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SNP discovery in the transcriptome of white Pacific shrimp Litopenaeus vannamei by next generation sequencing.

ABSTRACT: The application of next generation sequencing technology has greatly facilitated high throughput single nucleotide polymorphism (SNP) discovery and genotyping in genetic research. In the present study, SNPs were discovered based on two transcriptomes of Litopenaeus vannamei (L. vannamei) generated from Illumina sequencing platform HiSeq 2000. One transcriptome of L. vannamei was obtained through sequencing on the RNA from larvae at mysis stage and its reference sequence was de novo assembled. The data from another transcriptome were downloaded from NCBI and the reads of the two transcriptomes were mapped separately to the assembled reference by BWA. SNP calling was performed using SAMtools. A total of 58,717 and 36,277 SNPs with high quality were predicted from the two transcriptomes, respectively. SNP calling was also performed using the reads of two transcriptomes together, and a total of 96,040 SNPs with high quality were predicted. Among these 96,040 SNPs, 5,242 and 29,129 were predicted as non-synonymous and synonymous SNPs respectively. Characterization analysis of the predicted SNPs in L. vannamei showed that the estimated SNP frequency was 0.21% (one SNP per 476 bp) and the estimated ratio for transition to transversion was 2.0. Fifty SNPs were randomly selected for validation by Sanger sequencing after PCR amplification and 76% of SNPs were confirmed, which indicated that the SNPs predicted in this study were reliable. These SNPs will be very useful for genetic study in L. vannamei, especially for the high density linkage map construction and genome-wide association studies.

SUBMITTER: Yu Y

PROVIDER: S-EPMC3907553 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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SNP discovery in the transcriptome of white Pacific shrimp Litopenaeus vannamei by next generation sequencing.

Yu Yang Y Wei Jiankai J Zhang Xiaojun X Liu Jingwen J Liu Chengzhang C Li Fuhua F Xiang Jianhai J

PloS one 20140130 1

The application of next generation sequencing technology has greatly facilitated high throughput single nucleotide polymorphism (SNP) discovery and genotyping in genetic research. In the present study, SNPs were discovered based on two transcriptomes of Litopenaeus vannamei (L. vannamei) generated from Illumina sequencing platform HiSeq 2000. One transcriptome of L. vannamei was obtained through sequencing on the RNA from larvae at mysis stage and its reference sequence was de novo assembled. Th ...[more]

PMID: 24498047

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Project description:In the practical farming of Litopenaeus vannamei, the intensive culture system and environmental pollution usually results in a high concentration of ammonia, which usually brings large detrimental effects to shrimp, such as increasing the susceptibility to pathogens, reducing growth, decreasing osmoregulatory capacity, increasing the molting frequency, and even causing high mortality. However, little information is available on the molecular mechanisms of the detrimental effects of ammonia stress in shrimp. In this study, we performed comparative transcriptome analysis between ammonia-challenged and control groups from the same family of L. vannamei to identify the key genes and pathways response to ammonia stress. The comparative transcriptome analysis identified 136 significantly differentially expressed genes that have high homologies with the known proteins in aquatic species, among which 94 genes are reported potentially related to immune function, and the rest of the genes are involved in apoptosis, growth, molting, and osmoregulation. Fourteen GO terms and 6 KEGG pathways were identified to be significantly changed by ammonia stress. In these GO terms, 13 genes have been studied in aquatic species, and 11 of them were reported potentially involved in immune defense and two genes were related to molting. In the significantly changed KEGG pathways, all the 7 significantly changed genes have been reported in shrimp, and four of them were potentially involved in immune defense and the other three were related to molting, defending toxicity, and osmoregulation, respectively. In addition, majority of the significantly changed genes involved in nitrogen metabolisms that play an important role in reducing ammonia toxicity failed to perform the protection function. The present results have supplied molecular level support for the previous founding of the detrimental effects of ammonia stress in shrimp, which is a prerequisite for better understanding the molecular mechanism of the immunosuppression from ammonia stress.

Project description:A simple bioassay that quantifies feed intake as an estimation of relative attractability of feeds containing different ingredients in the Pacific white shrimp Litopenaeus vannamei is described. Fish meal (FM), fish protein hydrolysate (FPH), squid meal (SqM) and casein (CN) were assessed at the same dietary level for their relative influence on feed intake rates of Litopenaeus vannamei. A bland diet containing 92% whole wheat grain meal, 6% diatomaceous earth and 2% alginate with a known low attractability was used as the standard control or base diet. Ingredients were added to the bland base control diet at a level of 3% as fed. Shrimp were stocked into 80 L glass tanks (n= 20 per tank) in a recirculating aquaculture system. Tanks were randomly assigned to one of five diet treatments (3tanks/treatment). Experiments measuring the attractability of each feed were conducted twice daily at 0900 hours and 1330 hours over a five day period. For each experiment, 40 feed pellets (ca. 1 g) corresponding to the assigned treatment were provided to each tank. To calculate the rate of feed intake, pellets remaining in each tank were counted at six minute intervals for a seventy-two minute period. Differences in rate of feed intake among diets were evaluated using Cox Regression Analysis. This attractability assay required only small amounts of ingredients and incorporated ingredients into a bland feed, which significantly reduces the influence from other ingredients or compound in the pellets. All of the test protein ingredients, especially SqM, in the feeds significantly increased the feed consumption rate. The diet containing SqM was consumed at a significantly higher rate than those containing casein and FM but not FPH. FPH and CN containing diets were not significantly different but consumed at a higher rate than the diet containing FM. Results of these trials indicate that the presence of certain ingredients can increase feed intake, thereby increasing nutrient availability of the diets. This reported method to determine consumption of diets containing certain ingredients can be considered as a valid method to estimate attractability.

Project description:BackgroundEugenol is the most commonly used plant anesthetic to relieve the stressors during various aquaculture procedures. This study aims to investigate the pharmacokinetics of eugenol in Pacific white shrimp by immersion baths in a simulated transportation.ResultsThe pharmacokinetics of eugenol were firstly investigated in Pacific white shrimp by immersion baths of 300 mg L- 1 eugenol over 5 min (Treatment 1), 10 mg L- 1 eugenol during 24 h (Treatment 2) and a sequential immersion administration (Treatment 3). Concentrations of eugenol in hemolymph, hepatopancreas, and muscle were determined using Gas chromatography-tandem mass spectrometry (GC-MS/MS). After immersion bath of Treatment 1, the elimination half-life (t1/2z) values are 1.3 h and 11 h for hepatopancreas and muscles, indicating the rapid absorption and elimination of eugenol in shrimp. Under the Treatment 2 administration, the eugenol peak concentration is 6527.9 μg/kg in muscle, followed by 402.8 μg/kg in hepatopancreas, with the lowest concentration of 37.9 μg/L in hemolymph. Area under the curve (AUC0-∞) values lie in the order of muscle > hepatopancreas > hemolymph, suggesting that eugenol tends to accumulate in muscle by the immersion administration. Moreover, the average residence time (MRT0-∞) values of 38.6, 23.0 and 115.3 h for hemolymph, hepatopancreas and muscle are achieved, which may indicate that hepatopancreas is the main organ for elimination of eugenol. After combining the conditions in a sequential bath immersion of eugenol (Treatment 3), the maximum concentration (Cmax) values of eugenol are higher than those achieved in Treatment 2, indicating that accumulation of eugenol happened in haemolymph, hepatopancreas and muscle. In addition, the corresponding t1/2z values are 4.7, 14.9 and 47.6 h, respectively, suggesting the faster elimination from the tissues following sequential administration. After the immersion bath, eugenol concentrations in muscle of Pacific white shrimp are lower than 2.5 mg/kg at 2 h, 48 h and 24.5 h in Treatment 1 ~ 3.ConclusionsA withdrawal period of 2 h, 48 h and 24.5 h following a 300 mg L- 1 of eugenol over a 5-min, 10 mg L- 1 eugenol concentration during a 24-h and combined conditions in a sequential immersion bath were suggested.

Dataset Information

SNP discovery in the transcriptome of white Pacific shrimp Litopenaeus vannamei by next generation sequencing.

Publications

SNP discovery in the transcriptome of white Pacific shrimp Litopenaeus vannamei by next generation sequencing.

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