achieve the goals of Objective 2, experimental designs and methods of statistical analysis will be developed and evaluated. Current methods of QTL analysis mainly focus on QTL additive effects. Dominance effects exist widely in humans, experimental species, as well as in agricultural species. Likewise, gene interaction is a significant issue in species where a large number of genes have been found, such as in humans and mice. The number of known genes in farm animals is expected to increase rapidly and understanding gene interaction or epistasis in animal genetics and genome research will be important. New methods will be developed to detect QTL with dominance, epistatic and pleiotropic effects, to estimate the size of such non-additive effects, and to estimate the exact chromosomal locations of these QTL. Statistical methods and analytical tools also will be developed for map integration of markers and genes with non-Mendelian inheritance as well as for construction of sex-average and sex-specific maps. These studies will be carried out through participants from the IA and MN Stations. Furthermore, the use of markers such as SNPs permits the refinement of QTL location within small regions. The cattle population structure in the US, with large half-sib families and possible inbreeding, is problematic to fine mapping studies. Evaluation of current methods and the development of new methods of QTL fine mapping will be performed by participants of the IA, IL, and PA Stations. Collaboration with researchers participating in experiments of Objective 1 will enhance this component of the Project. New methodologies are also needed to analyze microarray experiments, since current methodologies have been developed and tested only in non-cattle systems. Furthermore, methods based on parametric and data-mining approaches need to be developed and compared to existing approaches. Participants from IL, MI, and PA will work with participants involved in studies under Objective 1 to develop appropriate designs for cDNA microarray and protein expression experiments and methodologies for subsequent data analyses. In addition, there is a dire need to develop and evaluate experimental designs with emphasis on identification of optimal designs for specific hypothesis testing and scenarios relevant to gene mapping, functional genomics, and proteomics studies. Participants of the IL, MI, MN, and PA Stations will work on solving these problems. Furthermore, it is essential that statistical methods are developed to control for false positive rates in QTL mapping, microarray data analysis, and cattle genomic analysis completed in Objective 1. Participants from the IA, IL, MI, and PA Stations will combine expertise to enhance this research initially developed at the IA Station. Each station will be responsible for evaluating and developing alternative methodologies consistent with its involvement with the other two Project objectives. Furthermore, statistical methods are needed whereby one can combine molecular, pedigree, and phenotypic information to enhance genetic improvement of cattle. Participants from the CA, IA, IL, MN, and PA Stations will develop, evaluate and enhance existing procedures or strategies to effectively incorporate molecular information into existing cattle breeding programs. Objective 3: Develop and deliver educational materials about bovine genomics research to consumers and stakeholders.

Dissemination of language-friendly, impact-full educational materials that are readily accessible by the consuming public and stakeholders will be critical for future acceptance and application of results from the proposed Multistate Research Project. This is especially true given the potential for a virtual explosion in new bovine genomics technologies and information that will result from this Project. With an outreach component firmly in place, the proposed Multistate Research Project will help people with vested, professional, and (or) societal interests in bovine genomics research to learn, form opinions and policies, and develop novel strategies and tools that will improve the health, reproduction, production, and product quality of dairy and beef cattle. Under Objective 3, Project participants will work to develop multiple educational materials (web site, posters, oral presentations, extension publications) for delivery to a wide variety of target audiences. The following outreach activities will occur during the proposed 5-year Project. Project participants from the MI Station who are also members of the NBFGC are developing a web site for use in public education about bovine functional genomics. This effort is being spear headed by Dr. T. Ferris, a member of the NBFGC who holds a dairy genetics extension position at Michigan State University. Therefore, it is proposed that the MI Station be the hub for development of a NC-1010 public education web site, with appropriate links to the developing NBFGC web site and EST database (http://gowhite.ans.msu.edu), as well as other bovine genome databases (http://sol.marc.usda.gov/genome/cattle/cattle.html; http://spinal.tag.csiro.au/cgd.html;http://www.hgsc.bcm.tmc.edu/bovine;http://locus.jouy.inra.fr), genome databases for other species, and other web sites of interest containing reliable genomics/proteomics information. The NC-1010 web site will also provide a platform for integration of cattle extension web sites at participating NC-1010 Stations. The main target audiences of the NC-1010 web site will be producers, veterinarians, university students, K-12 educators, consumers, and policy makers. However, participants of the proposed project are also interested in providing valuable educational pieces and links for professionals such as extension faculty and personnel, genomics and non-genomics research and teaching faculty, university administrators, and industry and government representatives. In this way the NC-1010 web site will provide not only education but also advertising of Project activities to strengthen participation from extension and research faculty other Stations and government/industry partners in the future. Dr. D. Bullock (KY station), who has recently joined the NC-1010 project (E-1 form to be sent to the current NC 209 Project Administrator in Feb., 2002), will be invaluable in facilitating development of such outreach activities. Dr. Bullock holds a beef genetics extension appointment at the University of Kentucky and serves on two national beef cattle improvement programs (the National Cattle Evaluation Consortium and the Beef Improvement Federation). Therefore, Dr. Bullocks participation will well connect NC-1010 with national extension efforts, especially as to the form and application of basic data and information that the Project will generate. Dr. Bullock will also communicate Project activities and results to these programs and, along with other Project participants, will extend invitations to the NC-1010 annual meetings to these and other key industry groups (e.g., NCBA, NAAB, AI companies, breed associations, Monsanto, Cargill, etc.). Another NC-1010 outreach activity will be held in conjunction with the annual Plant, Animal, and Microbe Genomes (PAMG) meetings in January 2005. This activity will be in the form of a hands-on Quantitative Genomics/Proteomics Workshop offered to PAMG participants, including members of other National Research Support and Multistate Research Projects. Advertising for the workshop will be done during the 2004 PAMG meetings and through PAMG announcements for the 2005 meeting. An assessment survey will be distributed to attendees at the end of the workshop so improvements can be made for the next series of outreach activities in year 2006 (see below). NC-1010 Project participants working under Objective 2 will be responsible for advertising, planning, and conducting this workshop (i.e., CA, IA, IL, MI, MN, PA Stations). Another series of outreach activities will occur at the annual joint meeting of the American Dairy Science Association and the American Association of Animal Scientists in summer 2006. These activities will occur in two formats. One will be a Multistate Research Project NC-1010 booth displaying extension-type bulletins (also to be available on CD) about the details and value of the cattle genomics research conducted under the NC-1010 Project. Bulletins highlighting the research activities of each participating Station will be available (to be written by participants from each Station) along with other written materials that summarize research findings to date and describe ways in which these findings may be applied in the US cattle industry. The 2006 NC-1010 Chair and Secretary will oversee drafting of these additional bulletins. At least two participants of the Project will man the booth to offer the reading materials displayed, answer questions about them, to help interested attendees locate and explore the Projects web site (on a lap top computer stationed at the booth), and to advertise an educational symposium on NC-1010 bovine functional genomics/proteomics research. Participants from at least four Stations working under Objectives 1 and 2 will deliver a series of brief lay presentations in this educational symposium on bovine functional genomics/proteomics. The goal of these presentations will be to help interested university administrators, research-teaching-extension faculty, graduate students, undergraduate students, industry representatives, and government officials better understand functional genomics/proteomics research in the context of improved health, reproduction, production, and product quality in cattle. The symposium will also highlight technological developments, research results, and applications of the genomics/proteomics research accomplished under Objectives 1 and 2 of the proposed Project. An assessment survey will be distributed to attendees of the symposium so improvements can be made for the next series of outreach activities in year 2007 (see below). A working group of Project participants will be assembled during the 2004 annual NC-1010 meeting and charged with advertising, planning, and conducting this series of outreach activities. Logical candidates for this outreach activity could include participants from the AZ, CA, KY, IA, IL, OH, MI, MN, PA, and VT Stations, and USDA-BARC. Advertisement about the booth and symposium will be done at the 2005 ADSA-AAAS and PAMG meetings and sent to other Multistate Research Project Administrators in 2005 to encourage contributions of similar educational materials from other species genome projects. A final outreach activity will occur at the annual meeting of the Conference of Research Workers in Animal Diseases (CRWAD) in November 2007. The target audience for these presentations is professionals in veterinary fields: clinicians, research-teaching-extension faculty, graduate students, university administrators, animal health industry representatives, and government representatives. CRWAD does an exceptional job of encouraging attendance at poster presentations by having no other concurrent sessions running when posters are on display. More than 600 CRWAD attendees typically view posters at this meeting. At least two posters will be presented, one that highlights the interconnected research performed under Objectives 1 and 2 and the other that demonstrates how the NC-1010 Project executed its outreach plan of Objective 3. A similar series of oral presentations (15-minutes each) will be delivered in the Immunology, Pathogenesis, Physiology, and Respiratory Diseases sections of the 2007 CRWAD program. A working group of Project participants will be assembled during the 2005 annual NC-1010 meeting and charged with advertising, planning, and conducting this series of outreach activities. Logical candidates for this outreach activity could be participants from AZ, KY, MI, and VT.

Measurement of Progress and Results

Outputs

• Objective 1: Identification & localization of candidate genes/markers associated with functional differences in health, reproduction, production, and product quality of cattle, including validation & refinement of QTL locations and enhanced genetic maps; Dissemination of functional cDNA microarrays to Project participants; Annotation of the publicly accessible MSU EST database with location, structure, function, and expression of identified candidate genes; Continuing development of unique genomic/proteomic resources and tools for future research and industry application; Provide well-defined and understood mechanisms that underlie key phenotypic traits related to health, reproduction, production and product quality in cattle; Multi-Station research publications on the location, structure, function, and expression of genes and proteins in various <I>in vivo</I> and <I>in vitro</I> models of health, reproduction, production, and product quality in dairy and beef cattle; and Identify a multi-State research project for extramural funding application.
• Objective 2: Public domain statistical and bioinformatics methodologies for the design and analysis of genomics/proteomics experiments, including computer programs; Recommendation of the statistical analysis and false positive control methods most suitable for different purposes (e.g. QTL detection, gene expression) and scenarios (e.g. data structure, magnitude of QTL effect); Support for the interpretation of results from the recommended statistical methods applied to cattle genomics/proteomics data; Advice on experimental designs adequate for a wide range of cattle genomic/proteomic studies; and Development, testing and implementation of strategies for using molecular information in applied cattle breeding programs.
• Outreach to a variety of target audiences through the Project web site and workshop/presentation activities at 3 International conferences.

Outcomes or Projected Impacts

• Identification of multiple candidate genes for susceptibility/resistance to mastitis, BRDC, Johnes disease, and metabolic diseases as targets of new, transferable drug discoveries.
• Identification of multiple candidate genes of reproductive efficiency as targets of novel, transferable management strategies.
• Annotation of existing EST databases with gene and protein structure, function, and expression data for the construction of metabolic pathways regulating health, reproduction, production, and product quality in cattle.
• Generation of state-of-the-art genomics, functional genomics, proteomics, reproductive, and bioinformatics technologies and resources to support future cattle research endeavors in multiple disciplines.
• Identification of the genetic and molecular bases of variation for development and health of the mammary gland, production and quality of milk, feed utilization, rate of growth, and meat quality for use in the genetic improvement of dairy and beef cattle.
• New opportunities to regulate expression of known and novel genes in tissues relevant to the health, reproduction, production, and product quality in cattle.
• New opportunities for the industry to improve cattle husbandry practices through unprecedented understanding of the biological systems involved in health, reproduction, production efficiency and product quality.
• New methodologies and technologies for the genetic improvement of cattle leading to improved disease resistance, conception rate, nutrient utilization, milk and meat production, and milk and meat quality.
• New opportunities to educate consumers and stakeholders in the processes, outcomes, values, and impacts of cattle genomics/proteomics research.
• Establishment of outreach practices that attract a wide variety of new researchers, teachers, extension specialists, industry representatives, and government workers to participate in Multistate Research Projects.

Milestones

(0):</b>: Objective 1 - Develop functional cDNA microarrays for distribution; Initiate cDNA microarray and proteomics experiments in two model systems each; Continued fine mapping using CDDR, DBDR, and other available resource populations</p> <p>Objective 2 - Microarray designs established; Establish methods to handle microarray false positives and data analysis; Establish statistical methods for mapping genes with non-Mendelian inheritance </p> <p> Objective 3 - Design and location of web site finalized

(0):</b>: Objective 1 - Establish gene & protein expression profiles in the 2003 model systems & initiate new experiments in at least two additional model systems each; SNP analysis and mapping of of ~ 50 candidate genes from model systems in 2003; Intron/exon boundary and promoter analyses of ~10 candidate genes from model systems studied in 2003 </p> <p> Objective 2 - Recommendations for optimal gene mapping and functional genomics study designs; Establish statistical methods for mapping QTL with non-additive effects</p> <p>Objective 3 - Web site contents fully developed

(0):</b>: Objective 1 - Establish gene and protein expression profiles in model systems studied in 2004; Identify proteins and genes expressed in model systems studied in 2004; SNP analysis and mapping of ~ 50 candidate genes from models systems of 2003-04</p> <p>Objective 2 - Establish statistical methods for combining molecular, pedigree, and phenotypic information in genetic improvement of cattle; Association testing of candidate genes for health/production traits; Begin to annotate EST database with new genomic/proteomic information. </p> <p>Objective 3 - Web site launched; and Outreach workshop at PAMG meeting.

(0):</b>: Objective 1 - SNP analysis and mapping of ~ 100 candidate genes from models studied in 2003-05; Intron/exon boundary and promoter analyses of ~ 20 candidate genes from 2003-05; Initiate antibody development for proteins identified in model systems studied.</p> <p>Objective 2 - Association testing of candidate genes for health/production traits; Annotate EST database with new genomic/proteomic information</p> <p>Objective 3 - Web site updated every 4 months; Outreach booth & symposium at ADSA-AAAS meeting

(0):</b>: Objective 1 - Continue development of antibodies for expressed genes and proteins in models studied; Intron/exon boundary and promoter analyses of ~ 20 new candidate genes found in model systems in 2003-06</p> <p>Objective 2 - Association testing of candidate genes for health/production traits; Annotate EST database with new genomic/proteomic information.</p> <P>Objective 3 - Outreach posters & oral presentations at CRWAD meeting; Summarize assessment data from outreach activities & publish results

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

Results and impacts of the research of this Project will be widely disseminated to the public through the outreach plan described in Objective 3. In addition, results will be disseminated to professional colleagues through scientific publications, articles in industry magazines, technical presentations at scientific and industry meetings, and through one-on-one personal contacts.

Organization/Governance

The membership of the regional Technical Committee will include the regional Administrative Adviser (non-voting), a representative from the Cooperative State Research, Education and Extension Service (non-voting), technical representatives from each participating State Agricultural Experiment Station (voting), and technical representatives from the USDA-ARS Beltsville Agricultural Research Center (voting). Each participating station/institute may have more than one representative on the Technical Committee, but each participating station/institute will be limited to one vote. All interested industry and government representatives are welcome to attend the annual meetings of NC-1010 but will not be included in the Technical Committee and cannot vote.

The Technical Committee shall elect two of its members annually to serve as Chairperson and Secretary. The two officers, along with the Administrative Adviser, will serve as an Executive Committee. The Chairperson of the Technical Committee will also serve as Chairperson of the Executive Committee. When necessary, the Executive Committee shall have the authority to act on behalf of the Technical Committee.

With the authorization of the Administrative Adviser, the Technical Committee, its Executive Committee, and its subcommittees will meet when necessary to coordinate, review, plan, and discuss research progress and outreach activities. A meeting of the full Technical Committee will be held annually to summarize and critically evaluate progress, coordinate the sharing of resources, analyze results, and plan future activities, reports, publications, and extramural grants. Annual meetings of the Technical Committee will be scheduled to occur in conjunction with annual PAMG meetings, or with other relevant meetings as appropriate. On years when the Technical Committee meeting is held in conjunction with PAMG, the Chairperson and secretary will organize the meeting. On years when the Technical Committee meeting is away from PAMG, volunteers from the Technical Committee will be sought to organize and host the meeting.

An annual report of research and outreach results shall be transmitted electronically by each cooperating station/institute to the Chairperson at least one week prior to the annual meetings. These reports will be compiled by the Chairperson into a consolidated report for dissemination at the annual meeting and approval by the Administrative Adviser. An appropriate number of copies of the consolidated report shall be transmitted to the Cooperative State Research, Education and Extension Service, USDA.

Literature Cited

Beckmann, J. Weissenbach, R. M. Myers, D. R. Cox, M. R. James, D. Bentley, P. Deloukas, E. S. Lander, and T. J. Hudson. 1997. Pieces of the puzzle:expressed sequence tags and the catalog of human genes. J. Mol. Med. 75:694.

Burton, J.L., P.S. D. Weber, J.B. Wells, S.A. Madsen, J. Yao, and P.M. Coussens. 2001. Immunogenomics approaches to understanding periparturient mastitis susceptibility in dairy cows. Acta Vet. Scand. 42(3)407-425.

Duggan, D.J., M. Bittner, Y. Chen, P. Meltzer, and J.M. Trent. 1999. Expression profiling using cDNA microarrays. Nature Genet. 21 (1,suppl.):10.

Jackson, M, W. Song, M-Y. Liu, L. Jin, M. Dykes-Hoberg, C-l. G. Lin, W. J. Bowers, H. J. Federoff, P. C. Sternweis, and J. D. Rothstein. 2001. Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins. Nature 410:89-93.

Jensen, O. N., M. Wilm, A. Shevchenko, and M. Mann. 1999. Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels. In: A. J. Link (Ed.) Methods in Molecular Biology: 2-D Proteome Analysis Protocols, vol. 112:p 513-530.

Kerr, D.E., K. Plaut, A.J. Bramley, C.M. Williamson, A.J. Lax, L. Moore, K.D. Wells, and R.J. Wall. 2001. Lysostaphin expression in mammary glands confers protection against Staphylococcal infections in transgenic mice. Nature Biotech. 19 (in press; http:/biotech.nature.com).

Lin, G. C-l, I. Orlov, A. M. Ruggiero, M. Dykes-Hoberg, A. Lee, M. Jackson, and J. D. Rothstein. 2001. Modulation of the neuronal glutamate transporter EAAC1 by the interacting protein GTRAP3-18. Nature 410:84-88.

Matthews, J. C., M. J. Beveridge, M. S. Malandro, J. D. Rothstein, M. Campbell-Thompson, J. Verlander, M. S. Kilberg, and D. A. Novak. 1998. Activity and protein localization of multiple glutamate transporters in gestation day 14 vs. day 20 rat placenta. Am. J. Physiol. 274:C603-C614.

Matthews, J. C., M. J. Beveridge, E. Dialynas, A. Bartke, M. S. Kilberg, and D. A. Novak. 1999. Placental cationic and anionic amino acid transporter expression in growth hormone overexpressing, null IGF-II or null IGF-1 receptor mice. Placenta 20:639-650.

Yao, J., J.L. Burton, P. Saama, S. Sipkovsky, and P.M. Coussens. Generation of EST and cDNA Microarray Resources for the Study of Bovine Immunobiology. Acta Vet. Scand. 42 (3):391-406.

Attachments

Land Grant Participating States/Institutions

AR, AZ, CA, DE, IA, IL, KY, MA, MI, MN, NC, NY, OH, SD, TN, TX, VT, WI

Non Land Grant Participating States/Institutions

USDA-ARS, USDA/ARS