Rex A. Dunham

Inheritance and usefulness of AFLP markers in channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and their F1, F2, and backcross hybrids.

Abstract Eight primer combinations were used to investigate the application of amplified fragment length polymorphism (AFLP) markers in catfish for genetic analysis. Intraspecific polymorphism was low among channel catfish or blue catfish strains. Interspecific AFLP polymorphism was high between the channel catfish and blue catfish. Each primer combination generated from 70 to more than 200 bands, of which 38.6–75.7% were polymorphic between channel catfish and blue catfish. On average, more than 20 polymorphic bands per primer combination were produced as quality markers suitable for genetic analysis. All AFLP markers were transmitted into channel catfish × blue catfish F1 hybrids, except rare markers that were heterozygous in the parents and therefore were segregating in F1 hybrids. The two reciprocal channel catfish × blue catfish F1 hybrids (channel catfish female × blue catfish male; blue catfish female × channel catfish male) produced identical AFLP profiles. The AFLP markers were inherited and segregated in expected Mendelian ratios. At two loci, E8-b9 and E8-b2, markers were found at significantly lower frequencies than expected with F2 and backcross hybrids which had been selected for increased growth rates. The reproducibility of AFLP was excellent. These characteristics of the catfish AFLP markers make them highly useful for genetic analysis of catfish, especially for construction of genetic linkage and quantitative trait loci maps, and for marker-assisted selection.

Transcriptome analysis of channel catfish (Ictalurus punctatus): genes and expression profile from the brain

Expressed sequence tag (EST) analysis was conducted using a complementary DNA (cDNA) library made from the brain mRNA of channel catfish (Ictalurus punctatus). As part of our transcriptome analysis in catfish to develop molecular reagents for comparative functional genomics, here we report analysis of 1201 brain cDNA clones. Of the 1201 clones, 595 clones (49.5%) were identified as known genes by BLAST searches and 606 clones (50.5%) as unknown genes. The 595 clones of known gene products represent transcripts of 251 genes. These known genes were categorized into 15 groups according to their biological functions. The largest group of known genes was the genes involved in translational machinery (21.4%) followed by mitochondrial genes (6.2%), structural genes (3.1%), genes homologous to sequences of unknown functions (2.3%), enzymes (2.7%), hormone and regulatory proteins (2.5%), genes involved in immune systems (2.1%), genes involved in sorting, transport, and metal metabolism (1.8%), transcriptional factors and DNA repair proteins (1.6%), proto-oncogenes (1.2%), lipid binding proteins (1.2%), stress-induced genes (0.7%), genes homologous to human genes involved in mental diseases (0.6%), and development or differentiation-related genes (0.3%). The number of genes represented by the 606 clones of unknown genes is not known at present, but the high percentage of clones showing no homology to any known genes in the GenBank databases may indicate that a great number of novel genes exist in teleost brain.

Expression Profile of the Channel Catfish Spleen : Analysis of Genes Involved in Immune Functions

Both qualitative and quantitative patterns of tissue-specific gene expression can be determined using gene profiling. Expressed sequence tag (EST) analysis is an efficient approach not only for gene discovery and examining gene expression, but also for development of molecular resources useful for functional genomics. As part of an ongoing transcriptome analysis of channel catfish (Ictalurus punctatus), EST analysis was conducted for gene annotations and profiling using a complementary DNA library developed from messenger RNA of the spleen. A total of 1204 spleen cDNA clones were analyzed. Of the 1204 clones, 665 clones (55.2%) were identified as orthologs of known genes from other organisms by BLAST searches and 539 clones (44.8%) as unknown gene clones. In total 147 novel genes were identified, and annotations were made to 118 of them. In addition, 389 novel EST clusters were identified. Expression profile was analyzed in relation to metabolic functional groups. A total of 28 known genes were involved in immune functions, of which 10 were identified for the first time in channel catfish. Microsatellite-containing clones were also identified that may be potentially useful for genome mapping. This work contributed to the Catfish Gene Index, and toward a Unigene set useful for functional genomics research concerning spleen gene functions in relation to disease defenses.

Enhanced Bacterial Disease Resistance of Transgenic Channel Catfish Ictalurus punctatus Possessing Cecropin Genes

The cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus promoter, was transferred to the channel catfish Ictalurus punctatus. Transgenic individuals (P1) were mated to produce individuals (F1) that exhibited enhanced disease resistance and survival when challenged with pathogenic bacteria. During the epizootic of Flavobacterium columnare in an earthen pond, the percentage of transgenic individuals containing preprocecropin B construct that survived (100%) was significantly greater (P <0.005) THAN THAT OF NONTRANSGENIC CONTROLS (27.3%). ALSO, WHEN CHALLENGED IN TANKS WITH EDWARDSIELLA ICTALURI, THE CAUSATIVE AGENT OF ENTERIC SEPTICEMIA OF CATFISH, THE PERCENTAGE OF TRANSGENIC INDIVIDUALS CONTAINING CATFISH IG LEADER CECROPIN B CONSTRUCT THAT SURVIVED (40.7%) WAS SIGNIFICANTLY GREATER (P <0.01) THAN THAT OF NONTRANSGENIC CONTROLS (14.8%). THERE WERE NO PLEIOTROPIC EFFECTS OF THE TRANSGENES, AND GROWTH RATES OF THE TRANSGENIC AND NONTRANSGENIC SIBLINGS WERE NOT DIFFERENT (P > 0.05). Inheritance of the transgene by the F1 generation, 20.2% to 30.7% was typical of that in studies with transgenic channel catfish.

Random amplified polymorphic DNA markers : usefulness for gene mapping and analysis of genetic variation of catfish

Abstract Catfish are the most important aquacultural species in the United States. A genetic linkage map is needed to improve efficiency of breeding by marker-assisted selection (MAS), and for identification, isolation and eventual cloning of commercially important genes. To identify DNA-based genetic polymorphism for constructing a genetic linkage map of catfish, we tested 100 random amplification of polymorphic DNA (RAPD) primers for their utility in identifying genetic polymorphism in catfish. The overall polymorphism was low among strains within a species for both channel catfish ( Ictalurus punctatus) and blue catfish ( Ictalurus furcatus) . However, considerably higher levels of polymorphism were detected between channel catfish and blue catfish. Among the 100 primers tested, 42 produced highly clean and reproducible RAPD profiles; 33 produced medium quality RAPD profiles; and 25 produced poorly reproducible RAPD profiles or non-polymorphic RAPD profiles. The 75 high and medium quality primers generated 462 polymorphic bands, an average of 6.1 bands per primer. The RAPD markers were highly reproducible in a size range from 200 to 1500 base pairs (bp). They were transmitted to F 1 hybrids as dominant markers. There was no difference in RAPD profiles between channel catfish×blue catfish F 1 hybrids or their reciprocal hybrids. The markers segregated in F 2 or backcross progeny with ratios as expected from Mendelian inheritance.

Genotype-environment interactions for growth of blue, channel and hybrid catfish in ponds and cages at varying densities

Channel catfish, Ictalurus punctatus; blue catfish, I. furcatus; and hybrid channel ♀×blue♂ catfish fry were grown in ponds at densities of 7410/ha–2 245 000/ha. Channel catfish fingerlings and hybrid fingerlings were grown in ponds at densities of 7410/ha–22 230/ha, or 7410/ha with the addition of 1000 or 2000 kg/ha of co-stocked larger fish and in cages at 275/m3. Genotype-environment interactions for growth rate occurred (P<0.05) between hybrid catfish and channel catfish fry and between blue catfish and channel catfish fry stocked at varying densities. Channel catfish fry grew faster (P<0.05) than hybrid fry at low densities, but hybrid catfish fry grew faster than channel catfish fry at higher densities. The relative growth advantage of channel catfish fry over blue catfish fry increased with density. The increased growth rate of hybrid catfish fry compared to channel catfish fry at high densities was observed in both communal and separate experiments. Hybrid catfish fingerlings grew faster to market-size than channel catfish fingerlings at all densities in ponds. Their relative advantage to channel catfish increased with density and the addition of larger competing fish in the ponds. The hybrid was also superior to channel catfish in both communal and separate high density experiments when grown from fingerlings to marketsize. Genotype-environment interactions occurred for growth rates in cages or ponds. Hybrid catfish grew faster (P<0.01) than channel catfish in ponds, but slower (P<0.01) than channel catfish in cages.

Transfer of the metallothionein-human growth hormone fusion gene into channel catfish

Abstract The metallothionein-human growth hormone fusion gene (MThGH), constructed by fusing the mouse metallothionein promoter with the human growth hormone gene, was microinjected into the cytoplasm of one-cell embryos of channel catfish Ictalurus punctatus. The 3-week-old fish that developed were analyzed for integrated copies of the MThGH gene. Two of 10 animals contained MThGH sequences that comigrated with genomic DNA of high molecular weight in southern blots. The MThGH sequences were organized in head-to-tail tandem arrays that are characteristic of foreign genes integrated into recipient cell genomes. These data strongly suggest that the MThGH genes are stably integrated into chromosomal DNA, and that transgenic channel catfish can be generated by cytoplasmic injection of early embryos.

Dress-out and fillet yields of channel catfish, Ictalurus punctatus, blue catfish, Ictalurus furcatus, and their F1, F2 and backcross hybrids

Abstract The channel catfish, Ictalurus punctatus, ♀×blue catfish, Ictalurus furcatus, ♂ F1 hybrid showed increased processing yields over the most commonly cultured catfish, channel catfish, in the US. The F1 hybrid had higher dress-out and fillet percentage (61.1% and 45.7%, respectively) than channel catfish (57.5%, 42.5%), blue catfish (58.9%, 44.4%), F2 hybrid catfish (57.3%, 42.5%), F1×channel catfish (57.3%, 42.7%), F1×blue catfish (58.3%, 42.4%), blue catfish×F1 (58.2%, 43.2%), and channel catfish×F1 (56.8%, 42.1%). Individual heterosis had a strong positive effect on dress-out and fillet percentage. Channel catfish additive genetic effects had a strong negative effect on dress-out and fillet percentage. Females had greater dress-out (58.4% to 57.9%) and fillet percentage (43.6% to 43.0%) than males (P

Assessing genetic diversity of domestic populations of channel catfish (Ictalurus punctatus) in Alabama using AFLP markers

Abstract Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity of domestic stocks of channel catfish ( Ictalurus punctatus ). Sixteen populations within Alabama (USA) were surveyed using five primer combinations. A total of 454 reproducible, polymorphic bands were detected from 282 individuals, with an average heterozygosity of 0.135. The percentage of polymorphic loci varied greatly within distinct populations, ranging from 18.26% to 100%. Research populations tended to have a lower percentage of loci polymorphic than farm populations. The estimated average F st value across all loci was 0.4456. However, this value dropped to 0.1763 when the Hicks Farm stock was excluded from the analysis. Cluster analysis by Jaccards pairwise similarity coefficient indicated high similarity among individuals, with some population-specific clusters and with the Hicks Farm stock being highly distinctive. Neis population-wise identity measures found all populations except the population from the Hicks Farm related with 96% similarity. The Hicks Farm stock was 34% similar to the other populations. The genotypic information derived from this study is consistent with the historical origins and management of catfish broodstocks. The results of this study suggest that many channel catfish farm strains in Alabama are genetically similar, but some very distinct differences exist. Such information has implications for future broodstock selection and management and for further analysis of interactions between domestic and wild populations of channel catfish.

Towards the Ictalurid Catfish Transcriptome: Generation and Analysis of 31,215 Catfish ESTs.

BackgroundEST sequencing is one of the most efficient means for gene discovery and molecular marker development, and can be additionally utilized in both comparative genome analysis and evaluation of gene duplications. While much progress has been made in catfish genomics, large-scale EST resources have been lacking. The objectives of this project were to construct primary cDNA libraries, to conduct initial EST sequencing to generate catfish EST resources, and to obtain baseline information about highly expressed genes in various catfish organs to provide a guide for the production of normalized and subtracted cDNA libraries for large-scale transcriptome analysis in catfish.ResultsA total of 17 cDNA libraries were constructed including 12 from channel catfish (Ictalurus punctatus) and 5 from blue catfish (I. furcatus). A total of 31,215 ESTs, with average length of 778 bp, were generated including 20,451 from the channel catfish and 10,764 from blue catfish. Cluster analysis indicated that 73% of channel catfish and 67% of blue catfish ESTs were unique within the project. Over 53% and 50% of the channel catfish and blue catfish ESTs, respectively, had significant similarities to known genes. All ESTs have been deposited in GenBank. Evaluation of the catfish EST resources demonstrated their potential for molecular marker development, comparative genome analysis, and evaluation of ancient and recent gene duplications. Subtraction of abundantly expressed genes in a variety of catfish tissues, identified here, will allow the production of low-redundancy libraries for in-depth sequencing.ConclusionThe sequencing of 31,215 ESTs from channel catfish and blue catfish has significantly increased the EST resources in catfish. The EST resources should provide the potential for microarray development, polymorphic marker identification, mapping, and comparative genome analysis.