2011 Annual Report
1a.Objectives (from AD-416)
A major constraint to increasing the production efficiency of the Nation's cool and cold water aquaculture industry is the lack of genetically improved strains of fish for aquaculture. There is only limited genetic information on traits that will enhance production efficiency and yield a better quality fish. Identification and characterization of genes affecting aquaculture production traits will facilitate the development of genetically improved strains to increase aquaculture production efficiency. The objectives of the research outlined in this agreement focus on the identification of genes affecting oocyte maturation and embryogenesis in rainbow trout. Characterization of these genes will result in more in-depth understanding of the biology of reproduction and embryonic development and will impact strategies for improving developmental characteristics in broodstock.
1b.Approach (from AD-416)
Collaborators at the USDA/ARS/NCCCWA and West Virginia University have worked to develop resources for conducting functional genomic research in rainbow trout. Resources which specifically target studies on embryogenesis include large volumes of expressed sequence tag data from oocyte and embryonic developmental stages and a cDNA panel representing oocytes thru day 50 post fertilization for gene expression analyses. Strategies will include analyses of gene expression and proteomic data in NCCCWA Broodstock to identify and characterize novel-oocyte specific genes, similar strategies will be employed to characterize gene expression throughout embryonic development.
A rainbow trout high density oligo-array representing 37,394 unique TCs was constructed using Agilent's SurePrint technology. Efforts to identify miRNAs in rainbow trout resulted in the discovery of a significant number of distinct and novel miRNAs. Using new sequencing technologies, we characterized the rainbow trout transcriptome (1.3 million reads from a double-haploid rainbow trout) and identified 22,022 to 47,128 putative SNPs from a reduced a representation library. These studies provided valuable resources for functional genome research in rainbow trout.
The cDNAs for rainbow trout cyclin B1 (CB1), cyclin B2 (CB2) and Cdc2 kinase were cloned and their expression profiles characterized during oogenesis and spermatogenesis. A number of novel oocyte-specific genes were identified from a rainbow trout oocyte cDNA library. One of them encodes a protein (OORP-T) with a conserved oxysterol binding protein (OSBP) domain, suggesting a role of this protein in the synthesis, transportation and metabolism of lipids during oogenesis and embryogenesis. Another novel transcript appears to be a noncoding mRNA-like transcript (RtGST-1). Expression of this transcript is extremely high in early previtellogenic ovaries, suggesting a role of this transcript in differentiation and/or development of germ cells. The expression patterns of 9 transcription factors were characterized during early embryonic development. Cloning and characterization of these key oocyte-expressed genes may lead to the discovery of new candidate genes associated with oocyte competence and embryonic survival in rainbow trout.
To identify genes that could potentially be used as molecular biomarkers to study protein turnover and select for germplasm with improved protein accretion in rainbow trout, microarray technology was used to identify genes/pathways involved in starvation-related protein turnover. A coordinated down- regulation of the protein biosynthesis genes in starved fish was observed. Further study using real-time PCR and enzyme activity assays revealed a reduction of calpastatin mRNA with an increase in the calpain catalytic activity in starved fish. Using microarray analysis, gene expression profiles between atrophy muscle from 2N fish and normal muscle from 3N sterile fish was compared. Muscle atrophy was associated with elevated expression of genes involved in the catheptic and collagenase proteolytic pathways, the aerobic production, utilization of ATP and growth arrest; and down-regulated expression of genes encoding enzymes of anaerobic respiration, muscle proteins as well as factors involved in RNA and protein biosynthesis/processing.
The ADODR is in frequent contact with the cooperator through phone calls, email, and site visits in addition to receipt of written reports.