Transcriptome Analysis of the Oriental River Prawn, <em>Macrobrachium nipponense</em> Using 454 Pyrosequencing for Discovery of Genes and Markers

<div><h3>Background</h3><p>The oriental river prawn, <em>Macrobrachium nipponense</em>, is an economically and nutritionally important species of the Palaemonidae family of decapod crustaceans. To date, the sequencing of its whole genome is unavailable as a non-model organism. Transcriptomic information is also scarce for this species. In this study, we performed <em>de novo</em> transcriptome sequencing to produce the first comprehensive expressed sequence tag (EST) dataset for <em>M. nipponense</em> using high-throughput sequencing technologies.</p> <h3>Methodology and Principal Findings</h3><p>Total RNA was isolated from eyestalk, gill, heart, ovary, testis, hepatopancreas, muscle, and embryos at the cleavage, gastrula, nauplius and zoea stages. Equal quantities of RNA from each tissue and stage were pooled to construct a cDNA library. Using 454 pyrosequencing technology, we generated a total of 984,204 high quality reads (338.59Mb) with an average length of 344 bp. Clustering and assembly of these reads produced a non-redundant set of 81,411 unique sequences, comprising 42,551 contigs and 38,860 singletons. All of the unique sequences were involved in the molecular function (30,425), cellular component (44,112) and biological process (67,679) categories by GO analysis. Potential genes and their functions were predicted by KEGG pathway mapping and COG analysis. Based on our sequence analysis and published literature, many putative genes involved in sex determination, including <em>DMRT1</em>, <em>FTZ-F1</em>, <em>FOXL2</em>, <em>FEM1</em> and other potentially important candidate genes, were identified for the first time in this prawn. Furthermore, 6,689 SSRs and 18,107 high-confidence SNPs were identified in this EST dataset.</p> <h3>Conclusions</h3><p>The transcriptome provides an invaluable new data for a functional genomics resource and future biological research in <em>M. nipponense</em>. The molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will be essential for accelerating aquaculture breeding programs with this species.</p> </div

Complete cDNA sequence and deduced amino acid sequence of <i>H. cumingii</i> ferritin.

<p>The IRE in the 5′ UTR is shaded. Seven residues in bold and italics represent a putative active site of ferroxidase. The iron associated residue Tyr27 is in bold and underlined. Iron-binding region signature 1 is in bold. The star represents the stop codon. The poly(A) signal is underlined.</p

Multiple alignments of <i>H. cumingii</i> ferritin with homolog from other mollusk species and human.

<p>Identical amino acid residues are darkly shaded, similar amino acids are lightly shaded, unrelated residues have a white background. <i>C. gigas</i>, AAP83794.1; <i>Crassostrea ariakensis</i>, ABE99842.1; <i>P. fucata</i>, AAQ12076.1; <i>Pinctada maxima</i>, ACS72281.1; <i>Chlamys farreri</i>, AAV66906.1; <i>A. irradians</i>, AAV66907.1; <i>Mytilus galloprovincialis</i>, ACM86786.1; <i>Sinonovacula constricta</i>, ACZ65230.1; <i>Venerupis decussatus</i>, ACB38006.1; <i>M. meretrix</i>, AAZ20754.1; <i>Haliotis rufescens</i>, ACZ73270.1; <i>H. discus hannai</i>, ABH10672.1; <i>H. diversicolor</i>, ABY87353.1; <i>Lymnaea stagnalis</i>, P42577.2; <i>Aplysia californica</i>, ABF21074.1; <i>Enteroctopus dofleini</i>, AAD29639.1; <i>Homo sapiens</i>, AAA52437.1.</p

Gene Ontology (GO) terms for the transcriptomic sequences of <i>M. nipponense</i>.

<p>Gene Ontology (GO) terms for the transcriptomic sequences of <i>M. nipponense</i>.</p

Clusters of orthologous groups (COG) classifications of unique <i>M. nipponense</i> sequences.

<p>1: RNA processing and modification; 2: chromatin structure and dynamics; 3: energy production and conversion; 4: cell cycle control, cell division, chromosome partitioning; 5: amino acid transport and metabolism; 6: nucleotide transport and metabolism; 7: carbohydrate transport and metabolism; 8: coenzyme transport and metabolism; 9: lipid transport and metabolism; 10: translation, ribosomal structure and biogenesis; 11: transcription; 12: replication, recombination and repair; 13: cell wall/membrane/envelope biogenesis; 14: cell motility; 15: posttranslational modification, protein turnover, chaperones; 16: inorganic ion transport and metabolism; 17: secondary metabolites biosynthesis, transport and catabolism; 18: general function prediction only; 19: function unknown; 20: signal transduction mechanisms; 21: intracellular trafficking, secretion, and vesicular transport; 22: defense mechanisms; 23: nuclear structure; and 24: cytoskeleton.</p

Distribution of simple sequence repeat (SSR) nucleotide classes among different nucleotide types found in the transcriptome of <i>M. nipponense.</i>

<p>Both contig and singleton sequences are used to predict the SSR loci.</p

Primers used and verified SNPs in the ESTs of <i>M. nipponense</i>.

<p>For each contig, the following are indicated: the number of SNPs verified/the number of SNPs predicted in the fragment/the number of additional SNPs using Sanger technology; n/a indicates that sequencing was not possible because the sequencing signal was interrupted.</p

Length distribution of <i>M. nipponense</i> transcriptomic ESTs.

<p>(<b>A</b>) <b>total transcriptomic reads, (B) contigs, (C) singletons.</b></p

Genotype frequencies of diploid copy number in ferritin gene in wild (PY) and cultured (JH) populations of <i>H. cumingii</i>.

<p>Genotype frequencies of diploid copy number in ferritin gene in wild (PY) and cultured (JH) populations of <i>H. cumingii</i>.</p

Neighbor-joining phylogenetic tree of ferritins from mollusk species.

<p><i>C. gigas</i>, AAP83794.1; <i>C. ariakensis</i>, ABE99842.1; <i>P. fucata</i>, AAQ12076.1; <i>P. maxima</i>, ACS72281.1; <i>C. farreri</i>, AAV66906.1; <i>A. irradians</i>, AAV66907.1; <i>M. galloprovincialis</i>, ACM86786.1; <i>S. constricta</i>, ACZ65230.1; <i>V.s decussatus</i>, ACB38006.1; <i>M. meretrix</i>, AAZ20754.1; <i>H. rufescens</i>, ACZ73270.1; <i>H. discus hannai</i>, ABH10672.1; <i>H. diversicolor</i>, ABY87353.1; <i>L. stagnalis</i>, P42577.2; <i>A. californica</i>, ABF21074.1; <i>E. dofleini</i>, AAD29639.1; <i>E. dofleini</i>, AAD29639.1.</p