Microsatellite primers for red drum (Sciaenops ocellatus)

In this note, we document polymerase-chain-reaction (PCR) primer pairs for 101 nuclear-encoded microsatellites designed and developed from a genomic library for red drum (Sciaenops ocellatus). Details of the genomic library construction, the sequencing of positive clones, primer design, and PCR protocols may be found in Karlsson et al. (2008). The 101 microsatellites (GENBA NK Accession Numbers EU015882-EU015982) were amplified successfully and used to genotype 24 red drum obtained from Galveston Bay, Texas (Table 1). A total of 69 of the microsatellites had an uninterrupted (perfect) dinucleotide motif, and 30 had an imperfect dinucleotide motif; one microsatellite had an imperfect tetranucleotide motif, and one had an imperfect and compound motif (Table 1 ). Sizes of the cloned alleles ranged from 84 to 252 base pairs. A ‘blast’ search of the GENBANK database indicated that all of the primers and the cloned alleles were unique (i.e., not duplicated)

Effect of starvation on global gene expression and proteolysis in rainbow trout (Oncorhynchus mykiss)

<p>Abstract</p> <p>Background</p> <p>Fast, efficiently growing animals have increased protein synthesis and/or reduced protein degradation relative to slow, inefficiently growing animals. Consequently, minimizing the energetic cost of protein turnover is a strategic goal for enhancing animal growth. Characterization of gene expression profiles associated with protein turnover would allow us to identify genes that could potentially be used as molecular biomarkers to select for germplasm with improved protein accretion.</p> <p>Results</p> <p>We evaluated changes in hepatic global gene expression in response to 3-week starvation in rainbow trout (<it>Oncorhynchus mykiss</it>). Microarray analysis revealed a coordinated, down-regulated expression of protein biosynthesis genes in starved fish. In addition, the expression of genes involved in lipid metabolism/transport, aerobic respiration, blood functions and immune response were decreased in response to starvation. However, the microarray approach did not show a significant increase of gene expression in protein catabolic pathways. Further studies, using real-time PCR and enzyme activity assays, were performed to investigate the expression of genes involved in the major proteolytic pathways including calpains, the multi-catalytic proteasome and cathepsins. Starvation reduced mRNA expression of the calpain inhibitor, calpastatin long isoform (CAST-L), with a subsequent increase in the calpain catalytic activity. In addition, starvation caused a slight but significant increase in 20S proteasome activity without affecting mRNA levels of the proteasome genes. Neither the mRNA levels nor the activities of cathepsin D and L were affected by starvation.</p> <p>Conclusion</p> <p>These results suggest a significant role of calpain and 20S proteasome pathways in protein mobilization as a source of energy during fasting and a potential association of the CAST-L gene with fish protein accretion.</p

Characterization of the Rainbow Trout Egg MicroRNA Transcriptome

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNA molecules that regulate post-transcriptional expression of target genes and play important roles in animal development. The objectives of this study were to characterize the egg miRNA transcriptome and identify novel egg-predominant miRNAs in rainbow trout. Small RNAs isolated from mature unfertilized rainbow trout eggs were subjected to deep sequencing using an Illumina Genome Analyzer. The massive sequencing produced 24,621,741 quality reads, among which, 266 known miRNAs were identified and 230 putatively novel miRNAs were predicted. The most abundantly known miRNAs are let-7 and miR-21, accounting for 24.06% and 18.71% of the known miRNAs, respectively. Other known miRNAs which are abundantly present in eggs include miR-24, miR-202, miR-148, miR-30, miR-10, miR-146, miR-25, and miR-143. Real time PCR analysis using cDNAs derived from 10 tissues validated 87 out of 90 selected putative miRNAs and identified three novel miRNAs predominantly expressed in rainbow trout eggs. Each of these novel egg-predominant miRNAs is predicted to target a significant number of genes, most of which are significantly down-regulated in naturally ovulated rainbow trout eggs based on analysis of publicly available microarray data sets. Quantitative real time PCR analysis also demonstrated low expression of a selected number of target genes in eggs relative to liver and muscle tissues. This study represents the first complete survey of miRNAs in fish eggs and provides a starting point for future studies aimed at understanding the roles of miRNAs in controlling egg quality and early embryogenesis in rainbow trout

Cloning and characterization of a novel oocyte-specific gene encoding an F-Box protein in rainbow trout (Oncorhynchus mykiss)

Background Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. The objectives of this study were to characterize the expression of a novel oocyte-specific gene encoding an F-box protein during ovarian development in rainbow trout, and identify its potential interacting partners in rainbow trout oocytes. Methods Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, a novel transcript represented by ESTs only from the oocyte library was identified. The complete cDNA sequence for the novel gene (named fbxoo) was obtained by assembling sequences from an EST clone and a 5′RACE product. The expression and localization of fbxoo mRNA and protein in ovaries of different developmental stages were analyzed by quantitative real time PCR, immunoblotting, in situ hybridization and immunohistochemistry. Identification of Fbxoo binding proteins was performed by yeast two-hybrid screening. Results fbxoo mRNA is specifically expressed in mature oocytes as revealed by tissue distribution analysis. The fbxoo cDNA sequence is 1,996 bp in length containing an open reading frame, which encodes a predicted protein of 514 amino acids. The novel protein sequence does not match any known protein sequences in the NCBI database. However, a search of the Pfam protein database revealed that the protein contains an F-box motif at the N-terminus, indicating that Fbxoo is a new member of the F-box protein family. The expression of fbxoomRNA and protein is high in ovaries at early pre-vitellogenesis stage, and both fbxoo mRNA and protein are predominantly expressed in early pre-vitellogenic oocytes. Several proteins including tissue inhibitor of metalloproteinase 2 (Timp2) were identified as potential Fbxoo protein binding partners. Conclusions Results suggest that the novel oocyte-specific F-box protein may play an important role in early oocyte development by regulating other critical proteins involved in oogenesis in rainbow trout

Characterization of the rainbow trout transcriptome using Sanger and 454-pyrosequencing approaches

<p>Abstract</p> <p>Background</p> <p>Rainbow trout are important fish for aquaculture and recreational fisheries and serves as a model species for research investigations associated with carcinogenesis, comparative immunology, toxicology and evolutionary biology. However, to date there is no genome reference sequence to facilitate the development of molecular technologies that utilize high-throughput characterizations of gene expression and genetic variation. Alternatively, transcriptome sequencing is a rapid and efficient means for gene discovery and genetic marker development. Although a large number (258,973) of EST sequences are publicly available, the nature of rainbow trout duplicated genome hinders assembly and complicates annotation.</p> <p>Results</p> <p>High-throughput deep sequencing of the Swanson rainbow trout doubled-haploid transcriptome using 454-pyrosequencing technology yielded ~1.3 million reads with an average length of 344 bp, a total of 447 million bases. <it>De novo </it>assembly of the sequences yielded 151,847 Tentative Consensus (TC) sequences (average length of 662 bp) and 224,391 singletons. A combination assembly of both the 454-pyrosequencing ESTs and the pre-existing sequences resulted in 161,818 TCs (average length of 758 bp) and 261,071 singletons. Gene Ontology analysis of the combination assembly showed high similarities to transcriptomes of other fish species with known genome sequences.</p> <p>Conclusion</p> <p>The 454 library significantly increased the suite of ESTs available for rainbow trout, allowing improved assembly and annotation of the transcriptome. Furthermore, the 454 sequencing enables functional genome research in rainbow trout, providing a wealth of sequence data to serve as a reference transcriptome for future studies including identification of paralogous sequences and/or allelic variation, digital gene expression and proteomic research.</p

Influence of resource pulses and perennial neighbors on the establishment of an invasive annual grass in the Mojave Desert

Invasion by exotic annual grasses is one of the most significant threats to arid ecosystems in the western USA. Current theories of invasibility predict plant communities become more susceptible to invasion whenever there is an increase in the amount of unused resources. The objective of this field study was to examine how resource pulses and temporal variation in resource demand by the native shrub vegetation influences establishment of the invasive annual grass Schismus arabicus. Water and nitrogen were applied as pulses in early spring, mid-spring, or continuously throughout the growing season to plots containing either Atriplex confertifolia or Atriplex parryi shrubs. The effect of resource pulses on Schismus density and biomass was highly dependent on the seasonal timing of the resource pulses and the identity of the neighbor shrub. When resource pulses coincided with high rates of resource capture and growth of the native vegetation, density and biomass of Schismus was reduced. Schismusestablishment was greater under continuous resource supply compared to pulsed resource supply, likely because more soil resources were available at a shallow depth when resources were supplied at a continuous low rate. These results suggest that the establishment of invasive annual grasses in arid systems can be influenced by the magnitude and spatial distribution of resource pulses in addition to the seasonal timing of resource pulses

Genomic structure and expression of uncoupling protein 2 genes in rainbow trout (Oncorhynchus mykiss)

Background Uncoupling protein 2 (UCP2) belongs to the superfamily of mitochondrial anion carriers that dissociate the respiratory chain from ATP synthesis. It has been determined that UCP2 plays a role in several physiological processes such as energy expenditure, body weight control and fatty acid metabolism in several vertebrate species. We report the first characterization of UCP2 s in rainbow trout (Oncorhynchus mykiss). Results Two UCP2 genes were identified in the rainbow trout genome, UCP2A and UCP2B. These genes are 93% similar in their predicted amino acid sequences and display the same genomic structure as other vertebrates (8 exons and 7 introns) spanning 4.2 kb and 3.2 kb, respectively. UCP2A and UCP2B were widely expressed in all tissues of the study with a predominant level in macrophage-rich tissues and reproductive organs. In fry muscle we observed an increase in UCP2B expression in response to fasting and a decrease after refeeding in agreement with previous studies in human, mouse, rat, and marsupials. The converse expression pattern was observed for UCP2A mRNA which decreased during fasting, suggesting different metabolic roles for UCP2A and UCP2B in rainbow trout muscle. Phylogenetic analysis including other genes from the UCP core family located rainbow trout UCP2A and UCP2B with their orthologs and suggested an early divergence of vertebrate UCPs from a common ancestor gene. Conclusion We characterized two UCP2 genes in rainbow trout with similar genomic structures, amino acid sequences and distribution profiles. These genes appeared to be differentially regulated in response to fasting and refeeding in fry muscle. The genomic organization and phylogeny analysis support the hypothesis of a common ancestry between the vertebrate UCPs

'Redlands for Regions': Producer demonstration sites of psyllid-resistant leucaena across north Queensland

Leucaena, a tree legume with potential to greatly improve cattle performance, has not been readily adopted in northern Queensland primarily due to prevalence of the psyllid (Heteropsylla cubana) insect in higher rainfall zones. Psyllids reduce edible biomass in leaves by 40–52%, combined with a 46–83% reduction of stem yield (Bray and Woodroffe 1991). Losses to the Central Queensland beef industry due to psyllid impact on animal performance are estimated at $2 M per year (Mullen et al. 1998). Cultivar Redlands is a psyllid-resistant leucaena variety recently developed by Meat and Livestock Australia (MLA) and the University of Queensland

Clinical Educators’ Perceptions of Students Following a Simulation-Based Learning Program

Purpose: Clinical education is a key component of speech-language pathology university curriculum, whereby students have the opportunity to apply theoretical knowledge and practical skills learned in the classroom into a real workplace. However, more recently the availability of high quality, consistent clinical placements and learning experiences across the range of practice areas in the discipline is reducing. Therefore, alternative clinical learning opportunities that enable students to develop skills and competencies are being explored. Recently, replacing clinical time with a simulated learning program has been shown to achieve equivalent levels of clinical competency in speech pathology. However, it is unknown how simulation impacts on student learning in traditional clinical placements. Therefore, this research explored clinical educators’ perceptions of students undertaking clinical placements in their workplace immediately following a five-day simulation-based learning program related to the same area of practice. Method: Thirty-five clinical educators who supervised students in the workplace immediately after they completed the simulation program participated in semi-structured interviews. All interviews were transcribed verbatim and analyzed using qualitative methods described by Graneheim and Lundman (2004). Result: The analysis identified four key themes related to the impact of students in the workplace, simulation priming students for learning, the importance of the transition from simulation-based learning to the workplace, and the role of simulation in clinical education programs. Conclusion: The use of simulation to support student learning and develop clinical skills and competencies in adult speech pathology practice is supported by workplace clinical educators. However, results of this study suggest that the simulation program needs to be embedded within the curriculum and clinical education program to enhance transition between learning experiences and maximize benefits of learning experiences in real workplace contexts

A first generation integrated map of the rainbow trout genome

Background Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. An integrated physical and genetic map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) programs for improving rainbow trout aquaculture production. Results The first generation integrated map of the rainbow trout genome is composed of 238 BAC contigs anchored to chromosomes of the genetic map. It covers more than 10% of the genome across segments from all 29 chromosomes. Anchoring of 203 contigs to chromosomes of the National Center for Cool and Cold Water Aquaculture (NCCCWA) genetic map was achieved through mapping of 288 genetic markers derived from BAC end sequences (BES), screening of the BAC library with previously mapped markers and matching of SNPs with BES reads. In addition, 35 contigs were anchored to linkage groups of the INRA (French National Institute of Agricultural Research) genetic map through markers that were not informative for linkage analysis in the NCCCWA mapping panel. The ratio of physical to genetic linkage distances varied substantially among chromosomes and BAC contigs with an average of 3,033 Kb/cM. Conclusions The integrated map described here provides a framework for a robust composite genome map for rainbow trout. This resource is needed for genomic analyses in this research model and economically important species and will facilitate comparative genome mapping with other salmonids and with model fish species. This resource will also facilitate efforts to assemble a whole-genome reference sequence for rainbow trout