High Polymorphism in the Dmrt2a Gene Is Incompletely Sex-Linked in Spotted Scat, Scatophagus argus

SIMPLE SUMMARY: Fishes have relatively younger chromosomes than mammals and birds, making them excellent models to study sex chromosome differentiation in teleosts. The spotted scat has a young chromosome, making it an ideal model to study the evolution of sex chromosomes in vertebrates. The doublesex and Mab-3-related transcription factor 1 (Dmrt1) is the candidate sex determination gene in spotted scat, while the differentiation of other sex-linked genes remains unknown. Dmrt2a is positioned close to Dmrt3 and farther away from Dmrt1 on the same chromosome, while Dmrt2b is not. Dmrt2a is highly expressed in testicular tissues, with several intronic and exonic mutations that do not affect gene translation. Using intronic markers, we found in 4 different populations that Dmrt2a is incompletely sex-linked. Incomplete sex linkage of markers suggests the absence of recombination depression in this region and indicates that the system of sex chromosomes is still young. This suggests that Dmrt2a might be necessary for developmental processes. Other possibilities are that the conservation differences between Dmrt1, Dmrt3, and Dmrt2a might be due to gene dosage effect, or specialization of truncated copies of Dmrt1 and Dmrt3 in the regulation of normal gene copies. The partial sex linkage of Dmrt2a suggests an excellent model for studying sex-linked gene differentiation during vertebrate evolution. Therefore, further studies are needed to identify the regulators of Dmrt2a expression, sequences of X and Y copies, and their functions in spotted scat. ABSTRACT: Unlike mammals and birds, many fishes have young sex chromosomes, providing excellent models to study sex chromosome differentiation at early stages. Previous studies showed that spotted scat possesses an XX-XY sex determination system. The X has a complete Dmrt3 copy (termed normal) and a truncated copy of Dmrt1 (called Dmrt1b), while the Y has the opposite (normal Dmrt1, which is male-specific, and a truncated Dmrt3 called Dmrt3△-Y). Dmrt1 is the candidate sex determination gene, while the differentiation of other sex-linked genes remains unknown. The spotted scat has proven to be a good model to study the evolution of sex chromosomes in vertebrates. Herein, we sequenced a neighbor gene of this family, Dmrt2, positioned farther from Dmrt1 and closer to Dmrt3 in the spotted scat, and analyzed its sequence variation and expression profiles. The physical locations of the three genes span across an estimated size of >40 kb. The open reading frames of Dmrt2a and its paralog Dmrt2b are 1578 bp and 1311 bp, encoding peptides of 525 and 436 amino acid residues, respectively. Dmrt2a is positioned close to Dmrt3 but farther from Dmrt1 on the same chromosome, while Dmrt2b is not. Sequence analysis revealed several mutations; insertions, and deletions (indels) on Dmrt2a non-coding regions and single-nucleotide polymorphisms (SNPs) on the Dmrt2a transcript. These indels and SNPs are sex-linked and showed high male heterogeneity but do not affect gene translation. The markers designed to span the mutation sites tested on four different populations showed varied concordance with the genetic sexes. Dmrt2a is transcribed solely in the gonads and gills, while Dmrt2b exists in the gonads, hypothalamus, gills, heart, and spleen. The Dmrt2a and Dmrt2b transcripts are profoundly expressed in the male gonads. Analyses of the transcriptome data from five other fish species (Hainan medaka (Oryzias curvinotus), silver sillago (Sillago sihama), Nile tilapia (Oreochromis niloticus), Hong Kong catfish (Clarias fuscus), and spot-fin porcupine fish (Diodon hystrix)) revealed testes-biased expression of Dmrt1 in all, similar to spotted scat. Additionally, the expression of Dmrt2a is higher in the testes than the ovaries in spotted scat and Hainan medaka. The Dmrt2a transcript was not altered in the coding regions as found in Dmrt1 and Dmrt3 in spotted scat. This could be due to the functional importance of Dmrt2a in development. Another possibility is that because Dmrt2a is positioned farther from Dmrt1 and the chromosome is still young, meaning it is only a matter of time before it differentiates. This study undeniably will aid in understanding the functional divergence of the sex-linked genes in fish.