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. 2014 Apr 8;9(4):e94046.
doi: 10.1371/journal.pone.0094046. eCollection 2014.

Characterization of the miiuy croaker (Miichthys miiuy) transcriptome and development of immune-relevant genes and molecular markers

Rongbo Che  1 , Yueyan Sun  1 , Dianqiao Sun  1 , Tianjun Xu  1
Affiliations

Characterization of the miiuy croaker (Miichthys miiuy) transcriptome and development of immune-relevant genes and molecular markers

Rongbo Che et al. PLoS One. .

Abstract

Background: The miiuy croaker (Miichthys miiuy) is an important species of marine fish that supports capture fisheries and aquaculture. At present commercial scale aquaculture of this species is limited due to diseases caused by pathogens and parasites which restrict production and limit commercial value. The lack of transcriptomic and genomic information for the miiuy croaker limits the ability of researchers to study the pathogenesis and immune system of this species. In this study we constructed a cDNA library from liver, spleen and kidney which was sequenced using Illumina paired-end sequencing to enable gene discovery and molecular marker development.

Principal findings: In our study, a total of 69,071 unigenes with an average length of 572 bp were obtained. Of these, 45,676 (66.13%) were successfully annotated in public databases. The unigenes were also annotated with Gene Ontology, Clusters of Orthologous Groups and KEGG pathways. Additionally, 498 immune-relevant genes were identified and classified. Furthermore, 14,885 putative simple sequence repeats (cSSRs) and 8,510 putative single nucleotide polymorphisms (SNPs) were identified from the 69,071 unigenes.

Conclusion: The miiuy croaker (Miichthys miiuy) transcriptome data provides a large resource to identify new genes involved in many processes including those involved in the response to pathogens and diseases. Furthermore, the thousands of potential cSSR and SNP markers found in this study are important resources with respect to future development of molecular marker assisted breeding programs for the miiuy croaker.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Analysis of Illumina short read assembly quality.
(A) Size distributions of de novo assembled contigs, scaffolds, and unigenes. (B) Gap distributions of assembled scaffolds and unigenes.
Figure 2
Figure 2. Evaluation and Validation of assembled transcripts.
(A) Coverage distribution of assembled unigenes. (B) Distribution of unique mapped reads of the assembled unigenes.
Figure 3
Figure 3. RT-PCR amplification and agarose gel (1%) electrophoresis of twelve transcripts.
The corresponding run lanes of Unigene 5, Unigene 62288, Unigene 10257, Unigene 68579, Unigene 10014, Unigene 68682, Unigene 20918, Unigene 69000, Unigene 10657, Unigene 4806, Unigene 20628 and Unigene 21126 are from 1 to 12, respectively. And the amplification lengths of these unigenes were given in table 2.
Figure 4
Figure 4. Comparison of the number of unigene annotations obtained from the different databases.
The number of unigene annotations hits from the NR, NT, Swiss-Prot and KEGG databases (E-value ≤1.0E-5), respectively.
Figure 5
Figure 5. Characteristics of homology search of assembled unigenes against NR databases.
(A) E-value distribution of BLAST hits for each unigene with a cutoff E-value of 1.0E-5. (B) Identity distribution of the top BLAST hits for each unigene. (C) BLASTx top-hit species distribution of gene annotations against NR databases. (D) Length of unigenes with hits compared with those without hits.
Figure 6
Figure 6. Gene Ontology classifications of assembled unigenes.
Unigenes were assigned to three classifications: (A) biological processes, (B) cellular components and (C) molecular functions. In total, 8,423 unigenes with BLAST matches to known proteins were assigned to gene ontology.
Figure 7
Figure 7. Histogram presentation of clusters of orthologous groups (COG) classification.
A total of 21,662 unigenes were assigned the Cluster of Orthologous Groups classification and classified into 25 functional categories (E-value ≤1.0E-5).
Figure 8
Figure 8. Pathway assignment based on KEGG.
(A) Distribution of assembled unigenes based on 6 main categories pathways. (B) Classification based on Immune System classification.
Figure 9
Figure 9. Frequency distribution of cSSRs based on motif sequence types.
The most abundant repeat motif in our cSSRs was AC/GT (41.90%), followed by AG/CT (15.91%), AGG/CCT (7.12%) and AGC/CTG (4.37%) among 284 motif sequence types.
Figure 10
Figure 10. Frequency distribution of SNPs based on different types.
A total of 8,510 putative single nucleotide polymorphisms (SNPs) included 2,328 transversions and 6,182 transitions.
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References

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Grants and funding

This study was supported by Nation Nature Science Foundation of China (31370049) and Important Science and Technology Specific Projects of Zhejiang Province (2011C14012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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