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Front Endocrinol (Lausanne)
2021 Nov 11;12:756530. doi: 10.3389/fendo.2021.756530.
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Application of SNP in Genetic Sex Identification and Effect of Estradiol on Gene Expression of Sex-Related Genes in Strongylocentrotus intermedius.
Han YL, Sun ZH, Chang S, Wen B, Song J, Zuo RT, Chang YQ.
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Sea urchin (Strongylocentrotus intermedius) is an economically important mariculture species in Asia, and its gonads are the only edible part. The efficiency of genetic breeding in sea urchins is hampered due to the inability to distinguish gender by appearance. In this study, we first identified a sex-associated single nucleotide polymorphism (SNP) by combining type IIB endonuclease restriction site-associated DNA sequencing (2b-RAD-seq) and genome survey. Importantly, this SNP is located within spata4, a gene specifically expressed in male. Knocking down of spata4 by RNA interference (RNAi) in male individuals led to the downregulation of other conserved testis differentiation-related genes and germ cell marker genes. We also revealed that sex ratio in this validated culture population of S. intermedius is not 1:1. Moreover, after a 58-day feeding experiment with estradiol, the expression levels of several conserved genes that are related to testis differentiation, ovary differentiation, and estrogen metabolism were dynamically changed. Taken together, our results will contribute toward improving breeding efficiency, developing sex-controlled breeding, and providing a solid base for understanding sex determination mechanisms in sea urchins.
Figure 1. The sex-associated SNP of the Strongylocentrotus intermedius. The female genotype is G/A heterozygous and the male genotype is G/G homozygous. NP, nutritive phagocytes; SPC, spermatocyte; Oo, oocyte; SPD, spermatid.
Figure 2. Sequence analysis of spata4 in S. intermedius. (A) Coding sequence and deduced amino acid sequences of spata4 in S. intermedius.
(B) Phylogenetic tree of Spata4 proteins.
Figure 3. Expression pattern of spata4 mRNA in S. intermedius. 18S was used as the control. Each bar represents mean ± standard deviation (SD) (n = 3). One-way ANOVA was used to determine statistical analysis. Asterisks (*) indicate significant differences (p ≤ 0.05).
Figure 4. RT-qPCR measured the mRNA levels of spata4 and other sex-related genes after RNAi. (A) The expression of testis differentiation genes, including spata4, dmrt1, and soxE. (B) The expression of ovary differentiation genes, including foxl2 and hsd17b8. (C) The expression of germ cell marker genes, including boule and nanos1. 18S was used as the control. The gene expression in the control group was set to 1. Each bar represents mean ± standard deviation (SD) from three individuals. Independent-sample t-test was used to perform statistical analysis. Asterisks (*) indicate significant differences (p ≤ 0.05) between knockdown and control.
Figure 5. The concentration of estradiol levels in S. intermedius. (A) Concentration of estradiol in excrement after 3 days of dietary supplementation of E2. (B) Concentration of estradiol in testis on different days after dietary supplementation of E2 (n = 6). E2, estradiol; d, day. One-way ANOVA was used to determine statistical analysis. Different letters indicate significant differences (p ≤ 0.05).
Figure 6. The significant Gene Ontology (GO) annotation of differentially expressed genes. (A) The GO cluster of upregulated genes. (B) The GO cluster of downregulated genes.
Figure 7. RT-qPCR measured the mRNA levels of sex-related genes and estrogen metabolism-related genes in the testis of E2-treated S. intermedius. (A) The expression of testis differentiation genes. (B) The expression of ovary differentiation genes. (C) The expression of germ cell marker gene. (D) The expression of steroidgenic enzyme gene. (E) The expression of estrogen metabolism gene (Emc4). 18S was used as the control. Each bar represents mean ± standard deviation (SD) (n = 3). One-way ANOVA was used to determine statistical analysis. Asterisks (*) indicate significant differences (p ≤ 0.05).
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