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

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.

Exploration of an XX/XY Sex Determination System and Development of PCR-Based Sex-specific Markers in Procambarus clarkii Based on Next-Generation Sequencing Data

“…Generally, the genetic system of sex determination for a species could be uncovered through descent identification or karyotype analysis ( Malecha et al, 1992 ; Ortega et al, 2017 ). Presently, high-throughput sequencing has been used as a rapid and efficient way to uncover the sex determination system of an organism ( Pan et al, 2015 ; Ou et al, 2017 ; Shi X. et al, 2018 ; Gao et al, 2019 ; Yang et al, 2020 ). P. clarkii lacks visual heterogeneity chromosomes ( Shi et al, 2019 ).…”

Non-invasive sex genotyping of paicheArapaima gigasby qPCR: An applied bioinformatic approach to identify sex differences

“…These studies designed three set of primers on a MSR which identified males with a success rate of 100%. Similar PCR protocols based on sex-specific markers have been developed for other fish species, for instance in the carps Hypophthalmichthys nobilis, Hypophthalmichthys molitrix (Liu et al, 2018) and the snakehead Channa argus (Ou et al, 2017). However, all these methods were based on end-point PCR, which does not enable the simultaneous detection of multiple genes and requires tissue samples for DNA extraction, affecting the desired non-invasive attribute.…”

The first de novo genome assembly and sex marker identification of Pluang Chomphu fish (Tor tambra) from Southern Thailand

“…To obtain candidate, specific DNA sequences on the W chromosome of T. tambra for use as a sex marker, we applied an NGS-based approach as previously proposed by Ou et al [32] . Briefly, we first mapped DNA sequences against the reference genome by aligning raw sequence reads from female fish to the male draft assembly with the BWA tool [20] , [21] .…”