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BACKGROUND: Sex determination mechanisms are complicated and diverse across taxonomic categories. Sea cucumber Apostichopus japonicus is a benthic echinoderm, which is the closest group of invertebrates to chordate, and important economic and ecologically aquaculture species in China. A. japonicus is dioecious, and no phenotypic differences between males and females can be detected before sexual maturation. Identification of sex determination locus will broaden knowledge about sex-determination mechanism in echinoderms, which allows for the identification of sex-linked markers and increases the efficiency of sea cucumber breeding industry.
RESULTS: Here, we integrated assembly of a novel chromosome-level genome and resequencing of female and male populations to investigate the sex determination mechanisms of A. japonicus. We built a chromosome-level genome assembly AJH1.0 using Hi-C technology. The assembly AJH1.0 consists of 23 chromosomes ranging from 22.4 to 60.4 Mb. To identify the sex-determination locus of A. japonicus, we conducted genome-wide association study (GWAS) and analyses of distribution characteristics of sex-specific SNPs and fixation index FST. The GWAS analysis showed that multiple sex-associated loci were located on several chromosomes, including chromosome 4 (24.8%), followed by chromosome 9 (10.7%), chromosome 17 (10.4%), and chromosome 18 (14.1%). Furthermore, analyzing the homozygous and heterozygous genotypes of plenty of sex-specific SNPs in females and males confirmed that A. japonicus might have a XX/XY sex determination system. As a physical region of 10 Mb on chromosome 4 included the highest number of sex-specific SNPs and higher FST values, this region was considered as the candidate sex determination region (SDR) in A. japonicus.
CONCLUSIONS: In the present study, we integrated genome-wide association study and analyses of sex-specific variations to investigate sex determination mechanisms. This will bring novel insights into gene regulation during primitive gonadogenesis and differentiation and identification of master sex determination gene in sea cucumber. In the sea cucumber industry, investigation of molecular mechanisms of sex determination will be helpful for artificial fertilization and precise breeding.
Fig. 1. Contact map of Hi-C interactions among chromosomes in A. japonicus generated by Juicebox software
Fig. 2. Circos plot of the global landscape of genomic features and genetic variations in AJH1.0 genome assembly. The tracks from outside to inside represent (1) 23 chromosomes of AJH1.0 genome assembly (Mb). (2) Distribution of GC content with a sliding window of 500Â kb. (3) Distribution of INDEL density with a sliding window of 500Â kb. (4) Distribution of SNP density with a sliding window of 500Â kb. (5) Distribution of repeat elements with a sliding window of 500Â kb. (6) Distribution of gene density with a sliding window of 500Â kb. (7) Relationship between syntenic blocks in AJH1.0 genome assembly
Fig. 3. Genome-wide association analysis and chromosome distribution of sex-associated SNPs. (A) Manhattan plots 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 theâââlog10 (P-value). The black threshold line represents P-valueâ=â1-e4, the red threshold line represents P-valueâ=â1-e6. (B) Chromosome distribution of sex-associated SNPs by GWAS. The x-axis depicts P-value thresholds (Pâ<â1-e6), and the y-axis depicts the percentages of sex-associated SNPs per unit chromosome length for each chromosome
Fig. 4. Principal component analysis of 30 females and 30 males using different sets of SNPs
Fig. 5. Identification of candidate sex determination regions on chromosome 4. (A) GWAS analysis associated with sex of A. japonicus. The x-axis depicts the physical location of SNPs, and the y-axis depicts theâââlog10 (P). The red threshold line represents P-valueâ=â1-e6. Red dots represent outlier values of P-value (Pâ<â1-e6) (B) Distribution of sex-specific SNP density with a sliding window of 2Â Mb. (C) Distribution of FST values estimated between females and males. The x-axis depicts the physical location of SNPs, and the y-axis depicts the FST values. The red threshold line represents FSTâ=â0.25
Fig. 6. Heatmap of the differentially expressed genes. (A) DEGs between female and male gonads within candidate sex determination region. (B) The protein-coding genes of DEGs between female and male gonads within candidate sex determination region. Orange represents high expression levels, while blue represents low expression levels
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