Identification of sex-specific sequences through 2b-RAD sequencing in Pseudobagrus ussuriensis

Abstract

Abstract Sex-specific markers and sequences are essential tools for sex-determination mechanism analyses and sex-control breeding of fishes. Pseudobagrus ussuriensis is a high-value aquaculture fish with great potential for all-male breeding, as males grow much faster than females. In the present study, reduced restriction site-associated DNA sequencing was performed in five female and five male P. ussuriensis using the 2b-RAD approach to identify sex-specific sequences. A total of 84.52 million clean reads were obtained, with 47.30 million in females and 37.22 million in males. After clustering of clean reads, 2,331,843 clean tags were obtained, with average numbers of 235,472 in females and 230,896 in males. Sexual comparison of these tags resulted in 349 potential male-specific tags, among which 22 were false positive (without differences between sequences of males and females), 46 contained insertions/deletions (InDels), 261 contained single nucleotide polymorphisms (SNPs), and 20 were male specific. A male draft genome containing 689,006 scaffolds with a size of 689.04 million base pairs (bp) was obtained to extend potential male-specific tags. Fourteen sex-specific sequences with lengths ranging from 85\u202fbp to 2494\u202fbp were screened out. The validation results indicated three male-specific markers and seven male-specific sequences, which were further confirmed in 300 individuals from two full-sib families and showed the expected 1:1 sex ratio. Annotation of these sex-specific sequences resulted in 12 candidate genes. Furthermore, at least eight of the sex-specific sequences were deduced to be located on the same chromosome, which may be the sex chromosome of P. ussuriensis. The results obtained in this study will be helpful for studies on mechanism analysis of sex determination and all-male breeding in P. ussuriensis.