The Genetic Basis Of Differences In Skin Color Traits Among Sinocyclocheilus Fishes By RNA-seq

Understanding the molecular mechanisms underlying phenotypic trait variation is one of the main themes in evolutionary biology.Animal skin color and color pattern are phenotypic traits with high variation among species,which are important traits for studying species diversity and adaptive evolution.The Sinocyclocheilus fishes that belong to the family Cyprinidae are endemic to China.Considering its special living environment,strong species differentiation,highly specialized adaptive phenotypic traits,and highly diversified skin color,there are marked differences in skin color traits among the currently recognized 68 species.Therefore,this fish genus is undoubtedly an excellent model for studying the molecular mechanisms of animal skin color variation.However,little is known about differences in skin color traits among Sinocyclocheilus fishes and their genetic basis behind.To dissect the differences in skin color traits among Sinocyclocheilus fishes and their genetic basis,8 representative species of Sinocyclocheilus fishes were chosen as subjects in this study.The 4 species of them belong to cave-dwelling type,including S.tianlinensis,S.brevibarbatus,S.broadihornes and S.rhinocerous,while the remaining 4 species belong to surface-dwelling type,including S.maculatus,S.qiubeiensis,S.jii and S.oxycephalus.Three biological duplicates of each species were collected.The comparative analysis among 4 pairs of surface-dwelling and cave-dwelling species that were composed of 8 species of Sinocyclocheilus fishes were carried out.First,to look into the differences in skin color traits among 8 species of Sinocyclocheilus fishes from the individual level and the tissue and cell level,microscopic observation and skin comparative histological study were carried out.Second,the skin transcriptomes of 8 species were sequenced by transcriptome sequencing(RNA-seq).To discover the differentially expressed genes between surface-dwelling and cave-dwelling type Sinocyclocheilus fishes,and to screened out the skin color related candidate genes with expression differences,the transcriptome analysis with or without reference genomes and joint analysis were carried out.The purpose of this study was to screen out the skin color related genes with a trend of common differential expression in at least 3 pairs of 4 pairs surface-dwelling and cave-dwelling type Sinocyclocheilus fishes,and to analyze the expression patterns and transcription changes of these skin color related genes that were relatively stable and credible.The main research results are obtained as follows:1.The skin color traits of 8 representative species of Sinocyclocheilus fishes can be classified into two main types,cave-dwelling and surface-dwelling type,and subdivided into five subtypes,including 1)Golden yellow with or without a few black spots(band),2)Golden yellow with a lot of dark spots,3)Brown with a few black spots(band),4)translucent(light)pink,and 5)translucent(shallow)grayish white.There are dendritic or snowflake melanocytes in the skin of both surface-dwelling and cave-dwelling type Sinocyclocheilus fishes.While the melanocyte dendrites in surface-dwelling type are short and thick,there are a lot of melanin particles,melanocytes are numerous,and the distribution of melanocytes is wide;the melanocyte dendrites in cave-dwelling type are thin and long,there are a few melanin particles,melanocytes are fewer,and the distribution of melanocytes is narrower.2.By transcriptome sequencing of 24 samples from 8 representative species of Sinocyclocheilus fishes,a total of 237.95 Gb of clean data was obtained.The clean data of each sample reached 6.61 Gb,and the percentage of Q30 scores base was more than 90.80%.Then,these clean data were assembled into unigenes,yielding 297,984 unigenes,and 131,766 unigenes were annotated.Moreover,the sequencing data were compared with the composite genome of S.graham,S.rhinocerous,and S.anshuiensis,10,013 novel genes were identified,of which 7,726 were successful annotated.3.Through the annotation and enrichment analysis of the KEGG pathway of differentially expressed genes,it was found that the differentially expressed genes in the skin of both surface-dwelling type and cave-dwelling type Sinocyclocheilus fishes were involved in the melanogenesis,Wnt signaling pathway,MAPK signaling pathway,purine metabolism,oxidative phosphorylation,and so on,which is highly likely to affect the skin pigmentation process among Sinocyclocheilus fishes.In these pathways,a total of 16 differentially expressed genes were down-regulated in cave-dwelling type Sinocyclocheilus fishes,and their downregulation trend was significant,including ADCY9,PRKACAA,GNA15.1,NRAS,FBXW11B,DAAM1,TAO,MAPK13,MAPKAPK2A,GUK1A,ENTPD2B,POLR2G,SDHDB,SDHB,QCR9 and ATP5MEB,of which 4 genes(ADCY9,PRKACAA,GNA15.1 and NRAS)enriched in melanogenesis may be the key genes limiting melanin synthesis in skin of cave-dwelling type Sinocyclocheilus fishes.4.Based on the reference of 97 skin color related genes of the zebrafish(Danio rerio),22 skin color related genes were found in 8 representative species of Sinocyclocheilus fishes,including ADRB2A,ALDOAA,ATP6AP1B,ATP6V0D1,CHD7,CTR9,ECE2,ERBB3B,KITA,MED23,MIB1,MYCBP2,OCRL,OPN1LW2,PAICS,PDHB,PSENEN,SMARCA4A,TRPM7,VPS18,VPS39 and YAP1.These 22 genes are related to the proliferation,migration,differentiation,survival and pigment synthesis of chromoplast,and have a significant downregulation trend in cave-dwelling type Sinocyclocheilus fishes,which may play an important role in the formation of skin color among Sinocyclocheilus fishes.In sunmmary,8 representative species of Sinocyclocheilus fishes(4 surface-and 4 cave-dwelling species)were chosen as subjects in this study.The differences in skin color traits were found out from the individual level and the tissue and cell level.The pathways and genes related to the formation of skin color in Sinocyclocheilus fishes were identified by RNA-seq,which established the link between the expression differences of skin color related genes and the variation of skin color traits in Sinocyclocheilus fishes,revealed the genetic basis of differences in skin color traits among Sinocyclocheilus fishes,and would provide new scientific evidences for fully dissecting the molecular mechanism of speciation and adaptive evolution of the genus Sinocyclocheilus fishes.