Identification of sex-specific markers using genome re-sequencing in the blunt snout bream (Megalobrama amblycephala)

Abstract

Background: The blunt snout bream (Megalobrama amblycephala) is an important economic freshwater fish in China with tender flesh and high nutritional value. With the cultivation of superior new varieties and the expansion of breeding scale, it becomes imperative to employ sex-control technology to cultivate monosexual populations of M. amblycephala, thereby preventing the deterioration of desirable traits. The development of specific markers capable of accurately identifying the sex of M. amblycephala would facilitate the determination of the genetic sex of the breeding population before gonad maturation, thereby expediting the processes of sex-controlled breeding of M. amblycephala.

Results: A whole-genome re-sequencing was performed for 116 females and 141 males M. amblycephala collected from nine populations. Seven candidate male-specific sequences were identified through comparative analysis of male and female genomes, which were further compared with the sequencing data of 257 individuals, and finally three male-specific sequences were generated. These three sequences were further validated by PCR amplification in 32 males and 32 females to confirm their potential as male-specific molecular markers for M. amblycephala. One of these markers showed potential applicability in M. pellegrini as well, enabling males to be identified using this specific molecular marker.

Conclusions: The study provides a high-efficiency and cost-effective approach for the genetic sex identification in two species of Megalobrama. The developed markers in this study have great potential in facilitating sex-controlled breeding of M. amblycephala and M. pellegrini, while also contributing valuable insights into the underlying mechanisms of fish sex determination.

Keywords: Megalobrama amblycephala; Genetic sex identification; Molecular marker; Whole-genome re-sequencing.

Fig. 1

Identification of phenotypic sex in blunt snout bream. (A)-(D) Gonad morphology of female and male individuals. (A)-(B) Individuals whose sex can be identified through anatomical observation; (C)-(D) Individuals whose sex cannot be identified through anatomical observation. (E)-(H) Sex identification of blunt snout bream by staining gonad tissue slices with hematoxylin and eosin. E, female, visible mature follicles can be observed in the ovary; F, male, spermatozoa were clearly visible in the testis; G, oocytes; H, spermatocytes

Fig. 2

Screening processes for male-specific fragments

Fig. 3

Sequences corresponding to the three sex-specific markers. The positions marked by black arrows are the forward and reverse primers. (A) Sequences amplified by M-marker 1; (B) Sequences amplified by M-marker 2; (C) Sequences amplified by M-marker 3

Fig. 4

PCR amplification results of three sex-specific primer pairs in EZ breeding population of blunt snout bream. Sex-specific primers and β-actin primers were incorporated into the same PCR reaction system, and the validity of the amplification results was verified by the presence or absence of control bands (152 bp). (A) Amplification of M-marker 1 resulted in a male-specific band at 173 bp. (B) Amplification of M-marker 2 resulted in a male-specific band at 275 bp. (C) Amplification of M-marker 3 resulted in a male-specific band at 436 bp. M, DL 2000 DNA marker

Fig. 5

Phylogenetic tree construction and PCR amplification results of three sex-specific primer pairs in other closely related species (M. terminalis, M. hoffmanni, and M. pellegrini). (A) A phylogenetic tree generated using single-copy orthologous genes. (B) Correspond to the results amplified by primer pairs of M-marker 1, M-marker 2 and M-marker 3. (C) Extended population verification of the sex-specific primer pair of M-marker 3 in M. pellegrini collected from Yibin. The amplified fragment of β-actin was 152 bp for the positive control, confirming the effectiveness of the amplification. M, DL 2000 DNA marker