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Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon.

ABSTRACT: BACKGROUND: The black tiger shrimp (Penaeus monodon) is one of the most important aquaculture species in the world, representing the crustacean lineage which possesses the greatest species diversity among marine invertebrates. Yet, we barely know anything about their genomic structure. To understand the organization and evolution of the P. monodon genome, a fosmid library consisting of 288,000 colonies and was constructed, equivalent to 5.3-fold coverage of the 2.17 Gb genome. Approximately 11.1 Mb of fosmid end sequences (FESs) from 20,926 non-redundant reads representing 0.45% of the P. monodon genome were obtained for repetitive and protein-coding sequence analyses. RESULTS: We found that microsatellite sequences were highly abundant in the P. monodon genome, comprising 8.3% of the total length. The density and the average length of microsatellites were evidently higher in comparison to those of other taxa. AT-rich microsatellite motifs, especially poly (AT) and poly (AAT), were the most abundant. High abundance of microsatellite sequences were also found in the transcribed regions. Furthermore, via self-BlastN analysis we identified 103 novel repetitive element families which were categorized into four groups, i.e., 33 WSSV-like repeats, 14 retrotransposons, 5 gene-like repeats, and 51 unannotated repeats. Overall, various types of repeats comprise 51.18% of the P. monodon genome in length. Approximately 7.4% of the FESs contained protein-coding sequences, and the Inhibitor of Apoptosis Protein (IAP) gene and the Innexin 3 gene homologues appear to be present in high abundance in the P. monodon genome. CONCLUSIONS: The redundancy of various repeat types in the P. monodon genome illustrates its highly repetitive nature. In particular, long and dense microsatellite sequences as well as abundant WSSV-like sequences highlight the uniqueness of genome organization of penaeid shrimp from those of other taxa. These results provide substantial improvement to our current knowledge not only for shrimp but also for marine crustaceans of large genome size.

SUBMITTER: Huang SW

PROVIDER: S-EPMC3124438 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon.

Huang Shiao-Wei SW Lin You-Yu YY You En-Min EM Liu Tze-Tze TT Shu Hung-Yu HY Wu Keh-Ming KM Tsai Shih-Feng SF Lo Chu-Fang CF Kou Guang-Hsiung GH Ma Gwo-Chin GC Chen Ming M Wu Dongying D Aoki Takashi T Hirono Ikuo I Yu Hon-Tsen HT

BMC genomics 20110517

<h4>Background</h4>The black tiger shrimp (Penaeus monodon) is one of the most important aquaculture species in the world, representing the crustacean lineage which possesses the greatest species diversity among marine invertebrates. Yet, we barely know anything about their genomic structure. To understand the organization and evolution of the P. monodon genome, a fosmid library consisting of 288,000 colonies and was constructed, equivalent to 5.3-fold coverage of the 2.17 Gb genome. Approximate ...[more]

PMID: 21575266

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Project description:BackgroundLarge insert paired-end sequencing technologies are important tools for assembling genomes, delineating associated breakpoints and detecting structural rearrangements. To facilitate the comprehensive detection of inter- and intra-chromosomal structural rearrangements or variants (SVs) and complex genome assembly with long repeats and segmental duplications, we developed a new method based on single-molecule real-time synthesis sequencing technology for generating long paired-end sequences of large insert DNA libraries.ResultsA Fosmid vector, pHZAUFOS3, was developed with the following new features: (1) two 18-bp non-palindromic I-SceI sites flank the cloning site, and another two sites are present in the skeleton of the vector, allowing long DNA inserts (and the long paired-ends in this paper) to be recovered as single fragments and the vector (~?8 kb) to be fragmented into 2-3 kb fragments by I-SceI digestion and therefore was effectively removed from the long paired-ends (5-10 kb); (2) the chloramphenicol (Cm) resistance gene and replicon (oriV), necessary for colony growth, are located near the two sides of the cloning site, helping to increase the proportion of the paired-end fragments to single-end fragments in the paired-end libraries. Paired-end libraries were constructed by ligating the size-selected, mechanically sheared pooled Fosmid DNA fragments to the Ampicillin (Amp) resistance gene fragment and screening the colonies with Cm and Amp. We tested this method on yeast and Setaria italica Yugu1. Fosmid-size paired-ends with an average length longer than 2 kb for each end were generated. The N50 scaffold lengths of the de novo assemblies of the yeast and S. italica Yugu1 genomes were significantly improved. Five large and five small structural rearrangements or assembly errors spanning tens of bp to tens of kb were identified in S. italica Yugu1 including deletions, inversions, duplications and translocations.ConclusionsWe developed a new method for long paired-end sequencing of large insert libraries, which can efficiently improve the quality of de novo genome assembly and identify large and small structural rearrangements or assembly errors.

Project description:Among the emerging diseases in shrimp aquaculture, monodon slow growth syndrome (MSGS) is a major concern in South and Southeast Asia. Shrimp farming in Thailand was severely affected during 2000-2002 due to MSGS, which caused an economic loss, of about US$ 300 million. MSGS is characterized by abnormally slow growth with coefficients of size variation of >35 %, that has impacted P. monodon production in Thailand. A new shrimp virus, Laem-Singh virus (LSNV) was identified to be associated in MSGS affected shrimp. LSNV a RNA virus of about 25 nm diameter is phylogenetically related to the insect-borne viruses in the families Barnaviridae, Tymoviridae and Sobemoviridae an important histopathological observation is exclusively noticed in growth-retarded shrimp. The LSNV infections have been confirmed in various organs of infected shrimp such as lymphoid organ, gills and nervous tissues by various diagnostic techniques such as reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, quantitative real-time RT-PCR and reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick (RT-LAMP-LFD) and these tools are available for the diagnosis of LSNV. Recently, an integrase containing element has been identified in absolute association with LSNV in stunted growth shrimp. The transmission of LSNV through horizontal and vertical routes has been experimentally demonstrated. The known natural host-range of LSNV includes P. monodon and other penaeid shrimp. The putative RdRp gene involved in replication of LSNV was targeted for dsRNA-mediated gene silencing and appeared to be effective in a dose-dependent manner. Since the discovery of LSNV in 2006 in Thailand, it has been added to the list of viruses to be excluded from domesticated specific pathogen-free stocks of P. monodon and it has been recommended that shrimp farmers avoid stocking post larvae positive for LSNV to prevent MSGS in their farms.

Dataset Information

Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon.

Publications

Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon.

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