| Salmo trutta fario,an introduced species,is the synonym of Salmo trutta and is only distributed in Yadong River and its tributaries in Tibetan Plateau in China.It has formed a stable local population in Yadong River now and has completely adapted to harsh environmental conditions.Nowadays,it has been listed as the second-level protected aquatic animal of Tibet Autonomous Region Government for its high economic value.Erythroculter ilishaeformis,commonly called culter fish,white fish and also scientifically named as Culter alburnus,is widely distributed in major river systems of China.This fish is an excellent aquaculture species for its significant economic and ecological values.It deserves valuable protection and utilization of germplasm resource and genetic breeding potential due to its various geographic populations.In this paper,the large-scale artificial breeding for Salmo trutta fario were studied and its molecular mechanisms of adaptation to the plateau water environment were investigated.Meanwhile,the genetic variation of breeding populations in Erythroculter ilishaeformis was analyzed and the molecular mechanisms of immune-related gene expression and regulation were also discussed by bioinformatics methods of transcriptome.The results indicated as follows: 1.Study on large-scale artificial breeding of Salmo trutta farioThe research of large-scale artificial breeding technology for Salmo trutta fario was carried out based on selection and cultivation of broodstock,fertilized egg collection,hatching management,and the temptation to eat feed.The results were:(1)The age of selected cultured healthy broodstock were 3 or above for the females,and 2 or above for the males.The weight were over 1.0kg for the females,and 2.0kg for the males.High quality feed was needed to increase the nutrition reserves of broodstock,and an appropriate increase in water flow in the inlet would promote the development of their gonads.(2)The balanced salt solution was adopted to increase the rate of fertilization,which was 96% in average.Correct egg and semen collection operation should be conducted.397 thousand larvae were incubated from 593 thousand eggs and a total of 312 thousand fries were gained after cultivation.(3)The best incubation temperature was 8~10?.The hatching rate from the heated was 81%,which was obviously higher than that of the unheated water(46%).During the incubation period,hatching water was replaced at fixed time and dead eggs were removed slightly to avoid the generation of Saprolegnia ferax that affected the development of surrounding normal eggs.(4)The suitable stocking density of larvae was about 10 thousand in each m3 of water and the operation of taming could be conducted after 20 days or so at water temperature 10~15 degrees centigrade.Filtered liquid of egg yolks was taken as feed for the first eating;then high quality feed specialized for trouts living cold water was fed.When feeding,oxygen filling and water flowing needed to be suspended for a certain period of time,and the number of times and time were adjusted according to the actual situation for full feeding of fries.Furthermore,the waste was cleaned regularly to guarantee enough dissolved oxygen in water.Significantly,the average body length of fries was increased by 67.16% at the end of the taming.2.Transcriptome analysis of Salmo trutta fario through high-throughput sequencingIn this chapter,whole mixed tissues(brain,heart,gill,liver,intestines,muscle,gonads,kidney,pancreas,and eyes)of Salmo trutta fario were sampled as the experimental material,and the transcriptome was sequenced based on Illumina HiseqTM 4000 high-throughput sequencing platform.We obtained 46,960,387 raw reads and the whole length was 14.1×10~9bp,and 35,998,548 clean reads were acquired after filtration.A total of 153,365 transcripts were assembled with an average length of 971 bp,of which 81,505(53.14%)transcripts had homologous sequences using BLAST search against NCBI-NR.Further functional analysis identified 38,518(25.12%)transcripts in Gene Ontology(GO)categories and 32,438(25.15%)transcripts in KEGG database.Analysis of GO term distribution at level 2 showed 19,10,10 subcategories in biological process,molecular function,and cellular component,respectively.Results of KAAS web server for KEGG annotation using BBH method revealed 13,394 transcripts in 280 KEGG pathways.More importantly,6 cold-related pathways with 365 genes were identified,and some relevant potential molecular mechanisms of function-related gene expression and regulation were further discussed.3.Genetic variation and relationship of breeding populations in Erythroculter ilishaeformisThe genetic difference and relationship of 4 breeding populations(HLJ,HH,CJ,CJX)in Erythroculter ilishaeformis was assessed by using the molecular marker of SSR.The results showed that it existed heterozygote deficiency in the 4 breeding populations,and there was a high degree of inbreeding within the populations.The genetic diversity of Erythroculter ilishaeformis was relatively low and had a simple genetic structure.The results of AMOVA indicated that the genetic variation was mainly derived from individuals within populations,and the variation among populations was 17.58 %(FST=0.1758,P<0.001).The largest genetic distance was found between HLJ and HH,and the lowest was observed between CJ and CJX.The four populations were assigned into four clusters on the basis of STRUCTURE.In conclusion,the results of less genetic variation and diversity in each population suggested that new individuals would be added in the current bloodstocks for breeding practice in future.4.Transcriptome analysis of immune-related genes and pathways in Erythroculter ilishaeformisIn this chapter,RNA-seq technology was applied to de novo assemble a comprehensive transcriptome,and to discover immune-related genes and pathways in Erythroculter ilishaeformis.A total of 102,017 transcripts were assembled with an average length of 945 bp,of which 45,482 protein coding sequences were predicted.BLAST search resulted in 53,575 and 45,602 transcripts with homologous sequences in NCBI-NR and UniProt,respectively.Further functional analysis identified 24,052 transcripts in Gene Ontology(GO)categories and 19,260 transcripts in KEGG pathways.Meanwhile,13 immune-related pathways with 524 immune-related genes were identified and the molecular mechanisms of immune function were discussed as well.Our data provided an integrated and comprehensive transcriptome resource for topmouth culter,which could be essential for further research in selective breeding of disease resistance,functional genomics,and genome editing of topmouth culter. |
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