# Full-Length Transcriptome Sequencing and Comparative Transcriptomics Reveal the Molecular Mechanisms Underlying Gonadal Development in Sleepy Cod (Oxyeleotris lineolata)

## TL;DR

This study uses full-length transcriptome sequencing to uncover the genetic mechanisms behind gonadal development in sleepy cod, an economically important fish species.

## Contribution

The study provides the first full-length transcriptome and identifies key genes and pathways involved in sex determination and gonadal differentiation in sleepy cod.

## Key findings

- A total of 49,113 non-redundant full-length transcripts were generated using PacBio Iso-Seq technology.
- 19,102 differentially expressed transcripts were identified between ovaries and testes, with key pathways including oocyte meiosis and arachidonic acid metabolism.
- qPCR validation confirmed the expression levels of 20 selected differentially expressed genes matched transcriptome results.

## Abstract

The sleepy cod Oxyeleotris lineolata is a species of freshwater goby, which is natively distributed throughout northern Australia. Due to its tender meat and delicious taste, it has become a freshwater fish of economic importance, with a relatively high price in Asian markets. We observed that sleepy cod presents as sexually dimorphic in its growth rate and body size, with the average body weight of mature males being significantly higher than that of mature females (p < 0.05). Therefore, the efficient development of all-male breeding will be helpful in increasing the yield and output value of this species. In this context, it is important to obtain more genetic information to understand the sex determination and gonadal differentiation mechanism of sleepy cod. The present study provides new genetic resources—including full-length transcriptome sequences and annotation information—as a coding genomic-level reference for sleepy cod—yielding valuable insights into the genetic mechanisms of sex determination and gonadal differentiation in this economically important species.

Sleepy cod (Oxyeleotris lineolata) is native to Australia and is now an economically valuable fish cultured in China and Southern Asian countries. Its growth rate exhibits as sexually dimorphic, with males generally growing more rapidly and attaining a larger body size compared to females. Thus, the effective development of sex control breeding can significantly contribute to increased yields and output value. Nevertheless, due to the lack of genomic and transcriptomic data, the molecular mechanisms underlying sex determination and gonadal differentiation in sleepy cod remain poorly understood. In this study, long-read PacBio isoform sequencing (Iso-Seq) was performed to obtain a full-length transcriptome from a pooled sample of eight tissues (kidney, brain, liver, muscle, heart, spleen, ovary and testis). A total of 30.41 G subread bases were generated and 49,113 non-redundant full-length transcripts with an average length of 2948 bp were produced. Using the full-length transcriptome as a reference, short-read Illumina sequencing was performed to investigate the differences in gene expression at the transcriptome level between ovaries and testes from 12-month-old individuals. A total of 19,102 differentially expressed transcripts (DETs) were identified, of which 8510 (44.55%) were up-regulated in the ovary and 10,592 (55.45%) were up-regulated in the testis. The DETs were mainly clustered into 241 KEGG pathways, in which oocyte meiosis and arachidonic acid metabolism were the most relevant pathways involved in gonadal differentiation. To verify the validity of the transcriptomic data, 20 DETs were selected to investigate the gonad expression profiles based on qPCR. The expression levels of all 20 screened genes were consistent with the transcriptome sequencing results. The present study provides new genetic resources—including full-length transcriptome sequences and annotation information—as a coding genomic-level reference for sleepy cod—yielding valuable insights into the genetic mechanisms of sex determination and gonadal differentiation in this economically important species.