| Hyriopsis schlegelii is one of the important freshwater cultured pearl mussels in China.Its cultivated pearl has large particle size,good gloss and good appearance.In this study,we explored the genetic basis of sex determination and sex differentiation and the effect of temperature on sex differentiation by exploring the karyotype of chromosomes,analyzing the full-length transcriptome of female and male mussels,and based on the gonadal transcriptome of five temperature-treated mussels.The main research results are as follows:(1)Hyriopsis schlegelii has 38 chromosomes,and the actual length of chromosomes ranges from 2.60 to 4.76 μm,with an average length of 3.51 μm.Among the 19 pairs of chromosomes,there were 13 middle centromeric chromosomes,5 submiddle centromeric chromosomes and 1 subtelocromeric chromosome.The karyotype was 2n=13m+5sm+1st and the number of chromosome arms NF = 74.Chromosome morphology and karyotype of female and male clams were similar,and there were no accessory and heteromorphic sex chromosomes.(2)Full-length transcriptome sequencing was performed on female and male mussel using third-generation SMRT sequencing technology,and 6577042 and5900972 sequences were obtained by female and male mussel.After Iso-seq standard correction and second-generation RNA-seq data correction,the transcripts were removed after redundancy.Female and male clams obtained 96867 and 111520 transcripts,respectively,with an average length of 4095 bp and 3876 bp.In the end,a total of 201,481 gonadal transcripts were obtained,and more than 99.5% of the transcripts were larger than 1 kpb,and the number of transcripts ranged from 2.5 kbp to 4.5 kbp.By comparison with NT,NR,KOG/COG,SSWISS-PROT,KEGG,PFAM and GO databases,more than 85% of the transcripts were annotated,and many gender-related genes,such as Sox9,Wnt4,Rsop1 and Sry,were screened out.By analyzing the structure of the full-length transcriptome,191199 CDS were predicted.A total of 217,648 SSRs were identified,and most of them were single nucleotide repeats and dinucleotide repeats.A total of 4021 TFs were predicted,among which 62 TFs were found to have zinc finger structure,among which ZF-C2H2 was the most.32,931 highly reliable lnc RNAs were found.(3)30-month-old mussels were cultured for two weeks at 15℃,20℃,25℃,30℃ and 33℃,respectively.After the experiment,the gonads were extracted and RNA-seq was performed.After comparing the RNA-seq data with the full-length transcripts,the gene expression level was analyzed.It was found that the expression level of the female samples at 25℃ was slightly lower than that of the other groups.According to DEseq2 software analysis,there were 6311,3013,6673,6798 and5685 genes differentially expressed by females and males at different temperatures,and the number of differentially expressed genes was the least at 20℃.Differential gene clustering analysis showed that there were similar expression patterns in the female group and the male group,and the expression patterns were similar at 15℃and 20℃,clustering into a cluster.The expression was similar at 25℃,30℃ and33℃.(4)GO enrichment analysis of differential genes showed that at 25℃,embryo development and cell proliferation and division related items,such as embryo development regulation,Notch signaling pathway,spindle microtubules and concentrated extrachromeres,appeared.KEGG enrichment showed that at 25℃,female related activities were actively expressed,Notch signaling pathway appeared,meiosis and cell cycle of egg cells began to appear.At 30℃,progesterone mediated oocyte maturation pathway also appeared in the enrichment pathway.Among the 802 genes differentially expressed in the five temperature groups,319 genes were up-regulated,and 483 genes were down-regulated.The gender-related genes were screened out,and four gender-regulated genes were identified,including Fem1,Tra1,Wnt4 and Rspo1.The q RT-PCR results showed that the expression levels of Wnt4 and Rspo1 were significantly higher in females(P < 0.01)than in males,while the expression levels of Fem1 and Tra1 were significantly higher in males(P < 0.01)than in females at different temperatures.The high expression of male-associated Fem1 in male mussels was high at 15℃ and 20℃.Female-related Wnt4 and Rspo1 were highly expressed at 25℃ to 33℃ in female mussels.Tra1,which is associated with controlling hermaphroditism,was highly expressed at 25℃ to 33℃ in female and male mussels.In summary,no heteromorphic sex chromosomes were found in Hyriopsis schlegelii;Through the analysis of full-length transcripts of female and male mussel,abundant gene function and gene structure information were obtained.Combined with differential gene enrichment analysis and gender-related gene expression,it was inferred that temperature above 25℃ would be beneficial to induce female development of Hyriopsis schlegelii.This study provides a basic reference for the mechanism and molecular characteristics of sex regulation and provides some data support for sex regulation and development of Hyriopsis schlegelii. |
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