Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication

A 10X rainbow trout bacterial artificial chromosome (BAC) library was constructed to aid in the physical and genetic mapping efforts of the rainbow trout genome. The library was derived from the Swanson clonal line (YY male) and consists of 184,704 clones with an average insert size of 137,500 bp (PFGE) or 118,700 bp (DNA fingerprinting). The clones were gridded onto 10 large nylon membranes to produce high-density arrays for screening the library by hybridization. The library was probed with 11 cDNAs from the NCCCWA EST project chosen because of interest in their homology to known gene sequences, seven known genes, and a Y-specific sex marker. Putative positive clones identified by hybridization were re-arrayed and gridded for secondary confirmation. FPC analysis of HindIII and EcoRV DNA fingerprinting was used to estimate the level of redundancy in the library, to construct BAC contigs and to detect duplicated loci in the semi-duplicated rainbow trout genome. A good correlation (R2 = 0.7) was found between the number of hits per probe and the number of contigs that were assembled from the positive BACs. The average number of BACs per contig was 9.6, which is in good agreement with 10X genome coverage of the library. Two-thirds of the loci screened were predicted to be duplicated as the positive BACs for those genes were assembled into two or three different contigs, which suggests that most of the rainbow trout genome is duplicated.     

A Second Generation Integrated Map of the Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Genome: Analysis of Conserved Synteny with Model Fish Genomes

DNA fingerprints and end sequences from bacterial artificial chromosomes (BACs) from two new libraries were generated to improve the first generation integrated physical and genetic map of the rainbow trout (Oncorhynchus mykiss) genome. The current version of the physical map is composed of 167,989 clones of which 158,670 are assembled into contigs and 9,319 are singletons. The number of contigs was reduced from 4,173 to 3,220. End sequencing of clones from the new libraries generated a total of 11,958 high quality sequence reads. The end sequences were used to develop 238 new microsatellites of which 42 were added to the genetic map. Conserved synteny between the rainbow trout genome and model fish genomes was analyzed using 188,443 BAC end sequence (BES) reads. The fractions of BES reads with significant BLASTN hits against the zebrafish, medaka, and stickleback genomes were 8.8%, 9.7%, and 10.5%, respectively, while the fractions of significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 6.2%, 5.8%, and 5.5%, respectively. The overall number of unique regions of conserved synteny identified through grouping of the rainbow trout BES into fingerprinting contigs was 2,259, 2,229, and 2,203 for stickleback, medaka, and zebrafish, respectively. These numbers are approximately three to five times greater than those we have previously identified using BAC paired ends. Clustering of the conserved synteny analysis results by linkage groups as derived from the integrated physical and genetic map revealed that despite the low sequence homology, large blocks of macrosynteny are conserved between chromosome arms of rainbow trout and the model fish species.     

Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress

Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. To accurately investigate the mRNA expression profiles of multiple Hsp70s in rainbow trout Oncorhynchus mykiss, we isolated full-length cDNA clones encoding Hsp70 from the fish and investigated their mRNA expression profiles during heat stress. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells. The induction levels of total Hsp70b mRNAs were consistently higher than Hsp70a counterparts during heat stress, although the expression profiles of the two genes were similar to each other in temperature shift and time course experiments. Interestingly, an mRNA species with a larger molecular size was expressed only under severe heat stress not less than 28 degrees C irrespective of Hsp70a and Hsp70b. These results suggest that the comprehensive identification of duplicated genes is a prerequisite to examining the gene expression profiles for tetraploid species such as rainbow trout.     

The beta 2-microglobulin locus of rainbow trout (Oncorhynchus mykiss) contains three polymorphic genes

Beta2-microglobulin (beta2m) associates with MHC and related class I H chains to form cell surface glycoproteins that mediate a variety of functions in defense. In humans, monomorphism of a single beta2m gene contrasts with the diversity and polymorphism of the class I H chain genes, and a similar picture was seen in almost all other species examined. In this regard, rainbow trout (Oncorhynchus mykiss) appeared unusual: trout beta2m genes gave a complicated and polymorphic pattern in Southern blots, and a minimum of 10 different mRNA encoding two distinct types of beta2m were expressed by a single fish. Characterization of genomic clones from the same fish now shows that the rainbow trout beta2m locus consists of two expressed genes and one partial gene that are closely linked. Four copies of the locus were identified and allelic variants of each gene defined, largely through comparison of the noncoding regions. A dramatic variation in the lengths of introns is caused by variable repetitive elements and accounts for the complex pattern seen in Southern blots. By comparison to noncoding sequences, the coding regions are conserved but the three loci differ within a cluster of codons that encode residues of beta2m that do not interact with class I H chains. Additional diversity in the trout beta2m genes appears to be due to somatic mutation that might be facilitated by the abundance of repetitive DNA elements within the 12 beta2m genes of an individual rainbow trout.     

Assignment of rainbow trout linkage groups to specific chromosomes

The rainbow trout genetic linkage groups have been assigned to specific chromosomes in the OSU (2N=60) strain using fluorescence in situ hybridization (FISH) with BAC probes containing genes mapped to each linkage group. There was a rough correlation between chromosome size and size of the genetic linkage map in centimorgans for the genetic maps based on recombination from the female parent. Chromosome size and structure have a major impact on the female:male recombination ratio, which is much higher (up to 10:1 near the centromeres) on the larger metacentric chromosomes compared to smaller acrocentric chromosomes. Eighty percent of the BAC clones containing duplicate genes mapped to a single chromosomal location, suggesting that diploidization resulted in substantial divergence of intergenic regions. The BAC clones that hybridized to both duplicate loci were usually located in the distal portion of the chromosome. Duplicate genes were almost always found at a similar location on the chromosome arm of two different chromosome pairs, suggesting that most of the chromosome rearrangements following tetraploidization were centric fusions and did not involve homeologous chromosomes. The set of BACs compiled for this research will be especially useful in construction of genome maps and identification of QTL for important traits in other salmonid fishes.     

Physical and genetic mapping of the rainbow trout major histocompatibility regions: evidence for duplication of the class I region

One of the most unexpected discoveries in MHC genetics came from studies dealing with the teleost MHC. Initially discovered in zebrafish, the MHC class I and II regions of all bony fish are not linked. Previous segregation analysis in trout suggested that the class I and II regions reside on completely different chromosomes. To learn more about MHC genomics in trout, we have isolated BAC clones harboring class Ia and Ib loci, a single BAC clone containing an MH class II gene ( DAB), as well as BAC clones containing the ABCB2 gene. Upon PCR and sequence confirmation, BAC clones were labeled and used as probes for in situ hybridization on rainbow trout metaphase chromosomes for determination of the physical locations of the trout MH regions. Finally, SNPs, RFLPs, and microsatellites found within the BAC clones allowed for these regions to be assigned to specific linkage groups on the OSU x Hotcreek (HC) and OSU x Arlee (ARL) genetic linkage maps. Our data demonstrate that the trout MH regions are located on at least four different chromosomes and the corresponding linkage groups, while also providing direct evidence for the partial duplication of the MH class I region in trout.     

Characterisation of a low pathogenic form of Gyrodactylus salaris from rainbow trout

Gyrodactylus salaris was isolated from rainbow trout in a Danish freshwater trout farm, and a laboratory population of this particular parasite form was established on rainbow trout. Challenge infections were performed using different salmonid strains and species, including East Atlantic salmon Salmo salar (from the Danish River Skjern?), Baltic salmon S. salar (from the Swedish River Ume Alv) and rainbow trout Oncorhynchus mykiss (from the Danish rainbow trout farm Fousing). These were compared to infection studies on the Norwegian Laerdalselva parasite form kept under exactly the same conditions in the laboratory. The Danish G. salaris form had low virulence towards both Atlantic and Baltic salmon, whereas rainbow trout proved susceptible to the parasite. The Danish G. salaris form was able to maintain a very low infection on East Atlantic salmon, but not on the Baltic salmon, which eliminated the infection within 2 wk. Rainbow trout developed infection intensities ranging up to several hundred parasites per host. The host colonization patterns of the parasite differed clearly from those of previous studies on microhabitats of the Norwegian form of G. salaris. A comparative study on morphological characters (opisthaptoral hard parts) from the Danish parasite form and Norwegian G. salaris showed no significant differences. Selected genes comprising internal transcribed spacers 1 and 2 (ITS), ribosomal RNA intergenic spacer (IGS) and cytochrome c oxidase subunit I (COI) regions were cloned and sequenced. Five sequenced ITS clones from 5 individuals of the Danish strain consistently revealed a single base substitution compared to ITS sequences from all other known species and strains of Gyrodactylus. Mitochondrial COI gene sequences demonstrated that the Danish G. salaris form is closely similar to the Laerdalselva parasite form found in Norway. The IGS sequences were highly variable, but very similar to those obtained from German isolates of G. salaris.     

Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava

Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava ( Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.Communicated by M.-A. Grandbastien     

Comparison of GNRH3 genes across salmonid genera

Genomic sequences of gonadotropin-releasing hormone genes were amplified and examined for sequence divergence among members of three different genera of the subfamily Salmoninae: rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and Arctic charr (Salvelinus alpinus). Sequences of GNRH3A and GNRH3B (formerly known as sGnRH1 and sGnRH2) were 97-99% similar in coding regions and 94-98% similar in non-coding regions among genera, but comparisons within species between GNRH3A and GNRH3B were only 90-92% similar in coding regions and 83-89% similar in non-coding regions. Polymorphisms in the parents of mapping families for each species allowed for linkage mapping of the GNRH3B gene in all three species and the GNRH3A gene in rainbow trout. GNRH3B maps to linkage group 6 in rainbow trout, linkage group 16 in Atlantic salmon and linkage group 25 in Arctic charr. GNRH3A mapped to linkage group 30 in rainbow trout.     

Molecular characterization of the MuRF genes in rainbow trout: Potential role in muscle degradation

Muscle growth is determined primarily by the balance between protein synthesis and degradation. When rates of protein synthesis are similar between individuals, protein degradation is critical in explaining differences in growth efficiency. Studies in mammals showed that muscle atrophy results from increased protein breakdown, and is associated with activation of the ubiquitin proteasome pathway, including induction of the muscle-specific ubiquitin protein ligase, MuRF1. Animals lacking MuRF1 are resistant to muscle atrophy. In fish, little is known about the role of the proteasome/MuRF pathway in muscle degradation. The objectives of this study were to: 1) clone and characterize MuRF genes in rainbow trout; and 2) determine expression of MuRF genes in association with starvation- and vitellogenesis-induced muscle atrophy in rainbow trout. We have identified full-length cDNA sequences for three MuRF genes (MuRF1, MuRF2, and MuRF3). These genes encode proteins with typical MuRF structural domains, including a RING-finger, a B-box and a Leucine-rich coiled-coil domain. RT-PCR analysis showed that MuRF genes are predominantly expressed in muscle and heart tissues. Real time PCR analysis revealed that expression of all MuRF genes is up-regulated during starvation and MuRF3 is up-regulated in vitellogenesis-associated muscle degradation. These results suggest that MuRF genes have an important role in fish muscle protein degradation. Further studies are warranted to assess the potential use of MuRF genes as tools to monitor fish muscle growth and degradation.