Identification of a candidate sex determination region and sex-specific molecular markers based on whole-genome re‑sequencing in the sea star Asterias amurensis

Abstract

Sex determination systems are diverse in echinoderms, however, our understanding is still very limited in this research field, especially for Asteroidea species. The northern Pacific seastar, Asterias amurensis, has attracted widespread concern due to its population outbreaks and high-risk invasions. Using whole-genome re-sequencing data from 40 females and 40 males, we identified a candidate sex determination region in A. amurensis. Based on the distribution characteristics of 525 sex-associated single nucleotide polymorphisms, identified by GWAS analysis, 119 sex-specific loci were isolated combining a custom Perl script, PCA analysis, and the selection signatures of fixation index FST, suggesting that a 7-12 Mb region on chromosome 10 is a candidate sex-determining region. The existence of female-specific sequences and the genotypes of sex-specific loci indicated that A. amurensis might utilize a ZZ/ZW sex-determination system. We also developed two pairs of sex-specific primers that could distinguish the genetic sex of this starfish with 100% accuracy. As the first study on sex determination in Asteroidea, it will provide novel insights into diverse sex determination systems in echinoderms and allow for in-depth studies on sex-related eco-physiological issues in A. amurensis.

Keywords: GWAS; sex determination; sex-specific markers; starfish; whole-genome re-sequencing.

Fig. 1.

Manhattan and QQ plot of genome-wide sex-associated SNPs in A. amurensis. (A) Manhattan plot displaying GWAS results between male and female A. amurensis. The X-and Y-axis represents the physical location of all SNPs across chromosomes 1-22 and corresponding -log₁₀ (P-value), respectively. The upper and lower dotted threshold lines represent ‘P-value = 1e-4’ and ‘P-value = 1e-8’, respectively. (B) QQ plot from SNP association analysis. The X-axis represents the expected observed -log₁₀ (P-value) under the assumption that P-values follow a uniform distribution and the Y-axis represents the observed -log₁₀ (P-value) from SNP association analysis. The blue region shows a 95% confidence interval under the null hypothesis of no association between the SNP and the sex.

Fig. 2.

Chromosome distribution of the selected loci. The X-axis refers to the significant sex-associated SNPs (P-value < 1e−8) (A), the sex-associated SNPs (P-value < 1e−4) (B), the loci with F_ST values greater than 0.25 (C), and the sex-specific loci isolated from the custom Perl script (D). The Y-axis represents the percentages of the selected loci per unit chromosome length for each chromosome.

Fig. 3.

Principal component analysis of 80 individuals using genome-wide SNPs excluding SNPs on chromosome 10 (A), SNPs on chromosome 10 (B), and chromosome 5 (C).

Fig. 4.

Identification of the candidate sex determination region on chromosome 10 of A. amurensis. (A) GWAS results on sex of A. amurensis. The X-and Y-axis represent the physical location of SNPs on chromosome 10 and corresponding -log₁₀ (P-value), respectively. The dotted threshold line represents ‘P-value = 1e-8’. The black dots represent SNPs located on 3’UTR, upstream and downstream of Aam_011084, Aam_011085, and Aam_011086. (B) Distribution of F_ST values. The X-and Y-axis represents the physical location of SNPs on chromosome 10 and corresponding F_ST values, respectively. The red dotted threshold line represents ‘F_ST value = 0.25’. (C) Distribution of sex-specific loci isolated from the Perl script. The X-and Y-axis represents the sliding window of 1 Mb and corresponding numbers of sex-specific loci.

Fig. 5.

The general pipeline for screening the sex-specific molecular markers in A. amurensis.

Fig. 6.

The validation of two pairs of sex-specific markers (Primer1 and Primer5) in two rounds of screening using agarose gel separation. The first round of screening used the gonad (A) and podia tissue (B) from 10 female and 10 male re-sequenced A. amurensis. The second round of screening used the podia tissue from another 10 females and 10 males (C). M represents DL 2000 DNA marker. The PCR amplification products using Primer1 and Primer5 are 477 bp and 650 bp, respectively. The PCR amplification product using Primer6 (positive control) is 195bp.