Progress 09/27/01 to 09/26/06
Outputs
Progress Report 4d Progress report. This termination report documents research conducted under a Specific Cooperative Agreement with Auburn University. Additional details can be found in the report for the inhouse project, 6402-31000-008-00D, Catfish Genetics, Breeding, and Physiology. The collaborative project has had several objectives. The very first was to identify DNA markers within genes of known functions, particularly those already mapped on the human and zebrafish linkage maps, are valuable for mapping the catfish genome because they provide landmarks for comparative genome mapping. The second was to characterize certain innate immune-related genes, in particular the CXCL8 gene that appear to have multiple versions of cDNAs that may be exploited as a marker for mapping of this gene. Third, after initial development of genetic linkage maps using various resource families, it is important to integrate the maps using the same resource DNA. For the first
objective, we took the approach of intron tagging. By amplification and sequence analysis of introns of known genes, polymorphic markers can be identified because intron sequences are highly variable and often contain microsatellites. On the basis of progress made in previous years, in the last year, we have completed the objective. We designed primers for 50 genes to amplify their introns. Of the 50 pairs of primers designed, 42 produced products. These PCR segments were sequenced. Sequence analysis of the 42 introns indicated that 8 (19%) included microsatellite sequences in them (Table 1). The sequences have been deposited to GenBank available for public use and their accession numbers are included in the publication. The information was transferred to the laboratory whose personnel conducted mapping analysis of the polymorphic markers. This work was published in Animal Genetics (Serapion et al., 2004). Interleukin-8 is a CXC type chemokine produced in response to
stimulation by pro-inflammatory cytokines or bacterial lipopolysaccharides. We cloned and characterized interleukin-8 cDNAs and its genomic segments containing all the exons and introns from channel catfish (Ictalurus punctatus). Multiple interleukin-8 cDNA clones were identified during analysis of expressed sequence tags (ESTs). Sequence analysis indicated presence of four types of alternatively spliced and two types of alternatively polyadenylated transcripts. Analysis of the genomic DNA of the locus confirmed that all four types of transcripts were likely derived from partial splicing. Channel catfish interleukin-8 gene has four exons and three introns with highly conserved splice sites as compared to interleukin-8 genes of other organisms. In spite of the structural conservation through evolution, the piscine interleukin-8 genes showed a much greater sequence divergence than their counterparts among mammals. RT-PCR indicated that two of the four splicing forms were
expressed at high levels whereas the other two aberrant splicing forms were not detectable. Expression of interleukin-8 gene was up-regulated in channel catfish and blue catfish after infection with pathogenic bacteria Edwardsiella ictaluri. This work was published in Developmental and Comparative Immunology (Chen et al., 2005). After consulting with CGRU scientist, an expanded objective was initiated: Integration of various linkage maps by using the same resource markers. In the last several months, experiments have been initiated to genotype microsatellite markers that were mapped using the channel catfish resource families. In this case, genotyping was conducted using PCR with channel catfish x blue catfish interspecific resource DNA. Several points are important for this objective: 1) integration of existing genetic maps will enhance the resolution of the linkage maps; and 2) use of the same set of microsatellite primers in different resource families will allow comparison
of the mapping populations and their impact on genetic distances, particularly between the intraspecific and interspecific families. To date, almost 50 microsatellite primer pairs have been tested and genotyped with 64 fish from an interspecific family.
Impacts
(N/A)
Publications
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report documents research conducted under Specific Cooperative Agreement with Auburn University. Additional details can be found in the report for the parent project 6402-31000-008-00D, Catfish Genetics, Breeding, and Physiology. The collaborative project has had several objectives. The very first was to identify DNA markers within genes of known functions, particularly those already mapped on the human and zebrafish linkage maps, are valuable for mapping the catfish genome because they provide landmarks for comparative genome mapping. The second was to characterize certain innate immune-related genes, in particular the CXCL8 gene that appear to have multiple versions of cDNAs that may be exploited as a marker for mapping of this gene. Third, after initial development of genetic linkage maps using various resource families, it is important to integrate the maps using the same resource DNA. For the first objective, we took the approach of
intron tagging. By amplification and sequence analysis of introns of known genes, polymorphic markers can be identified because intron sequences are highly variable and often contain microsatellites. On the basis of progress made in previous years, in the last year, we have completed the objective. We designed primers for 50 genes to amplify their introns. Of the 50 pairs of primers designed, 42 produced products. These PCR segments were sequenced. Sequence analysis of the 42 introns indicated that 8 (19%) included microsatellite sequences in them (Table 1). The sequences have been deposited to GenBank available for public use and their accession numbers are included in the publication. The information was transferred to the laboratory of Catfish Genetics Research Unit whose personnel conducted mapping analysis of the polymorphic markers. This work was published in Animal Genetics (Serapion et al., 2004). Interleukin-8 is a CXC type chemokine produced in response to
stimulation by pro- inflammatory cytokines or bacterial lipopolysaccharides. We cloned and characterized interleukin-8 cDNAs and its genomic segments containing all the exons and introns from channel catfish (Ictalurus punctatus). Multiple interleukin-8 cDNA clones were identified during analysis of expressed sequence tags (ESTs). Sequence analysis indicated presence of four types of alternatively spliced and two types of alternatively polyadenylated transcripts. Analysis of the genomic DNA of the locus confirmed that all four types of transcripts were likely derived from partial splicing. Channel catfish interleukin-8 gene has four exons and three introns with highly conserved splice sites as compared to interleukin-8 genes of other organisms. In spite of the structural conservation through evolution, the piscine interleukin-8 genes showed a much greater sequence divergence than their counterparts among mammals. RT-PCR indicated that two of the four splicing forms were expressed
at high levels whereas the other two aberrant splicing forms were not detectable. Expression of interleukin-8 gene was up-regulated in channel catfish and blue catfish after infection with pathogenic bacteria Edwardsiella ictaluri. This work was published in Developmental and Comparative Immunology (Chen et al., 2005). We initiated an expanded objective: Integration of various linkage maps by using the same resource markers. In the last several months, experiments have been initiated to genotype microsatellite markers that were mapped using the channel catfish resource families. In this case, genotyping was conducted using PCR with channel catfish x blue catfish interspecific resource DNA. Several points are important for this objective: 1) integration of existing genetic maps will enhance the resolution of the linkage maps; and 2) use of the same set of microsatellite primers in different resource families will allow comparison of the mapping populations and their impact on
genetic distances, particularly between the intraspecific and interspecific families. To date, almost 50 microsatellite primer pairs have been tested and genotyped with 64 fish from an interspecific family.
Impacts
(N/A)
Publications
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? D. Progress Report. This report documents research conducted under Specific Cooperative Agreement 6402-31000-006-06S, "Channel Catfish Molecular Markers," with Auburn University. Additional details can be found in the report for the parent project 6402-31000-006-00D, "Catfish Genetics, Breeding, and Physiology". DNA markers within genes of known functions, particularly those already mapped on the human and zebrafish linkage maps, are valuable for mapping the catfish genome because they provide landmarks for comparative genome mapping. However, they are often more difficult to develop. In this project, we took the approach of "intron tagging". By amplification and sequence analysis of introns of known genes, polymorphic markers can be identified because intron sequences are highly variable and often contain microsatellites. Sequencing 42 introns produced eight microsatellites that are associated
with the selected genes (Table 1) Thus, intron sequencing can be used to develop type I markers in catfish with a success rate of about 19%. While the efficiency was low, this approach may be useful for targeted microsatellite development within known genes. Because a large number of ESTs are available from catfish, large numbers of gene-derived type I markers could be developed with the assistance of intron-prediction software. Since last report a year ago, we have further characterized the microsatellite loci. Specifically, all the PCR conditions were tested for genotyping. Polymorphisms of the intron-contained microsatellites were analyzed by determination of their heterozygosities by genotyping 33-37 individuals using outbred catfish from commercial populations. The number of alleles and observed heterozygosities are summarized in Table 2. Of the eight gene-associated microsatellites, IpSTS00006 failed to produce scorable PCR products and was eliminated from further
analysis. Genotypes from the seven remaining gene- associated microsatellite loci were obtained from full-sib reference families 1 and 2, and linkage was determined using CRI-MAP. Six of the seven genes mapped to various linkage groups (Table 3) while IpSTS00008 was not polymorphic in either family. In addition to characterization of these microsatellite loci, we have cloned, sequenced, and characterized the chemokine gene for interleukin-8 from channel catfish. Chemokines are important mediators for innate immunity involved in recruitment, activation and adhesion of a variety of leukocyte types to inflammatory foci. While almost all chemokines have been identified from mammals, only a handful of fish chemokines have been identified. The catfish interleukin-8 gene has two alternatively spliced transcripts encoding 114 and 111 amino acids, respectively. The gene has four exons and three introns, typical of the CXC chemokine gene organization. In spite of the structural
conservation through evolution, the piscine interleukin-8 genes showed a much greater sequence divergence than their counterparts among mammals. RT-PCR indicated that both spliced forms were expressed. Expression of the IL-8 like gene was up-regulated 3-5 fold in channel catfish and blue catfish after infection with pathogenic bacteria Edwardsiella ictaluri.
Impacts
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Publications
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