An NGS-based approach for the identification of sex-specific markers in snakehead (Channa argus)

// Mi Ou 1, 2 , Cheng Yang 1, 2 , Qing Luo 3 , Rong Huang 1 , Ai-Di Zhang 1 , Lan-Jie Liao 1 , Yong-Ming Li 1 , Li-Bo He 1 , Zuo-Yan Zhu 1 , Kun-Ci Chen 3 and Ya-Ping Wang 1 1 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China 2 University of Chinese Academy of Sciences, Beijing 100049, China 3 Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China Correspondence to: Ya-Ping Wang, email: wangyp@ihb.ac.cn Kun-Ci Chen, email: chenkunci@aliyun.com Keywords: next generation sequencing; sex-specific molecular markers; snakehead Received: April 19, 2017 Accepted: August 07, 2017 Published: October 19, 2017 ABSTRACT We described a next generation sequencing (NGS)-based approach to identify sex-specific markers and subsequently determine whether a species has male or female heterogamety. To test the accuracy of this technique, we examined the snakehead ( Channa argus ), which is economically important freshwater fish in China. Males grow faster than females, and there is significant interest in developing methods to skew breeding towards all-males to increase biomass yields. NGS was conducted on DNAs of individual female and male, the male reads were spitted into 60 bp K-mers and aligned to the female reference genome assembled by female reads, unaligned male K-mers-60 were kept in next filter process. Meanwhile, DNA sample of 48 females was pooled and sequenced, this data was further used to filter out the previous unaligned male K-mers-60. Hence, numbers of candidate Y chromosome-specific sequences were screened out, their sex-specificity were validated in wild snakeheads through PCR amplification. Finally, three Y chromosome-specific fragments (Contig-275834, Contig-359642, and Contig-418354) were identified, and specific primers were obtained to distinguish the sex of snakehead. Additionally, a pair of primers of Contig-275834 (275834X/Y-F and 275834X/Y-R) was exploited to distinguish XX females, XY males, and YY super-males, whose amplification products of different lengths were produced for different sexes. Therefore, our work demonstrated the ability of NGS data in identification of sex-specific markers, and the pipeline adopted in our study could be applied in any species of sex differentiation. Furthermore, the sex-specific markers have tremendous potential for improving the efficiency of all-male breeding practices in snakehead.