| Salmon and trout live in fresh and pollution-free waters,which is the only aquatic product that can not meet the market demand in China.Due to the lack of high-quality aquaculture production of salmon and trout in China,one hundred thousand tons of production are imported every year,and the excellent germplasm of salmonids has also been monopolized by foreign enterprises for a long time.Therefore,the cultivation of high-quality salmon and trout germplasm with independent intellectual property rights is the best way for the sustainable development of salmonids aquaculture industry.Taimen(Hucho taimen)is an indigenous salmon in China,with the characteristics of fast growth,strong disease resistance and high nutritional value,it had great potential to become an excellent aquaculture species,and it is the first excellent cold-water fish with independent intellectual property rights in China which has broadly cultured.However,the currently cultured fishes are the offspring of wild stocks without breeding which causes some problems such as slow weight growth in low water temperature(less than 8℃)and at age of 1~2 year,acute stress response,and poor tolerance to high temperature,which hinders the development of aquaculture industry of taimen.Due to environmental deterioration and overfishing,it is imperative to carry out resource protection and breeding of Hucho taimen.Genetic resource is an important basis for population genetics,conservation genetics and genetic breeding,while few genetic resources are available for taimen.In this study,we carried out the de novo assembly of taimen transcriptome,construction of high-resolution genetic linkage map,QTL mapping of growth traits,identification of sex-specific markers and other related studies which provided necessary genetic resources for conservation genetics and genetic breeding of Hucho taimen.Furthermore,the specific markers for germplasm identification were obtained,which provided an effective tool for identification and protection of wild resources and cultured species.(1)We obtained 153 M clean reads(14.15 Gb)and used them to de novo assemble a high-quality transcriptome with a N50 size of 1060 bp.In the assembled transcriptome,82% of the transcripts were annotated,14666 transcripts had complete open reading frames,26559 transcripts covered more than 80% of the full-length protein sequence,and 30284 transcripts covered more than 50% of the full-length protein sequence.Compared with Atlantic salmon,155125 transcripts of Hucho taimen can be aligned in Atlantic salmon genome,covering 75.1% of Atlantic salmon transcripts.Compared with rainbow trout genome,131887 transcripts could be aligned in rainbow trout genome,covering 57.3% transcripts.Phylogenetic analysis showed that Salmonidae originated from 96 MYA,and Hucho taimen originated from 37 MYA.Gene family analysis showed that there were 313 unique gene families,which were enriched in the pathways,including focal adhesion(KEGG,dre04510,P < 0.05),GTP binding(GO: 0005525),protein deletion(GO: 0051697),transcription regulatory region DNA binding(GO: 0044212),small GTPase mediated signal transduction(GO: 0007264),C-terminal protein lipidation(GO: 0006501)and protein targeting to membrane(GO: 0006612).Based on the genome of rainbow trout and Atlantic salmon,128 positive selection genes were identified by transcriptome positive selection analysis.These genes were enriched in immune related pathways,including cytokine-cytokine receptor interactions(KEGG,dre04060),immune response(GO: 0006955),Chemokine activity(GO: 0008009)and cytokine activity(GO: 0005125).Combined with the specific gene families and positive selection analysis results of Hucho taimen,it showed that Hucho taimen had more immune related genes than rainbow trout and Atlantic trout,which explained the phenomenon that Hucho taimen had stronger disease resistance than rainbow trout and Atlantic trout.In addition,17841 microsatellite sequences with more than 6 repeats were obtained from the transcriptome.24 polymorphic microsatellite markers and 20 monomorphic markers were identified,and 68533 high-quality SNP markers were obtained.(2)Genetic linkage map is a necessary tool for genetic and genomic research.In order to construct a high-resolution linkage map of Hucho taimen,two full-sib families of F1 generation of Hucho taimen were selected as the mapping populations.Body length,body weight,body height and body width were measured.A total of 177 individuals from two full-sib families of Hucho taimen were sequenced using SLAF technique,and 58016 high-quality SNP markers were obtained.Lep-map3 software was used to construct the linkage map,and a high-resolution genetic linkage map containing 30535 SNPs was obtained.The genetic linkage map consists of 42 linkage groups,including 6734 loci.The length of each linkage group ranged from 66.257 cM to 135.533 cM.The total length is 4253.48 cM,with the average interval distance ranged from 0.468 cM to 1.110 cM,covering 98.74% of the genome.The linkage map was used to integrated the scaffold sequences of Hucho hucho,and the results showed that 65.1% of the scaffold sequences could be integrated into 42 pseudo-chromosomes.The length of each pseudo-chromosomes ranged from 14 M to 73 M,with a total length of 1620 M.The average colinear Pearson correlation coefficient was 0.893.The QTL fine mapping of body length,body weight,body height and body thickness was carried out by using the map,and 27 QTLs for 4 growth traits were obtained,including 18 QTLs for body weight,5 QTLs for body length,3 QTLs for body thickness,and 1 QTL for body height.The 95% confidence intervals of 5 QTLs were less than 10 cM.Based on the annotation of the Hucho hucho genome,4218 genes were screened in the QTL intervals.Among them,487 genes were significantly enriched in metabolism related pathways,which could be used as candidate genes related to growth traits of Hucho taimen aged 6-7 months.This studsy provides an effective tool for genetic research and molecular breeding of Hucho taimen.(3)Since it is difficult to discriminate Hucho taimen,Brachymystax lenok and their hybrids from morphology at juvenile stage,which easily results in germplasm contamination,it is essential to develop a simple operating method for distinguishing taimen(Hucho taimen),lenok(Brachymystax lenok)and their hybrids.In this study,specific molecular markers were screened from its genes and microsatellite markers.The results showed that the ITS marker(1-ITS1)was amplified a 334 bp fragment in Hucho taimen,while failed to amplify in Brachymystax lenok.To eliminate the effect of DNA degradation,artificial faults in experiments that caused the failure of PCR amplification,we introduced a pair of 12 S rRNA primers as the reference which was amplified a 251 bp fragment and established a duplex PCR method.If 1-ITS1 and 12 S rRNA were amplified successfully,the samples were classified to Hucho taimen,whereas if only 12 S rRNA was amplified,the samples were classified to Brachymystax lenok.For validation this method,60 samples of Hucho taimen and 60 samples of Branchymystax lenok were amplified,and the results showed that all samples were classified correctly,with accuracy of 100%.Additionally,a microsatelite marker was found that it could be used to classify the hybrids of taimen and lenok.Among the 20 SSR markers developed from transcriptome,Hta C1041 primer was identified to be able to distinguish Hucho taimen,Brachymystax lenok and their hybrids.The amplified band of Hucho taimen was 166 bp,and the amplified band of Brachymystax lenok was 158 bp,while the hybrid had two amplified bands.Since there are natural hybrids between Hucho taimen and Brachymystax lenok,158 wild Hucho taimen samples were tested by using this marker.It was found that one hybrid sample in the wild samples were identified,which confirmed the existence of the natural hybrid between Hucho taimen and Brachymystax lenok.This study proved that there is overlap in breeding time and breeding field of Hucho taimen and Brachymystax lenok at molecular level.(4)Sex-specific markers are an essential tool for mono-sexual fish cultivation,optimization of sex ratio of broodstock,and sex-controlled breeding.To identify sex-specific markers in Hucho taimen,the sex-related candidate sequences were obtained by alignment of the draft genome of Hucho taimen against the Y chromosome of rainbow trout(Oncorhynchus mykiss).Four pairs of primers were designed based on this candidate sequence to characterize the sex-specific markers.The results indicated that one pair of primers(ST2)might be the male-specific marker of Hucho taimen,with a PCR product of 153 bp in male samples but not in female samples.To eliminate the effects of DNA degradation and artificial faults in PCR amplification failure,a duplex PCR assay was established by introducing the 12S rRNA(251 bp)as a reference.Samples were classified into males when ST2(153 bp)and 12 S rRNA(251 bp)were amplified successfully,whereas samples were classified into females when only 12 S rRNA(251 bp)was amplified.ST4 primers can amplify two bands(306 bp and 255 bp)in male,but only one band in female(306 bp).The accuracy of sex identification by ST2 and ST4 primers was 100%.In this study,ST4 primers were used to amplify the samples of wild Hucho taimen.It was found that the sex ratio of wild Hucho taimen population was close to 1:1.It provides an effective tool for studying the population structure of wild germplasm of Hucho taimen.In conclusion,this study we obtained a high-quality transcriptome,and preliminarily revealed the evolutionary characteristics of the genome by comparative transcriptome analysis,and developed a number of SSR and SNP markers.We also constructed a high-resolution genetic linkage map for the first time,and carried out QTL analysis for growth traits of 1-year-old Hucho taimen,and obtained 27 QTLs and 487 candidate genes related to growth traits.Furthermore,we developed the molecular markers for species identification and sex-specific molecular markers were also developed.This study greatly enriched the genetic resources of Hucho taimen,and provide basic data and genetic tools for the study of population genetics,conservation genetics,genetic breeding and genetic management of Hucho taimen. |
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