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iScience
2024 Aug 18;2710:110852. doi: 10.1016/j.isci.2024.110852.
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Population genomic analysis reveals a polygenic sex determination system in Apostichopus japonicus.
Jiang C
,
Liu S
,
Yang Y
,
Cui W
,
Xu S
,
Rasoamananto I
,
Lavitra T
,
Zhang L
,
Sun L
.
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The sea cucumber Apostichopus japonicus, a key species in Chinese aquaculture, plays a significant evolutionary role within the Echinodermata phylum. However, the sex determination mechanism in this species remains poorly understood. Here, we conducted extensive sex surveys and sampling of eight wild populations, investigating the sex-related SNPs and insertion or deletions (indels) through bulk segregation analysis (BSA) and genome-wide association study (GWAS) analysis. Our findings suggest that A. japonicus employs a polygenic sex determination (PSD) system, with solute carrier family 8 (SLC8A) being the candidate gene for sex determination, encoding sodium-calcium exchanger (NCX1). The analysis of normalized sequencing depth reveals variations across chromosomes 6, 13, 14, 16, and 18, supporting the PSD system. We also identified 541.656 kb of male-specific sequences and screened five markers (C77185, C98086, C64977, C125, and C876) for molecular sex identification. Overall, this study provides new insights into A. japonicus sex determination, highlighting a complex multi-gene mechanism rather than a simple XX/XY system.
Figure 1. Sex distribution of individuals in different regions(A) Sampling site map.(B) Gonadal morphological differences between male and female sea cucumbers.(C) Sex ratios of sea cucumbers in different regions.(D) Statistical analysis of sex phenotype distribution. The red curve represents the density curve fitted based on the phenotype distribution plot, while the green curve represents the standard normal curve. “miss” refers to the missing rate of phenotype data, and “p_value” indicates the p value of the normality test.
Figure 2. Bulk segregation analysis for sex trait(A) Distribution of the two offspring pools Δ(All-index) on the chromosome. Δ(SNP-index) = SNP-index(S2) – SNP-index(S1).(B) Distribution of SNP-index in pool1 on chromosome 4. The y axis represents the index value, and the red line represents the average SNP-index.(C) Distribution of SNP-index in pool2 on chromosome 4. The y axis represents the index value, and the red line represents the average SNP-index.(D) Distribution of offspring Δ(SNP-index). The y axis represents the difference in index values between the two offspring pools. The red line represents the average SNP-index, the green line represents the 95% threshold line, and the purple line represents the 99% threshold line.(E) Distribution of indel-index in pool1 on chromosome 4. The red line represents the average indel-index.(F) Distribution of indel-index in pool2 on chromosome 4.(G) Distribution of offspring Δ(Indel-index).
Figure 3. Manhattan plots and quantile-quantile for the GWAS for sex trait(A) Manhattan plot of 50× sequencing depth depicting GWAS results associated with sex of A. japonicus. The x axis shows the physical location of SNPs across the 23 chromosomes, and the y axis shows the −log10 (p value). The black threshold line represents p value = 1-e8.(B) Q-Q plot of GWAS results associated with sex of A. japonicus.(C) KEGG enrichment pathways of genes annotated with significant SNPs across the entire genome.(D) KEGG enrichment pathways of genes annotated with significant SNPs on chromosome 4.(E) Part of the endocrine and other factor-regulated calcium reabsorption pathway, where the red boxes represent proteins expressed by genes annotated by SNPs, among which there are 12 annotated genes belonging to the solute carrier family 8 member genes (SLC8A), with SLC8A1 encoding NCX1.
Figure 4. Normalized depth of resequencing reads between female and maleThe red line and the green line respectively represent the normalized depth for female and male individuals on each chromosome, while the blue line represents the ratio of normalized depth (male to female).
Figure 5. Experimental flowchart for male-specific sequences
Figure 6. PCR amplification bands of 5 pairs of primers in 36 individuals(A) The amplification results of primer contig 77185 in DNA templates from 6 female and 6 male individuals.(B) The amplification results of primer contig 98086 in DNA templates from 6 female and 6 male individuals.(C) The amplification results of primer contig 64977 in DNA templates from 6 female and 6 male individuals.(D) The amplification results of primer contig 77185 in DNA templates from 12 female and 12 male individuals.(E) The amplification results of primer contig 98086 in DNA templates from 12 female and 12 male individuals.(F) The amplification results of primer contig 64977 in DNA templates from 12 female and 12 male individuals.(G) The amplification results of primer contig 125 in DNA templates from 18 female and 18 male individuals.(H) The amplification results of primer contig 876 in DNA templates from 18 female and 18 male individuals. Amplification conditions: annealing temperature set at 68°C, with 35 cycles.
Figure 7. Chromosome-level genome co-linearity between male and female(A) Distribution of SNPs and male-specific contigs proportions on the female and male genome. The scale on the heatmap represents the proportion (%) of SNPs or male-specific contigs on a single chromosome relative to the total quantity across all chromosomes.(B) Chromosome co-linearity of the male genome relative to the female genome.(C) Chromosome co-linearity of the female genome relative to the male genome. Scarf1-23 represents chromosome numbering in the male reference genome, while Chr1-23 represents chromosome numbering in the female reference genome.
