Comparative Transcriptomics And Molecular Evolution Of Major Hypoxia-related Gene Of Representative Species From The Schizothoracine Fish

Schizothoracine fishes are one of the main components of the fish fauna in the Qinghai-Tibet Plateau,which are also an ideal material to explore the impact of the uplift of Qinghai-Tibet Plateau and the environmental changes on species evolution.In view of its important position,widespread distribution,diversity of habitat water environment and strong adaptability to the plateau ecosystem,this study aims to take the representative species of Schizothoracine fishes in the Qinghai-Tibet Plateau as the research object including original grade(Schizothorax lantsangensis,Schizothorax macropogon,Schizothorax lissolabiatus and Schizothorax labiatus),special grade(Gymnodiptychus pachycheilus and Ptychobarbus dipogon),high specialization level(Oxygymnocypris stewartii,Platypharodon extremus,Schizopygopsis pylzovi,Schizopygopsis stoliczkai,Schizopygopsis younghusbandi,Gymnocypris waddelli,and Gymnocypris eckloni),mixed transcripts of liver,muscle,kidney and brain tissues were screened out by Illumina sequencing technology,carry on comparative transcriptome research.Based on the transcriptome data of 13 species from Schizothoracine fishes,the complete coding region sequences of globin family members,hypoxia-inducible factors,erythropoietin and erythropoietin receptors,and vascular endothelial growth factor were obtained by local BLAST and RT-PCR techniques.Finally,do molecular evolution analysis for every gene.In order to explore the molecular basis of Schizothoracine fishes to adapt to the unique environment of the plateau.The main results are as follows:(1)The 62,487,860-81,238,722 of clean reads were obtained by Illumina sequencing.Do de novo assembly for transcriptome,129,982-207,887 transcripts and 95,251-145,805 unigenes were obtained from 13 species.(2)Based on six public databases,the unigenes of 13 species were annotated,with a total annotation rate higher than 81%.Among them,the matching degree of unigene in Nt database is the highest,the lowest is 79.03%(Gymnocypris waddelli),and the highest is to 89.26%(Schizothorax lissolabiatus).(3)2,064 single-copy genes were identified from 13 species,of which 52 genes had a Ka/Ks ratio >1 and 187 genes had a Ka/Ks ratio between 0.5 and 1.GO functional enrichment analysis and KEGG metabolic pathway analysis revealed that nine candidate genes associated with innate immunity and adaptive immunity play an important role in the adaptive evolution of Schizothoracine fishes(4)The myoglobin gene(Mb)coding region of 13 species was 444 bp in length,encoding 147 amino acids.Molecular evolution analysis found that Mb experienced positive selection(? = 3.83),and four potential positive selection sites were detected.The branch-site model of increasing outer groups showed that the Mb of the Schizothoracine fishes experienced strong positive selection(?=140.69).(5)The neuroglobin(Ngb)coding region of 13 species is 480 bp in length,encoding 159 amino acids.The site models of 13 species showed that Ngb experienced positive selection(? = 3.69),and a potential positive selection sites were detected.The branch-site model of increasing outer groups showed that Ngb gene of the specialized and highly specialized Schizothoracine fishes experienced positive selection(? = 3.399).(6)The length in Cygb1 and Cygb2 coding regions of 13 Schizothoracine fishes were 525 bp and 540 bp,respectively,encoding 174 and 179 amino acids.The Cygb2 gene was lost in four species from the original grade.Positive selection was found in the Cygb1 gene of 13 species,and a positive selection site was detected.The branchsite model showed showed that Cygb2 gene of the specialized and highly specialized Schizothoracine fishes experienced positive selection.(7)The hemoglobin(Hb)of 13 species is consisted of embryonic Hbae and Hbbe genes with coding regions of 432 bp and 444 bp respectively,and adult Hba,Hbb1 and Hbb2 with coding regions of 432 bp,447 bp and 444 bp respectively.Molecular evolutionary analysis showed that five genes experienced positive selection(?> 1),and potential positive selection sites were detected.(8)The coding regions of Hif-1??,Hif-1?B,Hif-2?? and Hif-2?B genes were 2196 bp,2325 bp,2544 bp and 2511 bp in length,respectively.HIF-? experienced positive selection in all 13 species.No positive selection was found in the HIF-1?A gene after including the outer groups.(9)Molecular evolution analysis showed that both erythropoietin and erythropoietin receptor had positive selection effect,and potential positive selection site was detected.The branch-site model showed that the two genes of whole Schizothoracine fishes experienced positive selection,the branch model also supports this result.(10)Positive selection was found in vascular endothelial growth factor under the site model,and three potential positive selection site were detected.In the branch-site model,VEGFa of highly specialized Schizothoracine fishes experienced positive selection,which was the same as branch models.