Molecular identification of dmrt1 and its promoter CpG methylation in correlation with gene expression during gonad development in Culter alburnus.

Dmrt1, a member of the Dmrt family, is an important transcription regulator of gender determination. To study the biological function of dmrt1 in sexual differentiation and its potential implication in breeding technology, we obtained the full-length cDNA and proximal promoter sequence of dmrt1 in Culter alburnus, and analyzed the impact of promoter CpG methylation on the gene expression pattern of dmrt1 during gonad development. Dmrt1 was 922 bp in length and consisted a 150 bp 5'-UTR, a 28 bp 3'-UTR, and a 744 bp open reading frame (ORF). Based on the coding sequence of the dmrt1 gene, the deduced amino acid sequence was detected, and the protein structure of this gene was predicted in C. alburnus. The results indicate that the structure and function of dmrt1 were highly conservative compared to other vertebrates. The expression level of dmrt1 mRNA in different tissues was explored by qRT-PCR, which was only highly expressed in the testes and almost undetectable in other tissues. The CpG methylation pattern of the dmrt1 promoter was studied using DNA sequencing of sodium bisulfite in adult testes and ovaries, and it was found that dmrt1 promoter CpGs were not methylated in the testes, whereas hypermethylated in the ovaries. These findings demonstrate that DNA methylation can regulate sexual dimorphic expression of dmrt1, and therefore epigenetic modifications may play a critical role in the gonad differentiation of C. alburnus.

A Chromosome-Level Genome Assembly of the Dark Sleeper Odontobutis potamophila.

The dark sleeper, Odontobutis potamophila, is a commercially valuable fish that widely distributed in China and Southeast Asia countries. The phenomenon of sexual dimorphism in growth is conspicuous, which the males grow substantially larger and faster than the females. However, the high-quality genome resources for gaining insight into sex-determining mechanisms to develop sex-control breeding are still lacking. Here, a chromosomal-level genome assembly of O. potamophila was generated from a combination of Illumina reads, 10× Genomics sequencing, and Hi-C chromatin interaction sequencing. The assembled genome was 1,134.62 Mb with a contig N50 of 22.25 Mb and a scaffold N50 of 24.85 Mb, representing 94.4% completeness (Benchmarking Universal Single-Copy Orthologs). Using Hi-C data, 96.49% of the total contig bases were anchored to the 22 chromosomes, with a contig N50 of 22.25 Mb and a scaffold N50 of 47.68 Mb. Approximately 54.18% of the genome were identified as repetitive elements, and 23,923 protein-coding genes were annotated in the genome. The assembled genome can be used as a valuable resource for molecular breeding and functional studies of O. potamophila in the future.

Molecular characterization and expression profiles of transcription factor Sox gene family in Culter alburnus.

Sox protein family is characterized by the presence of the conserved high-mobility group (HMG) box. Sox transcription factors are involved in diverse developmental process in animals, including sex-determination, organogenesis, embryogenesis, neurogenesis, and cell fate decision. In this study, 23 Sox genes were identified based on the Culter alburnus whole-genome sequence and categorized into six subfamilies according to the conserved HMG-box domain. The duplicates of four members revealed that Sox genes in the teleost fishes underwent significant expansion. Moreover, their expression pattern in gonad tissues were analyzed by RNA-seq and qRT-PCR, and Sox9b was determined as a key gene that was essential for testis development. This current study will provide new insight into the role of Sox gene family in fish sex determination and differentiation.

Identification and functional analysis of the doublesex gene in the redclaw crayfish, Cherax quadricarinatus.

DM-domain (Zn-finger motif domain) genes play an important role in the sex determination and differentiation among animal kingdom. In the present study, the gene of Doublesex (Cqdsx) was identified and characterized for the first time in the redclaw crayfish, Cherax quadricarinatus. The full-length cDNA was 1271 bp, comprising a 155 bp 5'-untranslated region (5'-UTR), an 885 bp predicted open reading frame (ORF) encoding 294 amino acid polypeptides, and a 231 bp 3'-UTR. The deduced amino acid sequence of Cqdsx was predicted to contain a highly conserved DM domain and shared nearly 50% identity to DM-peptides from other species. The results of quantitative Real-time PCR in various tissues revealed that Cqdsx was strongly expressed in gonads, while was almost undetectable in gill, heart, hepatopancreas, muscle and intestine. Comparing expression level in different embryonic stages found that Cqdsx was gradually increased with the development of the embryos. In situ hybridization to gonad sections showed that intensive hybridization signals were mainly observed in oocytes and ovarian lamellae and weak signals were detected in spermatocyte. Additionally, Cqdsx gene exhibited higher transcript levels in the early stage of ovarian development. Furthermore, RNAi-targeting Cqdsx silencing induced a decrease of Cq-IAG trascripts, which regulate the male sexual differentiation in crustaceans. Taken together, these findings strongly suggest an essential role for Cqdsx in the female ovarian development/differentiation of the redclaw crayfish.