High-quality Genome Assembly And Identification And Functional Analysis Of Stress-related Genes And Long Non-coding RNAs In Turbot (Scophthalmus Maximus)

Turbot(Scophthalmus maximus)(Fish Base ID: 1348),a worldwide economically crucial cold-water flatfish species with delicious taste and high nutritional value,is now widely cultivated in Europe and China.In recent years,due to the high density intensive culture coupled with the increased frequency of extreme climate events,turbot is facing a variety of environmental stresses,which have a significant impact on its immune,growth,reproduction and other important economic traits,which has caused great economic losses to the breeding industry of turbot and has become one of the main bottlenecks greatly hindering the green,healthy and sustainable development of turbot culture industry.Therefore,it is urgent to cultivate new varieties of turbot with stress resistance or even comprehensive stress resistance by means of germplasm resources genetic improvement and molecular design breeding,so as to alleviate and eliminate the negative effects brought by environmental stress,improve the welfare and aquaculture benefit of turbot,and finally break through the bottleneck restricting the development of the turbot industry.However,the lack of high quality genome sequences,key stress responsive genes and the unclear molecular regulation mechanism of stress response greatly restrict the development of anti-stress strategies and the process of the genetic character improvement and molecular design breeding of turbot.To this end,in this study,a significantly improved,high-quality and chromosome-scale genome sequence was firstly obtained through a combination of PacBio long-read and Illumina short-read sequencing technologies.Based on this genome assembly,a large number of candidate stress responsive gene modules and key hub genes were screened by reanalysed all of the published biotic and abiotic stress-related RNA-seq datasets derived from the NCBI SRA database by constructing gene co-expression network.Furthermore,based on this reference genome,the stress-related mitogen-activated protein kinase(MAPK),mitogenactivated protein kinase kinase(MKK)and heat shock proteins 70(HSP70)gene families were identified at the whole genome level for the first time,respectively,and their roles in response to biotic and abiotic stresses were further clarified using the stress-related RNA-seq datasets,and the potential functions of the gene family members that may have comprehensive anti-stress functions were further verified by qPCR experiments.In addition,based on this genome sequence and all published RNA-seq datasets of turbot in the NCBI SRA database,long non-coding RNAs(lncRNAs)were identified at the whole genome level of turbot for the first time,and lncRNAs in response to each kind of stresses were screened out.Through above studies,high-quality genome sequence,a large number of stress responsive key hub genes,gene family members,and lncRNAs in response to stress in turbot were obtained,and the molecular mechanisms in response to biotic and abiotic stresses were preliminarily revealed in turbot.At the same time,these important genetic resources provided abundant basic data and important theoretical support for genetic improvement of germplasm resources and molecular-based design breeding programs for production of the turbot’s stress-resistant strains in aquaculture industry.(1)We present a significantly improved,high-quality and chromosome-scale genome assembly of turbot using a combination of PacBio long-read and Illumina shortread sequencing technologies.The genome assembly spans 538.22 Mb comprising 27 contigs with a contig N50 size of 25.76 Mb.Annotation of the genome assembly identified 104.45 Mb repetitive sequences,22,442 protein-coding genes and 3,345 nc RNAs.This genome assembly is the most well-annotated and high-quality genome sequence of turbot in the world to date.The acquisition of high-quality genome assembly and complete gene annotation information lay a solid foundation and provide important basic data for further research on the genomics,genetic character analysis,molecular mechanism,gene resources mining and molecular design breeding in turbot.(2)A total of 345 stress responsive candidate genes and multiple gene modules extremely significant related to each kind of stresses were identified by reanalyzed 14 published stress-related RNA-seq datasets consisting of 165 samples(date size of 1.11TB)using gene co-expression network analysis.These genetic resources not only provide the basic data for further research on the molecular regulation mechanism of stress response in turbot,but also will promote the progress of genetic improvement of germplasm resources and the molecular design breeding program for production of the stress-resistant strains in aquaculture industry of turbot.(3)The MAPK gene family performs crucial roles in cell division,migration,development,apoptosis,inflammatory response,and especially in abiotic and biotic stress responses.However,the study of the identification and function analysis of MAPK gene family has not been reported in turbot.In this study,15 MAPK gene family members(SmMAPKs)were identified at the whole genome level of turbot,and their basic chemical and physical properties and subcellular localization were illustrated.All SmMAPKs contained the serine/threonine protein kinases,catalytic domain(S＿TKc,SMART00220).Phylogenetic analysis revealed that SmMAPKs were classified into three subfamilies,namely,c-Jun NH2-terminal kinase(JNK),extracellular signal-regulated kinase(ERK),and p38.Conserved motif and gene structure analysis revealed high levels of conservation within and between subfamilies.Expression patterns of SmMAPKs under diverse abiotic and biotic stresses in distinct tissues were examined using multiple published stressrelated RNA-seq datasets.As a result,SmMAPKs showed obviously tissue-specific expression patterns.Furthermore,7 and 10 candidate stress-responsive MAPK gene family members were detected under abiotic and biotic stresses,respectively.Among them,SmMAPK4(ERK4),SmMAPK6(ERK3),SmMAPK11(p38b),and SmMAPK12b(p38g)showed extremely significant responses to abiotic(heat or oxygen or salinity stress)and biotic stresses(Enteromyxum scophthalmi or Vibrio anguillarum or Megalocytivirus infection stress),and SmMAPK15(ERK7/8)showed extremely significant responses to each kinds of stresses except for the Megalocytivirus infection stress,demonstrating their potential functions in comprehensive anti-stress especially SmMAPK15.qPCR analysis further verified that SmMAPK15 did indeed play important roles in response to both biotic and abiotic stresses,and can be used as a candidate MAPK gene family member with comprehensive stress resistance.These results demonstrate that MAPK gene family might play vital roles in response to various abiotic and biotic stresses in turbot.The MAPK gene family of turbot was systematically identified and analyzed for the first time in this study.Described in this study not only improves the understanding of the roles of MAPK genes in response to biotic and abiotic stresses,but also lays an important foundation for elucidating the molecular regulation mechanism of turbot in response to various environmental stresses.(4)MKKs are essential components of the highly evolutionarily conserved MAPK cascade,which play crucial roles in response to a variety of biotic and abiotic stresses and immune responses.However,very little information is available about the MKKs in turbot.In order to detect the roles of the MKKs in response to biotic and abiotic stresses in turbot,we first identified the MKK gene famlily members of turbot at the whole genome level through bioinformatics methods,and then analyzed their expression patterns in different tissues and under different biotic and abiotic stresses conditions using multiple stress-related RNA-seq datasets.As a result,a total of 9 MKK gene family members of turbot(SmMKKs)were identified,and they were unevenly distributed on 7chromosomes.Physicochemical characteristics,secondary structure and subcellular localization of the MKK proteins of turbot were predicted,respectively.Phylogenetic analysis revealed that SmMKKs were classified into 5 subfamilies.Conserved motif,intron-exon structure and multiple sequence alignment not only provided evidences for the classification of MKK subfamilies,but also revealed high levels of conservation in evolution within and between subfamilies.Expression patterns of SmMKKs in distinct tissues and under diverse abiotic and biotic stresses were examined using multiple published RNA-seq datasets.As a result,SmMKKs showed obviously tissue-specific expression.In addition,SmMKK6 a was extremely significant differentially expressed after infection with both E.scophthalmi and Megalocytivirus.After heat stress,SmMKK6 a also showed extremely significant differential expression.Furthermore,SmMKK4 a,SmMKK4 b,SmMKK6 a and SmMKK7 were extremely significant differentially expressed after high-or low-salinity stresses.Among these stressresponsive MKK genes,SmMKK6 a showed extremely significant response to both abiotic and biotic stresses,demonstrating its potential functions in comprehensive antistress.qPCR analysis further verified that SmMKK6 a did indeed play important roles in response to biotic and abiotic stresses,and can be selected as a candidate MKK gene family member with comprehensive stress resistance.This study may be the first study to systematically identify and analyze the MKK gene family in turbot.The results not only demonstrate that SmMKKs play crucial roles in response to various biotic and abiotic stresses,but also provide important basic data and theoretical support for the development of molecular design breeding for comprehensive anti-stress in turbot.(5)HSP70s play essential roles in many biological processes,especially in response to various environmental stresses.To date,no systematic identification and functional analysis of HSP70 gene family have been conducted on turbot.Herein,we identified 16HSP70 genes at the genome-wide level of turbot,and their biochemical properties,subcellular localization and protein structure were illustrated,respectively.Gene structure,motif composition and phylogenetic relationship analysis provided strong evidence for the evolution,classification and functional diversity of the HSP70 gene family members.16 turbot HSP70 genes were unevenly distributed on 9 chromosomes.Molecular evolution analysis indicated that the duplicated HSP70 genes in turbot were all under purifying selection.Expression profiles of the HSP70 genes in diverse tissues under five different stresses(heat,salinity,parasitic infection,bacterial and viral infections)were comprehensively investigated by examining multiple RNA-seq datasets.Results showed that most of the HSP70 genes were expressed in stress-specific and tissue-specific expression patterns under both abiotic and biotic stresses.In addition,10,6,8,10,and 9HSP70 genes showed significantly up-or down-regulated expression after heat,salinity,and parasitic,bacterial,and viral infection stress,respectively.Among them,hsp70(hspa1a),hspa1 b and hspa5,showed significant responses to all five diverse stresses,indicating their potential roles in comprehensive anti-stress.In addition,qPCR analysis showed that hsp70(hspa1a),hspa1 b and hspa5 were significantly or extremely significant upregulated after both heat and Edwardsiella tarda infection stresses,further validating their involvement in comprehensive anti-stress.This may be the first study that systematically identified and analyzed the HSP70 gene family in turbot.These findings not only provid new insights into the biological functions of HSP70 in adapting to various environmental stresses in turbot,but also provid important target genes for the molecularbased design breeding of comprehensive stress resistance in turbot.(6)lncRNAs are a type of non-coding RNA with transcript length greater than 200 bp and lacking protein-coding ability.An increasing number of studies have shown that lncRNAs played important roles in stress response in a variety of organisms,such as plants,animals.However,identification and functional analysis of lncRNAs have not been reported in turbot to date.In view of the important roles of lncRNAs in response to various stresses,lncRNAs were identified at the whole genome level for the first time in turbot in this study.Based on the high-quality genome sequence and all published 24RNA-seq datasets(1.88 TB)collected from the NCBI SRA database,a non-redundant lncRNA dataset consisting of 12,999 lncRNAs(including 29,187 transcripts)was identified.Tissue expression analysis showed that lncRNAs,especially linc RNAs,had stronger tissue specific expression than coding genes.In addition,482,1,351,1,060,875,420,and 1,689 differentially expressed lncRNAs were identified under V.anguillarum,E.scophthalmi,and Megalocytivirus infection,and heat,oxygen,salinity stress,respectively,using multiple stress-related RNA-seq datasets.Among them,9 lncRNAs(lnc＿MSTRG.17190,lnc＿MSTRG.28492,lnc＿MSTRG.22156,linc＿MSTRG.12463,lnc＿MSTRG.7047,linc＿MSTRG.7983,lnc＿MSTRG.7990,linc＿MSTRG.13517 and lnc＿MSTRG.9703)showed differential expression under both biotic and abiotic stresses,which could be selected as candidates for comprehensive stress resistance.In addition,the analysis of lncRNA expression patterns further demonstrated the comprehensive stress resistance of these 9 candidate lncRNAs.