IDENTIFICATION AND STUDY OF GENES AFFECTING ECONOMICALLY IMPORTANT TRAITS IN DOMESTIC AND BIOMEDICAL ANIMALS
Melissa S. Ashwell
My research program is focused on identifying and understanding genes affecting economically important traits in dairy cattle and swine. Our approach involves genetic and genomic methods as the primary tools for our studies. We are currently utilizing DNA marker genotyping for quantitative trait loci (QTL) detection and fine-mapping studies, DNA microarrays, and Serial Analysis of Gene Expression (SAGE) libraries. Once the genes involved in these processes have been identified, additional genetic, molecular biological and biochemical approaches will be applied to characterize the mechanisms involved in these complex processes.
DESCRIPTION OF ONGOING RESEARCH
Detection of quantitative trait loci affecting economically important traits in dairy cattle. Traditional genetic selection methods have been effective in improving milk production in dairy cattle without the need for DNA marker information. The same is not true for lowly heritable traits that are expressed later in age, such as health and reproduction, which are of utmost importance to US producers. Fertility of dairy cows has gotten more and more attention in the last decade. During the past twenty years, substantial increases in milk, milk fat, and milk protein yields have been realized, but with those increases have come an unfavorable increase in the interval from calving to conception. Other factors, such as disease status and energy balance, also contribute to the decrease in reproductive efficiency. Detection of genes affecting economically important traits, such as reproduction, disease resistance and body conformation, would allow producers the opportunity to improve these traits while maintaining milk production levels. The long-term goals of this project are to identify genes associated with these traits that can be applied in marker-assisted selection programs. We are accomplishing this goal through detection and fine-mapping of previously identified quantitative trait loci (QTL). Currently we are fine-mapping QTL on chromosomes 15 and 18 that affect daughter pregnancy rate, somatic cell score (an indirect measure of mastitis) and udder conformation traits. This project involves the genotyping of genetic markers in large, complex multi-generation Holstein families. Use of complex families increases the statistical power to detect and fine-map QTL. We are currently building haplotypes of the sires’ gametes and will then analyze them with the phenotypes to narrow the confidence intervals for each of the QTL. Once the QTL regions are narrowed to approximately 10 cM, the bovine genome sequence will be interrogated to identify positional candidate genes. Any identified gene will be sequenced to scan for mutations that may be responsible for the observed effects on these economically important traits.
Transcript profiling of mice divergently selected for high and low body fat at fixed body weight. Mouse lines have been used to identify the genetic basis of growth and fatness traits and serve as models for diabetes and obesity in humans and genetic improvement in agriculturally important animals such as pigs. Research with different lines of mice has led to the identification of mutations in genes (i.e., leptin, leptin receptor) that have major effects on fat and lean accretion but do not explain much of the obesity observed in humans. Mice selected for high and low body fat while maintaining constant body weight would be advantageous to dissect out the genes only affecting fat deposition. The overall goal in animal production is to select animals for fast, lean growth. Therefore, it would be advantageous to identify and control the genes specific to fat accumulation while maintaining fast growth.
Two divergently selected lines of mice were developed, the LF (Low Fat) line and the HE (High Epididymal fat) line. The LF line was selected for low epididymal fat pad weight as a percentage of body weight at 12 weeks of age. The HE mice were selected using a restricted index to increase epididymal fat weight while holding body weight unchanged at 12 weeks of age. Male mice of similar age and weights were used for this project. The right epididymal fat pad, one liver lobe and the right gastrocnemius muscle were collected and RNA extracted. Microarrays and real-time PCR are being used to identify and confirm genes that are differentially expressed in the two mouse lines that may shed more light on the genetic basis of fat and lean accretion.
Transcript profiling and sequencing of genes involved in osteoarthritis using an in vitro porcine injury model. Osteoarthritis results when the cartilage layer covering the bony surfaces of a joint begins to degrade. In the US alone, more than 21 million people suffer from this disease. While the final stages of the degenerative process are quite well documented, there is very little known about the early tissue changes that start the cartilage down the degenerative pathway. Since early tissue changes can not easily be observed in human cases, animal models provide an important tool to study the early phenomena that initiate the degeneration leading to osteoarthritis. Since most cases of osteoarthritis are idiopathic in nature, it is difficult to find a way to reproducibly initiate the disease process. Joint injury, however, is a known predisposing factor for this condition making injury models one of the more useful tools in osteoarthritis research. If we can gain a better understanding of the early degenerative changes that precede full blown osteoarthritis we will be better able to treat the disease and prevent the debilitating changes that occur further down the road. One step in this research is to determine changes in gene expression that initiate the degenerative processes leading to diseases such as osteoarthritis. Differentially expressed genes are being identified through production of SAGE libraries. Serial Analysis of Gene Expression (SAGE), is a powerful technique to determine gene expression without previous knowledge of the genes to be identified, allowing us to also identify potential novel transcripts in porcine cartilage. To date two libraries have been produced—one from impacted tissue and a second from non-impacted tissue. Sequencing of the libraries is ongoing and has already identified differentially expressed genes. Future research will include real-time PCR validation of these genes and evaluation of their expression levels under different treatment conditions, such as shear forces.
RECENT PUBLICATIONS (since 2000)
Sonstegard, T.S., Garrett, W.M., Ashwell, M.S., Bennett, G.L., Kappes, S.M. and Van Tassell, C.P. (2000) Comparative map alignment of BTA27 and HSA4 and 8 to identify conserved segments of genome containing fat deposition QTL. Mammalian Genome11: 682-688.
Van Tassell, C.P., Ashwell, M.S. and Sonstegard, T.S. (2000) Detection of putative loci affecting milk, health, and conformation traits in a US Holstein population using 105 microsatellite markers. Journal of Dairy Science 83: 865-1872.
Ashwell, M.S., Ashwell, C.M., Garrett, W.M. and Bennett, G.L. (2001) Isolation, characterization and mapping of the bovine signal peptidase subunit 18 gene. Animal Genetics 32: 232.
Ashwell, M.S., Van Tassell, C.P. and Sonstegard, T.S. (2001) A genome scan to identify quantitative trait loci affecting economically important traits in a US Holstein population. Journal of Dairy Science 84: 2535-2542.
Sonstegard, T.S., Van Tassell, C.P. and Ashwell, M.S. (2001) Dairy cattle genomics: tools to accelerate genetic improvement. Journal of Animal Science 79: E307-315.
Ashwell, M.S., Sonstegard, T.S., Kata, S. and Womack, J.E. (2002) A radiation hybrid map of bovine chromosome 27. Animal Genetics 33: 75-76.
Connor, E.E., Ashwell, M.S. and Dahl, G.E. (2002) Characterization and expression of the bovine growth hormone-releasing hormone (GHRH) receptor. Domestic Animal Endocrinology. 22:189-200.
Sadler, R.S., Anderson, M.A. and Ashwell, M.S. (2002) Isolation of DNA from buccal and vaginal samples using the BuccalAmp kit. Epicentre Forum 9: 13.
Van Tassell, C.P., Sonstegard, T.S., Kappes, S.M., Ashwell, M.S. and Connor, E.E. (2002) Genetics, Cattle Genomics. In: Roginski, H., Fuquay, J.W. and Fox, P.F. (eds) Encyclopedia of Dairy Science. Elsevier Science, New York, vol. 2: 1219-1224.
Ashwell, M.S., Schnabel, R.D., Sonstegard, T.S., and Van Tassell, C.P. (2002) Fine-mapping of QTL affecting protein percent and fat percent on BTA6 in a popular U.S. Holstein family. World Congress of Genetics Applied to Livestock Production Proceedings. 31:123-126.
Ashwell, M.S. (2002) Genomic tools and techniques used in livestock species. American College of Veterinary Internal Medicine Forum Proceedings. 20:261-263.
Ashwell, M.S., Sonstegard, T.S. and Van Tassell, C.P. (2002) Mapping genes related to disease resistance and milk production in Holsteins. American College of Veterinary Internal Medicine Forum Proceedings. 20:264-266.
Vallejo, R.L., Li, Y.L., Rogers, G.W. and Ashwell, M.S. (2003) Genetic diversity and background linkage disequilibrium in North American Holstein cattle population. Journal of Dairy Science. 86: 4137-4147.
Van Tassell, C.P., Sonstegard, T.S. and Ashwell, M.S. (2004) Mapping quantitative trait loci for dairy form in regions of chromosome 27 in two families of Holstein. Journal of Dairy Science. 87: 450-457.
Ashwell, M.S., Heyen, D.W., Sonstegard, T.S., Van Tassell, C.P., Da, Y. VanRaden, P.M., Ron, M. Weller, J.I. and Lewin, H.A. (2004) Detection of quantitative trait loci affecting female fertility and milk production in ten Dairy Bull DNA Repository families. Journal of Dairy Science. 87: 468-475.
Connor, E.E., Sonstegard, T.S., Keele, J.W., Bennett, G.L., Williams, J.L., Papworth, R., Van Tassell, C.P. and Ashwell, M.S. (2004) Physical and linkage mapping of mammary-derived expressed sequence tags in cattle. Genomics. 83: 148-152.
Connor, E.E., Sonstegard, T.S., Ashwell, M.S., Bennett, G.L. and Williams, J.L. (2004) An improved comparative map of bovine chromosome 27 targeting dairy form QTL regions. Animal Genetics. 35: 265-269.
Schnabel, R. D. , Kim, J.-J., Ashwell, M.S., Sonstegard, T.S., Van Tassell, C.P. Connor, E.E. and Taylor, J.F. (2005) Fine-mapping milk production quantitative trait loci on BTA6: Analysis of the bovine osteopontin gene. Proceedings of the National Academy of Sciences of the USA. 102: 6896-6901.
Schnabel, R.D., Sonstegard, T.S., Taylor, J.F. and Ashwell, M.S. (2005) Whole genome scan to detect QTL for milk production, conformation, fertility and functional traits in two U.S. Holstein families. Animal Genetics. 36:408-416.
Ashwell, M.S., Heyen, D.W., Weller, J.I., Ron, M., Sonstegard, T.S., Van Tassell, C.P., and Lewin, H.A. (2005) Detection of quantitative trait loci influencing conformation traits and calving ease in Holstein-Friesian cattle. Journal of Dairy Science. 88: 4111-4119.
Connor, E.E., Ashwell, M.S., Schnabel, R., and Williams, J.L. (2006) Comparative mapping of bovine chromosome 27 with human chromosome 8 near a dairy form QTL in cattle. Cytogenetics and Genome Research. 112:98-102.
Muncie, S.A., Cassady, J.P., and Ashwell, M.S. (2006) Refinement of quantitative trait loci on bovine chromosome 18 affecting health and reproduction in US Holsteins. Animal Genetics. 37:273-275.
Blowe, C.D. Boyett, K.E., Ashwell, M.S., Eisen, E.J., Robison, O.W., and Cassady, J.P. (2006) Characterization of ESR, RBP4, and follistatin in a line of pigs previously selected for increased litter size. Journal of Animal Science, submitted.