Sex-Specific Markers Of The Farmed Ayu(Plecoglossus Altivelis) Screened And Developed By Next-Generation Sequencing

Ayu(Plecoglossus altivelis)is a migratory annual fish raised in seawater and cultivated in freshwater.It is fresh and tasty with a sweet smell,and regarded as a treasure in the water.However,the sex-determination mechanism is poorly understood.Whole-genome sequencing technology can comprehensively and accurately analyze the base sequence of the genome,thereby deciphering the information it contains and revealing the complexity and diversity of the genome.Type IIB restriction endonuclease site - related DNA sequencing(2b-RAD-seq)is a popular method to simplify sequencing.Due to its simplicity and reproducibility,it is considered to be an effective and valuable method to identify sex-specific DNA fragments and sex determination systems.In this study,high-throughput whole-genome sequencing and 2b-RAD-seq were used to screen sex-specific tags,determine the sex- related locus of ayu,and finally established a method for sex identification of ayu.We first performed candidate whole-genome sequencing on 1 male and 1female ayu,and performed 2b-RAD-seq on 10 male and 10 female ayu,and mapped the tags to the male and female genomes respectively,and screened for sex-specific tags.The results showed that124 male-specific tags were screened,but no female-specific tags were screened.Further analysis showed that these candidate male-specific tags were located on 14 male scaffolds.Among them,7female allelic scaffolds were found through whole-genome comparison,and 2 female allelic scaffolds were identified though polymerase chain reaction(PCR)and chromosome walking.Female scaffolds,the remaining 5 scaffolds were preliminarily identified as candidate male-specific scaffolds through genome-wide comparison and PCR methods.Among the 5 candidate scaffolds,scaf162525,scaf184183,and scaf192610 encoded a total of 45 candidate genes,of which 9 had functional annotations.This included the anti-Müllerian hormone type-2 receptor(Amhr2),Amhr2 by located on scaf192610 was identified as a Y-linked duplicate copy of the Amhr2 a gene on the autosome.Whether it was a sex-determining gene still needs further research.Besides,based on the highly heterogeneous sequences of scaf175983,scaf123942,scaf147,and scaf143776,and the female equivalent locus,four sets of primers were designed to establish a multiplex PCR method for male and female ayu identification.The results showed that these 4 male-specific markers can accurately identify the sex of 12 male and 12 female ayu.To further confirm the accuracy of multiplex PCR,we also identified 60 male and 60 female ayu of its offspring for verification.Finally,the accuracy of gender identification was 100%.In conclusion,this study screened 5 male fish-specific scaffolds,which indicated that ayu belonged to the XX/XY sex-determination system.A multiplex PCR detection based on male-specific markers can be used to perform molecular sexing in ayu culture.