Characterization of the rainbow trout spleen transcriptome and identification of immune-related genes

Abstract

Resistance against diseases affects profitability of rainbow trout. Limited information is available about functions and mechanisms of teleost immune pathways. Immunogenomics provides powerful tools to determine disease resistance genes/gene pathways and develop genetic markers for genomic selection. RNA-Seq sequencing of the rainbow trout spleen yielded 93,532,200 reads (100 bp). High quality reads were assembled into 43,047 contigs. 26,333 (61.17%) of the contigs had hits to the NR protein database and 7024 (16.32%) had hits to the KEGG database. Gene ontology showed significant percentages of transcripts assigned to binding (51%), signaling (7%), response to stimuli (9%) and receptor activity (4%) suggesting existence of many immune-related genes. KEGG annotation revealed 2825 sequences belonging to "organismal systems" with the highest number of sequences, 842 (29.81%), assigned to immune system. A number of sequences were identified for the first time in rainbow trout belonging to Toll-like receptor signaling (35), B cell receptor signaling pathway (44), T cell receptor signaling pathway (56), chemokine signaling pathway (73), Fc gamma R-mediated phagocytosis (52), leukocyte transendothelial migration (60) and NK cell mediated cytotoxicity (42). In addition, 51 transcripts were identified as spleen-specific genes. The list includes 277 full-length cDNAs. The presence of a large number of immune-related genes and pathways similar to other vertebrates suggests that innate and adaptive immunity in fish are conserved. This study provides deep-sequence data of rainbow trout spleen transcriptome and identifies many new immune-related genes and full-length cDNAs. This data will help identify allelic variations suitable for genomic selection and genetic manipulation in aquaculture.

Keywords: KEGG; annotation; full-length cDNA; immune-related genes; spleen transcriptome; spleen-specific genes.

Figure 1

Comparison of contig length with and without BLASTX hits to the NR database.

Figure 2

Gene ontology (GO) assignments (2nd level GO terms) for the spleen transcriptomic sequences of rainbow trout. (A) Refers to biological process, (B) refers to molecular function and (C) refers to cellular component.

Figure 3

Gene Ontology (2nd level GO terms) comparison of rainbow trout whole reference and spleen transcriptomes. GO comparison shows a higher representation of signaling, response to stimuli, binding and receptor activity in spleen compared to whole reference transcriptome. (A) Refers to biological process, (B) refers to molecular function and (C) refers to cellular component.

Figure 4

BLASTX top-hit species distribution of gene annotations showing high homology to fish species with known genome sequences.

Figure 5

Number of all annotated sequences obtained from the different KEGG immune pathway mapping.

Figure 6

Toll-like receptor signaling pathway showing the annotated and non-annotated proteins. Proteins appearing in our transcriptome are represented in green color and absent proteins in white color.

Figure 7

B cell receptor signaling pathway showing the annotated and non-annotated proteins. Proteins appearing in our transcriptome are represented in green color and absent proteins in white color.

Figure 8

T cell receptor signaling pathway showing the annotated and non-annotated proteins. Proteins appearing in our transcriptome are represented in green color and absent proteins in white color.

Figure 9

Chemokine signaling pathway showing the annotated and non-annotated proteins. Proteins appearing in our transcriptome are represented in green color and absent proteins in white color.

Figure 10

Fc gamma R-mediated phagocytosis showing the annotated and non-annotated proteins. Proteins appearing in our transcriptome are represented in green color and absent proteins in white color.

Figure 11

Leukocyte transendothelial migration showing the annotated and non-annotated proteins. Proteins appearing in our transcriptome are represented in green color and absent proteins in white color.

Figure 12

Natural killer cell mediated cytotoxicity showing the annotated and non-annotated proteins. Proteins appearing in our transcriptome are represented in green color and absent proteins in white color.

Figure 13

Mean of RPKM of the spleen-specific genes in 13 tissues of rainbow trout. The mean RPKM values of the spleen-specific genes were 5491, 973, and 400 in spleen, kidney and fat, respectively.