A simple and rapid method for fish sex identification based on recombinase-aided amplification and its use in Cynoglossus semilaevis

Zhichao Nie,Peng Lü,Yishuai Tu,Xiqing Li,Cong Tang,Feng Li,Kun Zhao,Jun Wang,Qiang Zhou,Yin Li,Rusong Zhang,Zhanzhuang Zeng,Hong Zhang,Min Tu,Zhenni Liu

doi:10.1038/s41598-021-89571-z

Abstract

Fish sex identification is a basic technique of great importance for both fish genetic studies and fisheries. Due to the sexual reversal phenomenon in many fish species, a simple and rapid molecular identification method for fish genetic sex is urgently needed to suit versatile detection scenarios, such as point-of-need applications. In this study, we took Cynoglossus semilaevis as an example, established a recombinase-aided amplification (RAA)-based method for sex identification, and combined the RAA-detection with two result visualization approaches with distinct features, capillary electrophoresis (CE) and lateral flow dipstick (LFD). Specific primers and probe were designed to specifically detect the sex chromosome W of C. semilaevis in order to distinguish the genetic sex between males, pseudo-males and females. To evaluate the performance of our methods, the genetic sex for twenty-eight males, sixty-eight pseudo-males and fifty-four females were examined with the RAA-based method and classical PCR-based genotyping method, demonstrating the consistent results of sex identification between both methods. The RAA-LFD method is operationally simple, rapid (~ 30 min) and holds great potential for point-of-need applications of fish sex identification, including fishery fields. The method presented here could be effective for identifying fish gender with the ZW karyotype.

Highlights

Fish sex identification is a basic technique of great importance for both fish genetic studies and fisheries
C. semilaevis sex chromosomes Z and W was chosen for recombinase-aided amplification (RAA) amplification and for establishing the RAA-based fish sex identification method, because two sex identification markers have been reported previously in this region[2, 22] (Fig. 2A)
Because the forward primer and the probe are labeled by FAM and biotin at their 5’ends, respectively, the amplicons can be captured by lateral flow dipstick (LFD) and separately detected on the control and test lines (Fig. 2C)

Summary

Introduction

Fish sex identification is a basic technique of great importance for both fish genetic studies and fisheries. Males and females can differ strikingly in morphology, and reproductive development behavior While these phenomena, known as sexual dimorphism, are widespread in many species, the genetic mechanisms regulating such a ubiquitous pattern are diverse. Chinese half-smooth tongue sole (Cynoglossus semilaevis) has been recognized as an ideal model for fish sex determination and identification because of its typical mode of sex determination, the small genome and relatively large amount of studies on sex-determination genetics[1, 3]. Another key factor for C. semilaevis as a research. New technologies, such as single nucleotide polymorphism (SNP), Insertion/deletion polymorphism (IDP) and penta-primer amplification refractory mutation system (PARMS) have been developed as fish sex identification m ethods[21, 22]

Methods

Results

Conclusion