Marine Biotechnology

, Volume 14, Issue 5, pp 620–633 | Cite as

Expressed Sequences and Polymorphisms in Rohu Carp (Labeo rohita, Hamilton) Revealed by mRNA-seq

  • Nicholas Robinson
  • Pramoda K. Sahoo
  • Matthew Baranski
  • Kanta Das Mahapatra
  • Jatindra N. Saha
  • Sweta Das
  • Yashowant Mishra
  • Paramananda Das
  • Hirak K. Barman
  • Ambekar E. Eknath
Original Article

Abstract

Expressed genes and polymorphisms were identified in lines of rohu Labeo rohita selected for resistance or susceptibility to Aeromonas hydrophila, an important bacterial pathogen causing aeromoniasis. All animals were grown in a common environment and RNA from ten individuals from each line pooled for Illumina mRNA-seq. De novo transcriptome assembly produced 137,629 contigs with 40× average coverage. Forty-four percent of the assembled sequences were annotated with gene names and ontology terms. Of these, 3,419 were assigned biological process terms related to “stress response” and 1,939 “immune system”. Twenty-six contigs containing 38 single nucleotide polymorphisms (SNPs) were found to map to the Cyprinus carpio mitochondrial genome and over 26,000 putative SNPs and 1,700 microsatellite loci were detected. Seventeen percent of the 100 transcripts with coverage data most indicative of higher-fold expression (>5.6 fold) in the resistant line pool showed homology to major histocompatibility (MH), heat shock proteins (HSP) 30, 70 and 90, glycoproteins or serum lectin genes with putative functions affecting immune response. Forty-one percent of these 100 transcripts showed no or low homology to known genes. Of the SNPs identified, 96 showing the highest allele frequency differences between susceptible and resistant line fish included transcripts with homology to MH class I and galactoside-binding soluble lectin, also with putative functions affecting innate and acquired immune response. A comprehensive sequence resource for L. rohita, including annotated microsatellites and SNPs from a mixture of A. hydrophila-susceptible and -resistant individuals, was created for subsequent experiments aiming to identify genes associated with A. hydrophila resistance.

Keywords

Labeo rohita Transcriptome expression Single-nucleotide polymorphism Disease resistance Aeromonas hydrophila 

Notes

Acknowledgements

This work was supported by the Norwegian Research Council (project number 183833), Department of Biotechnology of the Government of India, the Central Institute of Freshwater Aquaculture and Nofima. The authors would like to thank Steve Carson who provided computer help and advice, Thomas Moen for his assistance with analysis scripts, and the staff at Genotypic Technology Pty Ltd in Bangalore India (especially Anand Damodaran) who prepared the RNA and organised for the sequencing of samples. The authors are thankful to Dr. S Ayyappan, Director General, Indian Council of Agricultural Research, for his guidance and constant support.

Supplementary material

10126_2012_9433_MOESM1_ESM.doc (150 kb)
ESM 1 Gene ontology annotations for biological processes assigned to L. rohita contigs (DOC 150 kb)
10126_2012_9433_MOESM2_ESM.doc (169 kb)
ESM 2 Top 100 most highly differentially expressed transcripts in resistant line fish. M, copy number ratio. N, number of BLASTx hits with E-value exceeding 0.001 out of top 10 hits. E-value, minimum E-value out of the top ten hits. Similarity, mean BLASTx similarity. Sequence length, contig sequence length (DOC 169 kb)
10126_2012_9433_MOESM3_ESM.doc (182 kb)
ESM 3 SNPs showing allele frequency differences >0.95 between resistant and susceptible line animals. SNP position, base position where SNP occurs along contig sequence. N, number of times allele was sequenced (DOC 182 kb)
10126_2012_9433_MOESM4_ESM.xlsx (16 kb)
ESM 4 Contigs and SNPs mapping to the mitochondrial genome. SNP position in each contig and in the overall mitDNA reference sequence is indicated in base pairs (XLSX 15 kb)
10126_2012_9433_MOESM5_ESM.xlsx (12 kb)
ESM 5 Occurrence of detected microsatellite unit types showing the number of repetitions (5–41 times) (XLSX 12 kb)
10126_2012_9433_MOESM6_ESM.xlsx (90 kb)
ESM 6 Microsatellite repeat array, contig location, primer sequence and gene identification. C, complex repeating units. P, perfect repeating units. Start and end indicate positions in base pairs of microsatellites along each contig (XLSX 90 kb)

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Nicholas Robinson
    • 1
    • 3
    • 4
  • Pramoda K. Sahoo
    • 2
  • Matthew Baranski
    • 1
  • Kanta Das Mahapatra
    • 2
  • Jatindra N. Saha
    • 2
  • Sweta Das
    • 2
  • Yashowant Mishra
    • 2
  • Paramananda Das
    • 2
  • Hirak K. Barman
    • 2
  • Ambekar E. Eknath
    • 2
  1. 1.NofimaÅsNorway
  2. 2.Central Institute of Freshwater AquacultureBhubaneswarIndia
  3. 3.Flinders UniversityAdelaideAustralia
  4. 4.MelbourneAustralia

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