Tissue Structure and Full-Length Transcriptome Analysis of the Scaly Sublayer of Cyprinus carpio var. Quanzhounensis

Cyprinus carpio var. Quanzhounensis, native to Quanzhou County, Guilin City, Guangxi, has a dark brown body color, translucent gill cover, and abdominal skin, and is an important farmed species in the local integrated rice-fishery industry. A comparative study on the skin of Cyprinus carpio var. Quanzhounensis revealed a lack of reflective guanine crystals on the body surface and a significantly higher melanin content than that of C. carpio var. Jian, which was tentatively considered the direct cause of variations in body color of this group. Iridocytes are a pigment cell species that contain regularly arranged guanine crystals, which are the key material basis for the metallic luster of the fish body surface. In species such as medaka (Oryzias latipes) and zebrafish (Danio rerio), the absence of guanine crystals is considered a manifestation of the absence of iridocyte differentiation and therefore is ideal for studying the mechanism of pigment cell differentiation. The pnp4a, Gbx2, sox10, tfec genes and other iridocyte-related genes have been mined using mutant materials, and we have uncovered the differentiation mechanism that regulates the formation of iridescent cells. C. carpio var. color has abundant genetic variation in body color and is a good material for studying the mechanism of body color determination in fish. The roles of ASIP and MC1R in the aggregation and distribution of melanin and formation of black spots in C. carpio var. color were verified. The guanine crystalline deletion trait of C. carpio var. Quanzhounensis may be loaded with regulatory mechanism diversity and mutation loci related to guanine crystal formation or iridescent cell differentiation. In addition, as a rice-fish culture species, the living environment of C. carpio var. Quanzhounensis harvestmen differ significantly from pond and net-pen culture species, requiring high resistance to disease, adversity, and transport. It is unclear whether the absence of guanine crystals in their skin leads to changes in their basal physiological state, and in-depth studies are beneficial for accurate assessment of culture performance. To reveal the structural basis and transcriptomic characteristics of guanine crystalline deficiency traits in the skin of C. carpio var. Quanzhounensis in this study, we selected the subscale tissues with the most significant differences in guanine crystalline distribution as the control material, and transmission electron microscopy was used to observe the tissue structure and full-length transcriptome sequencing to understand the structural and transcriptomic characteristics of guanine crystalline deficiency in the skin of C. carpio var. Quanzhounensis. The results of this study provide information for the analysis of body color traits, identification of economic traits, and utilization of germplasm resources. Transmission electron microscopy of the subscale tissue sections revealed two significant differences in the histological structure of C. carpio var. Quanzhounensis, and C. carpio var. Jian. First, guanine crystals were absent in C. carpio var. Quanzhounensis, whereas guanine crystals were widely present in the tissues of C. carpio var. Jian and cascading cavities were observed in the sections after guanine crystals were dislodged. Second, the number and density of melanin particles in the tissues of C. carpio var. Quanzhounensis harvestmen were significantly higher than those of C. carpio var. Jian, showing smaller, darker, and more numerous particles, which is consistent with the darker color and lack of silvery reflective material on the body surface of C. carpio var. Quanzhounensis harvestmen. The transcriptome characteristics were analyzed using Oxford Nanopore (ONT) sequencing technology, and 2.88~3.26 Gb of high-quality data were obtained for each sample. The number of full-length sequences after filtering ribosomal RNA for all sample data was 2 203 826~2 412 500, and the proportion of full-length sequences for each sample was 87.06%~88.57%. The comparison rate was 90.35%~92.46%. Variable splicing events in the transcripts were counted; 3 075 variable splicing events and 57 624 variable polyadenylation events were detected; and 15 615 new coding region sequences and 771 long-stranded non-coding RNAs were predicted. The number of exon jumps and intron retention in variable splicing events differed significantly (P<0.01) between species, and the number of transcripts with five polyadenylation sites differed significantly (P<0.01) between species, indicating that variable splicing and polyadenylation are involved in regulatory processes related to trait formation. A total of 15 615 open reading frames (ORFs), including 9 890 complete ORFs, were predicted in this study. A total of 841 differentially expressed transcripts were screened in this study; 183 transcripts were upregulated and 658 transcripts were downregulated in C. carpio var. Quanzhounensis compared to C. carpio var. Jian (JH), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed enrichment in extracellular matrix-receptor interactions and adherent spots. KEGG pathway analysis showed that it was enriched for extracellular gaps, transcription factor complexes, integrin complexes, and other terms. The most significantly enriched KEGG pathway and GO terms were closely associated with the extracellular matrix, and it is speculated that these transcriptional changes may lead to changes in the composition, density, and conformation of the skin extracellular matrix.