Source: UNIVERSITY OF MARYLAND submitted to
GENOMIC RESOURCES FOR EXAMINING DISEASE RESISTANCE IN RAINBOW TROUT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0193839
Grant No.
2003-35205-12820
Project No.
MDR-2002-03472
Proposal No.
2002-03472
Multistate No.
(N/A)
Program Code
43.1
Project Start Date
Nov 15, 2002
Project End Date
Nov 14, 2005
Grant Year
2003
Project Director
Hansen, J. D.
Recipient Organization
UNIVERSITY OF MARYLAND
(N/A)
BALTIMORE,MD 21201
Performing Department
CENTER OF MARINE BIOTECHNOLOGY
Non Technical Summary
Salmonid species are plagued by a variety of viral and bacterial pathogens leading to acute and chronic disease situations that hamper the full potential of the US salmonid aquaculture industry. Despite considerable research aimed at protecting fish from pathogens, very little is actually known about the molecular genetic mechanisms dictating resistance or susceptibility of salmonid fish to disease. A major goal of our research is to elucidate the genomic content and organization of the rainbow trout major histocompatibility genes as an initial assessment for their role in disease resistance. This region has long been considered as the gateway to immune responsiveness. We will characterize and map the major histocompatibility complex (MHC) gene families from homozygous, diploid lines of trout in an effort to design genotyping methods that are suitable for identifying trout that are resistant or susceptible to pathogenic infection. Through this effort, we will completely sequence the MHC class I and II regions from homozygous trout representing for the first time the genomic sequence for this important genetic area in an agriculturally significant species. In our second aim we will develop focused immuno-microarray technology that can be used for the identification of molecular pathways and candidate genes that are relevant to fish health and the production of effective vaccine technologies. Finally, we will develop of a sensitive screening tool for our two large genomic insert libraries (BAC) that will be used for the rainbow trout genomic mapping project .
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3040810108050%
3110810109050%
Knowledge Area
311 - Animal Diseases; 304 - Animal Genome;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1080 - Genetics; 1090 - Immunology;
Goals / Objectives
Sequencing of the MHC class I and II genomic regions from homozygous lines of rainbow trout. Development of focused immuno-microarrays for assessing gene transcription profiles during infection. Development of PCR-based superpools for screening the Swanson and OSU-142 homozygous BAC DNA libraries
Project Methods
BAC contigs spanning the MHC class I and II genomic regions from the OSU-142 and Swanson homozygous lines of rainbow trout have been developed. Shotgun DNA libraries will be constructed from the individual BAC clones and completely sequenced at level ensuring complete coverage for each BAC clone. DNA contigs will be assembled using the Phred/Phrap/Consed system and the assembled contigs will then be completely annotated. For the DNA microarrays, differentially expressed genes from acute viral infections will be isolated using PCR suppression hybridization. Target sequences (long oligos) will be printed on glass slides using an automated arrayer. Microarrays will then be hybridized with Cy-3/Cy-5 cDNA probes from experimental and control tissues for transcriptional profiling. Finally, PCR superpools will be developed using automated technology for the rapid screening of rainbow trout BAC DNA libraries.

Progress 10/01/02 to 09/30/03

Outputs
Major histocompatibility class I and II molecules play an essential role in managing and eliminating pathogens by presenting self and foreign peptides (viral, bacterial) to the cellular arm of the immune system. Class IA molecules are expressed on all nucleated cells providing protection against endogenous pathogens, whereas class II proteins are only found on professional antigen presenting cells. If the protein fragment is deemed to be foreign, the cellular arm of the immune system (T-cells) will either directly kill the infected cell and/or direct a more potent antibody response against the pathogen. The human major histocompatibility complex (MHC) is the most gene-dense, polymorphic region in the genome encoding over 220 genes within a relatively short region (4 million bp) on chromosome 6. Our lab and others have previously demonstrated that bony fish differ from all other vertebrate classes in that the MHC class I and II genes are not linked. Recently we have made an important discovery in that rainbow trout possess a duplicated block of MH class I pathway genes that are found on two separate trout chromosomes (Chrs. 14q and 18q). The class I pathway is critical for alerting killer T-cells that a host cell is infected. We then found that TAP1 (ABCB2: transporter associated with processed antigen), a class I pathway member, is not found on either chromosome 14 or 18, but instead is located on the telomeric end chromosome 3p. Finally, we were able to determine that the class IIA and B genes, which are responsible for presenting peptides to helper T-cells, are map to centromeric arm of chromosome 17q. In short, this study determined that the trout MH genes are located on at least four different. The above study laid the groundwork for completely sequencing the MH regions in trout. To this end, we have assembled three sets of overlapping BAC clones that span the two class I regions and the region where TAP1 resides. BAC clones are DNA vectors that are capable of accommodating large pieces of genomic DNA for sequencing portions or entire chromosomes. We have completely sequenced and annotated one of these clones (BAC 24), which spans the core of the class IA region on Chr. 18. In addition we have sequenced over 1,200,000 bp of raw sequence reads for a second BAC clone (BAC 11) that partially overlaps BAC clone 24. BAC clones 24 and 11 span roughly 200,000 bp of continuous genomic DNA. The class IB region is also being sequenced. A thorough analysis of the class IB region is essential as class IB genes are implicated in the activation of natural killer cells (NK cells). NKs are likely the first responders to viral infection. Finally, we have also coordinated the construction of a new screening system for the Swanson BAC genomic library. This PCR-based super pool system has been used with good success to identify genetic markers associated with growth (production) and disease resistance and has been distributed to three other salmonid research laboratories in the US.

Impacts
Detailed information regarding the genomic composition of the major histocompatility regions in rainbow trout will aid in our of host-pathogen interactions in salmonids. By defining these chromosomal regions and variations of the genes themselves (including how they are regulated), salmon researchers will be able to use this information for the development of selective breeding programs for the US aquaculture and sport fishing industries.

Publications

  • Phillips, R., Zimmerman, A., Noakes, A.M., Palti, Y., Morasch, M.R.W., Eiben, L., Ristow, S.S., Thorgaard, G.H. and Hansen, J.D. 2003. Physical and genetic mapping of the rainbow trout major histocompatibility regions: evidence for duplication of the class I region. Immunogenetics. 55:561-569.
  • Landis, E.L. and Hansen, J.D. 2004. Annotation of two tapasin promoters from rainbow trout. In press-Marine Biotechnolgy.
  • Palti, Y., Gahr, S.A, Hansen, J.D and Rexroad , C.E. Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication. 2004. In press-Animal Genetics.