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Genome Sequencing of the Japanese Eel (Anguilla japonica) for Comparative Genomic Studies on tbx4 and a tbx4 Gene Cluster in Teleost Fishes - PubMed

  • ️Tue Jan 01 2019

Genome Sequencing of the Japanese Eel (Anguilla japonica) for Comparative Genomic Studies on tbx4 and a tbx4 Gene Cluster in Teleost Fishes

Weiwei Chen et al. Mar Drugs. 2019.

Abstract

Limbs originated from paired fish fins are an important innovation in Gnathostomata. Many studies have focused on limb development-related genes, of which the T-box transcription factor 4 gene (tbx4) has been considered as one of the most essential factors in the regulation of the hindlimb development. We previously confirmed pelvic fin loss in tbx4-knockout zebrafish. Here, we report a high-quality genome assembly of the Japanese eel (Anguilla japonica), which is an economically important fish without pelvic fins. The assembled genome is 1.13 Gb in size, with a scaffold N50 of 1.03 Mb. In addition, we collected 24 tbx4 sequences from 22 teleost fishes to explore the correlation between tbx4 and pelvic fin evolution. However, we observed complete exon structures of tbx4 in several pelvic-fin-loss species such as Ocean sunfish (Mola mola) and ricefield eel (Monopterus albus). More interestingly, an inversion of a special tbx4 gene cluster (brip1-tbx4-tbx2b- bcas3) occurred twice independently, which coincides with the presence of fin spines. A nonsynonymous mutation (M82L) was identified in the nuclear localization sequence (NLS) of the Japanese eel tbx4. We also examined variation and loss of hindlimb enhancer B (HLEB), which may account for pelvic fin loss in Tetraodontidae and Diodontidae. In summary, we generated a genome assembly of the Japanese eel, which provides a valuable genomic resource to study the evolution of fish tbx4 and helps elucidate the mechanism of pelvic fin loss in teleost fishes. Our comparative genomic studies, revealed for the first time a potential correlation between the tbx4 gene cluster and the evolutionary development of toxic fin spines. Because fin spines in teleosts are usually venoms, this tbx4 gene cluster may facilitate the genetic engineering of toxin-related marine drugs.

Keywords: Japanese eel (Anguilla japonica); fin spine; genome sequencing and assembly; pelvic fin; tbx4; tbx4 gene cluster; teleost fish.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

A 17-mer distribution of the Japanese eel genome sequencing. Only the sequencing data from short-insert libraries (500 and 800 bp) were used for the k-mer analysis. The x-axis is the sequencing depth of each unique 17-mer, and the y-axis is the percentage of these unique 17-mers. The peak depth (K_depth) is 37, and the corresponding k-mer number (N) is 37,982,773,125. We therefore calculated the genome size (G) to be ~1.03 Gb based on the following formula [12]: G=N/K_depth.

Figure 2
Figure 2

Structures of the tbx4 genes in various vertebrate species. Green boxes and lines represent exons and introns, respectively. Numbers inside the boxes are the exact amino acid numbers, indicating their similarity among various species.

Figure 3
Figure 3

Similarity of the T-box domain in the tbx4 genes of various vertebrate species. The blue color represents conserved sites. The yellow color represents the similarity >80% and white shows less conserved sites. The colored dots beneath the sequence alignment indicate conservation track, ranging from blue (non-conserved) to red (the most conserved). The red box highlights the NLS region of the tbx4 genes. A red arrow indicates a nonsynonymous mutation in the Japanese eel tbx4 gene.

Figure 4
Figure 4

Visualization of the tbx4 NLS regions in different vertebrate species using Bioedit (Tom Hall Ibis Therapeutics, Carlsbad, CA, USA). Red characters represent the codons and amino acids of nonsynonymous mutations.

Figure 5
Figure 5

Alignment of the TBX4 protein sequences of Japanese eel against zebrafish. The red box highlights the NLS regions (same as Figure 4).

Figure 6
Figure 6

Phylogenetic and synteny comparisons of the tbx4 genes in vertebrates. The figure in the left is a Bayesian tree. Numbers on the branches are bootstrap supports (black) obtained from the phyML-3.1 reconstruction. Spotted gar was used as the outgroup. The figure in the right represents the synteny of tbx4. Distances between genes and the gene length are not drawn to scale. The red branches and the two black boxes highlight two remarkable inversions of the brip1-tbx4-tbx2b-bcas3 cluster in teleost species. The five-point star represents the determined pelvic fin loss in the examined species.

Figure 7
Figure 7

The brip1-tbx4-tbx2b-bcas3 cluster. Colored boxes and lines represent genes and intergenic regions, respectively. The distance between two adjacent genes is indicated underneath the lines, while the length of exons is drawn to scale. Genes in the same orientation as tbx4 are marked above the horizontal lines; however, genes in the opposite orientation are placed below the lines.

Figure 8
Figure 8

A VISTA plot to compare the HLEB sequences of Acanthopterygii fishes against the reported 873-bp HLEB from three-spined stickleback (Gasterosteus aculeatus). Sequence identity along the y-axis, ranging from 50% to 100%, is shown in 100-bp sliding windows across the examined region (x-axis, bp). The pink shadows stand for regions with <100-bp continuous bases at ≥70% identity.

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