Single-cell transcriptome sequencing of skin tissue in blue discus fish （Symphysodon spp.）

【Objective】To profile and compare the expression patterns of the genes involved in formation of body color between the skin tissues of blue discus fish and wild Alenquer discus fish through a single-cell transcriptome sequencing， which provided the data basis for further study of body color and breeding， as well as the technical reference for the single-cell transcriptome studies in discus fish and other fishes. 【Method】Using the single-cell transcriptome sequencing technology， transcriptome sequencing was performed on the skin tissues of blue discus fish（bred strain） and Alenquer discus fish （wild type）. The expression matrix of single-cell data was generated by Cell Ranger alignment against reference genomes after fastp quality control filtration， which was used for dimensionality reduction clustering to construct transcriptome atlas by Seurat and indentification of cell types referencing the marker genes. The differentially expressed genes （DEGs） between the two samples were identified by DEGseq， which was annotated through the GO functional analysis and KEGG pathway enrichment. 【Result】Through single-cell transcriptome sequencing， up to 815237286 and 728886552 raw reads were generated in blue discus and Alenquer discus， respectively. A total of 17035 cells were captured after the integration of the two samples’ data， which built 18 clusters（C0-C17） after filtering and dimensionality reduction. Referred to known and potential marker genes， these clusters were divided into 13 cell types and their subtypes， which encompassed most of the cell types in fish skin. Based on pseudo-bulk treatment， 20 DEGs were detected from the 2 samples （8 were highly expressed in blue discus and 12 were highly expressed in Alenquer discus）， mainly annotated to GO functional terms of gas transport and oxidative metabolism. In the single-cell dimension， two cell clusters of melanocytes and iridophore were selected for inter-sample difference analysis， which detected eight DEGs in melanocytes， involving in response to heat， cytoplasm， unfolded protein binding， growth factor activity and metal ion binding. Twentyfive DEGs were found in iridophore， which were mainly employed in the signaling pathwasy，such as apoptosis， Salmonella infection and MAPK， cell survival， stress response and signaling. 【Conclusion】TSPAN3A， PTGES， ATF3， JUNK， GADD45GA， HSP70L and FOSAB genes play an important role in regulation of pigment biosynthesis and pigmentation of blue discus fish skin. The inactive synthesis of melanin probably results in more vivid and gorgeous body color in blue discus than Alenquer discus.