Genome and Phylogenetic Analysis of Genes Involved in the Immune System of Solea senegalensis - Potential Applications in Aquaculture

Abstract

Global aquaculture production continues to increase rapidly. One of the most important species of marine fish currently cultivated in Southern Europe is Solea senegalensis, reaching more than 300 Tn in 2017. In the present work, 14 Bacterial Artificial Chromosome (BAC) clones containing candidate genes involved in the immune system (b2m, il10, tlr3, tap1, tnfα, tlr8, trim25, lysg, irf5, hmgb2, calr, trim16, and mx), were examined and compared with other species using multicolor Fluorescence in situ Hybridization (mFISH), massive sequencing and bioinformatic analysis to determine the genomic surroundings and syntenic chromosomal conservation of the genomic region contained in each BAC clone. The mFISH showed that the groups of genes hmgb2-trim25-irf5-b2m; tlr3-lysg; tnfα-tap1, and il10-mx-trim16 were co-localized on the same chromosomes. Synteny results suggested that the studied BACs are placed in a smaller number of chromosomes in S. senegalensis that in other species. Phylogenetic analyses suggested that the evolutionary rate of immune system genes studied is similar among the taxa studied, given that the clustering obtained was in accordance with the accepted phylogenetic relationships among these species. This study contributes to a better understanding of the structure and function of the immune system of the Senegalese sole, which is essential for the development of new technologies and products to improve fish health and productivity.

Keywords: Solea senegalensis; aquaculture; bacterial artificial chromosome; immune system; syntenic conservation.

FIGURE 1

Results of mFISH of the BACs isolated with the following candidate genes: (a) tlr8 (blue), lysg (green), tap1 (pink), tnfα (orange); (b) irf5 (blue), calr (green), b2m (red); (c) trim 16 (green), mx (orange), calr (pink); (d) irf5 (blue), tlr3 (green); tnfα (orange), mx (pink); (e) hmgb2 (blue), lysg (green), b2m (pink); (f) trim25 (blue), tnfα (green), tap1 (orange), b2m (pink); (g) il10 (blue), tnfα (green), tap1 (orange), b2m (pink). In those cases where two or more probes co-localize in one chromosome, a diagrammatic representation is included.

FIGURE 2

BAC clones analyzed in the chromosomes of S. senegalensis. On chromosome 1 the BAC clone calr was localized; on chromosome 2 there were four BAC clones, with the genes b2ml, b2m, irak3, trim25, hmgb2, and irf5. On chromosomes 4 and 12 the BAC clone 30J4 was localized, with the genes tlr8, tlr7, and nlrc3. The BAC clones 4N21 and 29D4, with the lysg and tlr3 candidate genes, respectively, were found on chromosome 13. On chromosome 16, the BAC clones 53D20 and 71N11 were localized, with the genes tap, hla-drb1, tnfα and pycard. Lastly on chromosome 19 the BAC clones 42P4, 12K6 and 31C1 were localized, with the genes il10, il19, mx, trim16, ikbke.

FIGURE 3

Circos analysis in the species C. semilaevis. On the left side the distribution of the BAC clones of Senegalese sole distributed in chromosomes can be observed. Each BAC clone is represented in a different color. The genes found by annotation are indicated within each BAC clone and their corresponding localization in the C. semilaevis chromosomes is denoted by crossing lines. The BAC clones analyzed are given in Table 1.

FIGURE 4

Circos analysis in the species S. maximus. On the left side the distribution of the BAC clones of Senegalese sole distributed in chromosomes can be observed. Each BAC clone is represented in a different color. The genes found by annotation are indicated within each BAC clone and their corresponding localization in the S. maximus chromosomes are denoted by crossing lines. The BAC clones analyzed are given in Table 1.

FIGURE 5

Micro-synteny of 11 BAC clones. Genes of the species C. semilaevis, S. maximus, S. lalandi dorsalis, S. dumerili, G. aculeatus, O. niloticus, and D. rerio are represented in different colors. White color indicates non-synteny genes. (a) BAC clone il10; (b) BAC clone tlr3; (c) BAC clone hmgb2; (d) BAC clone lysg; (e) BAC clone nlrc3; (f) BAC clone b2m; (g) BAC clone tap1; (h) BAC clone trim25; (i) BAC clone calr; (j) BAC clone tnf alfa; (k) BAC clone tlr8.

FIGURE 6

Phylogenetic tree constructed from five immune-system genes (tlr3, tlr8, nlrc3, calr, ikbke) of thirty-four different species.