Comparative Genomics Studies on the dmrt Gene Family in Fish

Abstract

Doublesex and mab-3-related transcription factor (dmrt) genes are widely distributed across various biological groups and play critical roles in sex determination and neural development. Here, we applied bioinformatics methods to exam cross-species changes in the dmrt family members and evolutionary relationships of the dmrt genes based on genomes of 17 fish species. All the examined fish species have dmrt1-5 while only five species contained dmrt6. Most fish harbored two dmrt2 paralogs (dmrt2a and dmrt2b), with dmrt2b being unique to fish. In the phylogenetic tree, 147 DMRT are categorized into eight groups (DMRT1-DMRT8) and then clustered in three main groups. Selective evolutionary pressure analysis indicated purifying selections on dmrt1-3 genes and the dmrt1-3-2(2a) gene cluster. Similar genomic conservation patterns of the dmrt1-dmrt3-dmrt2(2a) gene cluster with 20-kb upstream/downstream regions in fish with various sex-determination systems were observed except for three regions with remarkable diversity. Synteny analysis revealed that dmrt1, dmrt2a, dmrt2b, and dmrt3-5 were relatively conserved in fish during the evolutionary process. While dmrt6 was lost in most species during evolution. The high conservation of the dmrt1-dmrt3-dmrt2(2a) gene cluster in various fish genomes suggests their crucial biological functions while various dmrt family members and sequences across fish species suggest different biological roles during evolution. This study provides a molecular basis for fish dmrt functional analysis and may serve as a reference for in-depth phylogenomics.

Keywords: comparative genomics studies; dmrt genes; fish; phylogenetic evolution; synteny analysis.

FIGURE 1

Characterization and phylogenetic analysis of the dmrt family in vertebrates. (A) Various structures of dmrt genes. The genomic structures of dmrt1–dmrt5 are based on the data of Japanese pufferfish, whereas those of dmrt6–dmrt8 are derived from the relevant data of mouse. (B) A Bayesian phylogenetic tree of 147 dmrt sequences. The phylogenetic analysis was performed using MrBayes v3.2.6. Amino acid replacement model selection was calculated using ProtTest with the best-fit model of JTT+I+G+F. The tree is rooted with the N. vectensis dmrtA.

FIGURE 2

Phylogenetic tree and synteny conservation of the fish dmrt1-dmrt3-dmrt2(2a) gene clusters. (A) The rectangular Bayesian phylogenetic tree, rooted with human and mouse sequences. Numbers besides the nodes are Bayesian posterior probabilities (colored). (B) The synteny of examined dmrt1-dmrt3-dmrt2(2a) gene clusters. Full names of relevant genes are provided in Supplementary Table S3.

FIGURE 3

Ka (A) and Ka/Ks (B) values for the target coding sequences. Paralog genes in the spotted gar were used as the references. **p < 0.01; ***p < 0.001; and ****p < 0.0001 (based on a Wilcoxon test). Four methods, including GMYN, GYN, MYN, and YN, were used.

FIGURE 4

Species variances of the regulatory regions in the dmrt1-dmrt3-dmrt2(2a) gene cluster. (A) A VISTA plot of the dmrt1-dmrt3-dmrt2(2a) gene clusters among six examined fish species. Peaks of similarity in pair-wise sequence alignments between zebrafish (D. rerio) are compared with Chinese tongue sole (C. semilaevis), Japanese medaka (O. latipes), southern platyfish (X. maculatus), Asian sea bass (L. calcarifer), European sea bass (D. labrax), and largemouth bass (M. salmoides). Blue peaks represent coding exons and pink peaks denote non-coding sequences. The horizontal axis shows relative positions in the zebrafish genomic sequence, whereas the vertical axis indicates percentage of identity (50–100%). Three distinct regions, named as Region 1, Region 2, and Region 3, were identified. (B) Protein sequence alignments of the Regions 2 and 3 in 17 examined fish species. (C) Numbers of different regulatory elements in six representative fish species. Additional details regarding the sequences of each regulatory element are provided in Supplementary Table S4.

FIGURE 5

Synteny of dmrt genes in various fish species. The synteny analyses were performed in eight vertebrate species (M. salmoides, O. niloticus, T. rubripes, O. latipes, I. punctatus, L. oculatus, A. carolinensis, and H. sapiens). Chr., chromosome; LG, linkage group. The most conserved surrounding genes of dmrt genes were shown in green and bold fonts. The most conserved were shown in red fonts.

FIGURE 6

Hypothetical evolutionary history of the dmrt genes in fish through fish-specific (3R) genome duplications. This figure was constructed based on figures in this study. The common ancestor of chordate might possess four ancestral genes, including dmrt4/5, dmrt2a/2b, dmrt93B, and dmrt1/6. A common ancestor of vertebrata may have possessed four dmrt family genes, dmrt1/6, dmrt2a/2b, dmrt3, and dmrt4/5. The syntenies of kank1-dmrt1-dmrt3-dmrt2a, dmrt4-elavl2-caap1, kank4-lrp8-dmrt2b, and dmrt5- elavl4 are conserved after three rounds of whole genome duplication in the ancestral vertebrates. dmrt6 is lost in most fish species.