| [Objectives] Capitulum mitella belongs to the Arthropoda, Pollicipedidae, and Capitulum, which is similar in size to the Tortoise’s foot. C. mitella is widely distributed in subtropical and tropical coastal high tide zone, mainly distributed in the sea area south of the Yangtze Estuary. Due to the great environmental differences in the sea areas where they live, different geographical populations of C. mitella will have different environmental adaptability under multiple environmental stresses. This study for the first time applied high-throughput sequencing to C. mitella in the Yangtze River estuary offshore of Zhoushan, Zhejiang Province, the southeast coast of Ningde, Fujian Province, and Zhuhai, Guangdong Province in the South China Sea to compare transcriptomic analysis. Differentially expressed genes (DEGs) among three geographical populations of C. mitella were screened to explore how the different geographic populations adapted to the environment. [Methods] The C. mitella samples were collected from intertidal reefs in Ningde, Fujian Province (120°04′04″ E, 26°52′34″ N), Zhoushan, Zhejiang Province (122°27′04″ E, 30°43′32″ N), and Zhuhai, Guangdong Province (114°02′33″ E, 22°05′55″ N) from September to November 2021. Morphological identification and mitochondrial molecular marker cytochrome c oxidase subunit I (COI) amplification analysis showed that all of them were C. mitella.RNA was extracted for library construction and transcriptome sequencing. Clean reads were obtained after raw data quality control. Trinity (v2.4.0) was used to assemble clean reads to obtain Unigenes, and the functional cluster analysis of Unigenes was conducted. Screening of differentially expressed genes (DEGs) and significant differences in expression of the threshold for P < 0.05 and | log2FoldChange | over 1 using DESeq2 (1.6.3). To analyze the functional characteristics and physiological and biochemical metabolic pathways involved in DEGs, GO and KEGG enrichment analysis of DEGs was conducted using GOseq (1.10.0) and KOBAS (v2.0.12), and the threshold of significant enrichment in DEGs was q < 0.05. Orthologs were identified to analyze the environmental adaptability of different geographical C. mitella populations using OrthoMCL (v2.0.3). Then, the Ka/Ks values of orthologs genes were calculated by the Branch Site model. If Ka/Ks > 1, the genes were evolving rapidly; if Ka/Ks = 1, the genes were under neutral selection; if 0 < Ka/Ks < 1, the gene is selected for purification. GO and KEGG enrichment for genes under positive selection pressure, and gene function and metabolic pathway were annotated, the threshold of significant enrichment was P < 0.05. [Results] In this study, a total of 60.9 Gb of clean reads were obtained from three C. mitella populations, and 61 899 Unigenes were assembled (Table 1). A total of 651 Unigenes were significantly expressed between Zhuhai and Ningde populations, there were 3 738 DEGs between Zhoushan and Ningde populations, and 3 619 DEGs between Zhoushan and Zhuhai populations (Fig. 4). Functional annotation analysis showed that DEGs were significantly enriched in ribosome biogenesis (GO0042254, q = 8.26 × 10﹣53) and structural constituent of ribosome (GO0003735, q = 9.14 × 10﹣52) between Zhoushan and Zhuhai populations (Fig. 5). DEGs were further enriched in protein processing in endoplasmic reticulum (ko04141, q = 2.88 × 10﹣4), antigen processing and presentation (ko04612, q = 1.16 × 10﹣35) and ECM-receptor interaction (ko04512, q = 4.16 × 10﹣2) (Fig.6). Among them, genes such as agrin (q = 1.72 × 10﹣3) and solute carrier family 8 (q = 3.93 × 10﹣5) may be involved in regulating osmolality balance. Compared with Zhoushan and Zhuhai populations, DEGs function involved in antigen processing and presentation (ko04612) and protein processing in endoplasmic reticulum (ko04141), among which cathepsin B (q = 6.36 × 10﹣3) and high temperature protein G (q = 9.97 × 10﹣6) were significantly expressed. Heat shock protein family, including HSP70 (q = 1.45 × 10﹣6) and HSP40 (q = 4.58 × 10﹣7) were also significantly expressed in the Zhoushan population (Table 2). Analysis of positive selection pressure among different geographical populations by orthologs screening showed that 7 genes under positive selection pressure were obtained from 254 orthologs including D-aspartate oxidase (Ka/Ks = 2.076), histone deacetylase (Ka/Ks = 1.145), baculoviral IAP resting-containing protein (Ka/Ks = 1.008), GTP-binding nuclear protein Ran (Ka/Ks = 1.001), mediator of RNA polymerase II transcription subunit (Ka/Ks = 2.867), translation initiation factor 2B (Ka/Ks = 1.001), and pyrroline-5-carboxylate reductase (Ka/Ks = 1.131) (Table 3). They are all involved in growth hormone, reproductive, nutritional, nervous, immune system regulation, and neuronal protection. They also participate in physiological processes such as growth and development, tissue differentiation, metabolism, and anti-apoptosis. [Conclusion] This study will help to understand the adaptation mechanism of C. mitella to multiple external environments and provide a basic foundation of genetic diversity, germplasm resources protection, and the development of different geographical populations of C. mitella. |
