Project description:Abstract. The molecular pathways in embryonic vertebrates leading to gonad formation in each sex are incompletely understood. The purpose of this study was to identify novel genes that could be associated with sex-specific gonadal differentiation in a fish, the rainbow trout (Oncorhynchus mykiss). This study was facilitated by a custom microarray based on 7,681 genes derived from embryonic rainbow trout gonad cDNA libraries and public databases. Gonad samples for total RNA isolation were obtained from pvasa-green fluorescent protein (pvasa-GFP) transgenic rainbow between 300 and 700 degree days of development post-fertilization. The transgenic fish permitted the collection of gonads from embryonic rainbow trout during the period of molecular sex differentiation in advance of any morphologically distinguishable characteristics of sex. A bioinformatic method was used with the microarray data that looked for strong associations in gene expression patterns between known sex differentiation genes (the target genes) and novel genes (the target-associated genes) previously not allied with sex differentiation in fishes. The expression patterns of representative targets genes from both sexes and their target-associated genes were independently confirmed by real-time reverse transcription-polymerase chain reaction to support the validity of the bioinformatics method employed. Numerous, novel genes were identified in the gonads of embryonic female and male rainbow trout that could be involved in sex-specific differentiation pathways in this fish.
Project description:Embryonic development rate is a key trait significantly associated and genetically linked with both growth rate and sexual maturity in rainbow trout. To identify candidate genes underlying embryonic development rate, whole genome expression microarray analyses were conducted using embryos from a fourth generation backcross family; each backcross generation involved the introgression of the fast-developing alleles for the major development rate QTL (from the Clearwater clonal line [CW]) into a slow-developing clonal line (Oregon State University [OSU]). Embryos were collected at 15, 19, and 28 days post-fertilization. Microsatellite markers (One112ADFG, OMM1009 and OmyFGT12TUF) linked to the major embryonic development rate QTL region were used to determine the QTL genotype. The sex marker OMY-Y1 was used to determine the genotypic sex of each embryo. cDNA from 48 individual embryos were used for microarray expression analysis. An ANOVA modeling approach was used to detect differential gene expression between embryos possessing fast-developing genotypes and slow-developing genotypes. A total of 182 features have been identified with significant differences between embryonic development rate genotypes. Quantitative PCR was conducted on ten representative genes using the rainbow trout homologous sequences and microarray results were validated.
Project description:Abstract. The molecular pathways in embryonic vertebrates leading to gonad formation in each sex are incompletely understood. The purpose of this study was to identify novel genes that could be associated with sex-specific gonadal differentiation in a fish, the rainbow trout (Oncorhynchus mykiss). This study was facilitated by a custom microarray based on 7,681 genes derived from embryonic rainbow trout gonad cDNA libraries and public databases. Gonad samples for total RNA isolation were obtained from pvasa-green fluorescent protein (pvasa-GFP) transgenic rainbow between 300 and 700 degree days of development post-fertilization. The transgenic fish permitted the collection of gonads from embryonic rainbow trout during the period of molecular sex differentiation in advance of any morphologically distinguishable characteristics of sex. A bioinformatic method was used with the microarray data that looked for strong associations in gene expression patterns between known sex differentiation genes (the target genes) and novel genes (the target-associated genes) previously not allied with sex differentiation in fishes. The expression patterns of representative targets genes from both sexes and their target-associated genes were independently confirmed by real-time reverse transcription-polymerase chain reaction to support the validity of the bioinformatics method employed. Numerous, novel genes were identified in the gonads of embryonic female and male rainbow trout that could be involved in sex-specific differentiation pathways in this fish. Embryonic gonads were removed by dissection from known genetic female (XX) and male (XY) rainbow trout (Oncorhynchus mykiss) from a transgenic population where expression of GFP is controlled by vasa-gene regulatory elements (Yoshizaki et al. 2000; Takeuchi et al. 2002). These fish were maintained in incubators containing flowing freshwater (10°C) at the Ooizumi Research Station, Yamanashi, Japan. Ten female or male transgenic rainbow trout were randomly selected every 5 days, beginning at 30 days (i.e., 300 degree days = incubation temperature in °C x number of days) post fertilization through until 70 days (i.e., 700 degree days) post fertilization. The gonads from each sex, at each sampling time, were pooled and immediately frozen for subsequent total RNA isolation.
Project description:Transcriptional profiling of rainbow trout liver cells comparing liver cells from small fish with liver cells from large fish at two time periods.
Project description:Transcriptional profiling of rainbow trout muscle cells comparing muscle cells from small fish with muscle cells from large fish at two time periods.
Project description:Purpose:Our data significantly advance understanding of heat stress regulatory mechanism of miRNA in the head kidney of rainbow trout Methods:miRNAs of rainbow trout were involved in heat stress were identified by high-throughput sequencing of six small RNA libraries of the kidney tissues under control (18℃) and heat-treated (24℃) conditions Results:high-throughput sequencing was performed to identify miRNAs responsive to heat stress. We obtained 41,991,119 and 43,882,123 raw reads and 39,756,736 and 42,538,331 clean reads from under control (18℃) and heat-treated (24℃) .A total of 392 conserved miRNAs and 989 novel miRNAs were identified, of which 78 miRNAs were expressed in different response to heat stress. In addition to, including 393 negative correlation miRNA-target gene pairs Conclusions:through high-throughput sequencing of the six libraries from head kidney tissue of rainbow trout, the expression level of miRNA has significant changes after heat stress.
Project description:Gynogenetic development in fish is induced by activation of eggs with irradiated spermatozoa followed by exposure of the activated eggs to the temperature or high hydrostatic pressure (HHP) shock that prevents 1st cell cleavage. Produced specimens are fully homozygous fish also known as Doubled Haploids. Gynogenetic DH individuals might be used aquaculture and developmental biology unfortunately; the potential application of DHs is limited by a rather low survival rate of such specimens. However, observed variation in the survival rates of the gynogenetic embryos originated from different clutches suggests that eggs from some females have increased ability for gynogenetic development than others. Taking into account that first 10 cell cleavages in the fish embryos rely on the maternal RNA, it is tempting to assume that the ova showing such a vast difference in potential for gynogenesis may have also had different biological characteristics including alterations in maternal gene expression profiles. If so, then genes that up- or down –regulated expression in eggs increases competence for gynogenetic development in trout might be considered as candidate genes for gynogenesis in rainbow trout. Thus, the main goal of the project is identification of genes that increase ability of rainbow trout eggs for gynogenetic development. Within the project, we tried to verify following hypotheses: 1. Eggs from different females have different potential for gynogenesis in rainbow trout. 2. Eggs with different ability for gynogenetic development with all maternal inheritance have different biological characteristics including morphology and anti-ROS enzyme activities. 3. Eggs with increased competence for gynogenesis have altered transcriptomic profiles. 4. There are some particular genes that altered expression in trout eggs enable development of gynogenetic embryos. Gynogenetic rainbow trout specimens were produced in the course of activation of eggs with UV-irradiated spermatozoa and High Hydrostatic Pressure shock (HHP) applied around 1st cell cleavage. Eggs from several females were used in the experiment. Survival rates of gynogenetic rainbow trout was monitored since fertilization. Quality of eggs was examined by assessment of their morphology and activity of anti-ROS (reactive oxygene species) enzymes. Transcriptome of eggs showing increased and decreased developmental competence for gynogenesis was analyzed using RNA-seq approach and results compared to find out any alterations related to survival of gynogenetic trout.
Project description:Transcriptional profiling of rainbow trout liver and muscle cells comparing small fish with large fish within a population of neomale offspring.