Research paperCandidate sex-associated gene identification in Trachinotus ovatus (Carangidae) using an integrated SLAF-seq and bulked segregant analysis approach
Introduction
Trachinotus ovatus (family Carangidae) is widely distributed in Southeast Asia, Australia, Japan, eastern Africa, and the Atlantic Ocean (Tan et al., 2017). In recent years, the aquaculture of T. ovatus has spread rapidly throughout China (Guo et al., 2018, Xie et al., 2019) due to its fast growth and high-quality flesh (Tan et al., 2017). However, the sexes of T. ovatus are difficult to discriminate based on appearance, even at maturity (Jiang et al., 2015). Thus, studies of sex-related genetic markers are crucial in this species. Although the genome sequence of T. ovatus is available (Zhang et al., 2019), data on its sex-related genes are limited (Zhang et al., 2019). To establish a rapid technique for T. ovatus sex discrimination, it is necessary to identify sex-related genes and their linked markers.
Bulked segregant analysis (BSA) (Klein et al., 2018, Ryo et al., 2018, Zhang et al., 2018, Gao et al., 2019) identifies molecular markers linked closely to a gene or Quantitative trait locus (QTL) controlling a trait of interest. BSA uses a pair of bulked DNA samples from individuals with distinct, extreme phenotypes (Wang et al., 2018, Zhang et al., 2018). Although BSA has been widely used in crop research, this technique is rarely used in aquatic studies; in one of the rare examples, BSA was used to identify sex-specific molecular markers in the pirarucu (Arapaima gigas) (IG et al., 2013). Next-generation sequencing techniques, such as specific-locus amplified fragment sequencing (SLAF-seq), have promoted the development of BSA. SLAF-seq is a rapid, high-throughput, highly accurate strategy for identifying single-nucleotide polymorphisms (SNPs) (Sun et al., 2013, Shen et al., 2017, Chang et al., 2018, Zheng, 2018).
An integrated BSA and SLAF-seq approach has been used to assess SNPs in various crops, including rice (Xu et al., 2015), pepper (Wang et al., 2018, Zhang et al., 2018), cotton (Zhen et al., 2015, Cunpeng et al., 2018), and cucumber (Zhang et al., 2015). However, this combined approach has seldom been used in aquatic species.
Here, SLAF-seq, coupled with BSA, was employed to map sex-associated traits of T. ovatus using pooled DNA from an F2 generation with distinctive sex phenotypes, which was obtained by crossing two strains of T. ovatus, R404 and R403. The main objective of this study was to identify candidate sex-related genes and SNPs in T. ovatus to be used for molecular sex discrimination in this species.
Section snippets
Ethics statement
All experiments involving T. ovatus were performed as approved by the Animal Care and Use Committee of the Guangxi Academy of Fisheries Sciences (Nanning, China).
Sample collection and morphological sex determination
Parents and 200 six-month-old F2 T. ovatus were provided by the Guangxi Academy of Fisheries Sciences. The animals were, euthanized with an overdose of MS222 (300 mg/L), and muscle tissue samples were collected from the parents and the F2 individuals and immediately frozen in liquid nitrogen for DNA extraction and SLAF library
Analysis of the sequence data
We generated 635.49 Mb and 123.13 Mb of clean reads from the SLAF libraries of the parents and the F2 pools, respectively. The average read length was 50 bp. All four libraries had average Q30 values (i.e., nucleotides with a quality score >30) greater than 93% and GC percentages greater than 40%, indicating that most of the bases were high quality (Table 1). Over 98.65% of the clean reads mapped to the reference genome, indicating a high level of sequencing accuracy.
Identification of SNPs and Indels
A total of 3,153,153 SNPs
We identified 132 candidate sex-related genes in T. ovatus using SAF-seq combined with BSA
Among the 132 candidate genes identified in T. ovatus, six have previously been implicated in gonad development, sex differentiation, or gamete formation: XPA binding protein 2 (xab2) participates in human spermatogenesis (Shen et al., 2013); GIPC PDZ domain containing family member 1 (gipc1) was implicated in sex-determination in Yellowtail (Takashi et al., 2015); hydroxysteroid (17-beta) dehydrogenase 1 affects mouse spermatogenesis and ovarian development in pigs (Harini et al., 2009, Yuan
Conclusions
We constructed four SLAF libraries from each parent and the two F2 pools. F2 pools were composed of DNA collected from 50 individuals of each sex. Approximately 3,153,153 high-quality single-nucleotide polymorphism (SNP) markers and 135,363 high-quality insertion-deletion (Indel) markers were identified. Six candidate regions within chromosome 14, encompassing 132 candidate genes, were identified as closely related to sex. Based on annotations, six genes were predicted to be closely associated
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant No. 31660740), the Fundamental Research Funds for non-profit research institutes of the Guangxi Zhuang Autonomous Region (Grant No. GXIF-2016-19), Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture Opening Fund (Grant No.GXKEYLA2019-03), and the China Agriculture Research System (Grant No. CARS-47-z17). We thank LetPub (www.letpub.com) for linguistic assistance with our manuscript.
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These authors contributed equally to this work and share first authorship.