| Salmonidae is an ancient fish that has existed more than 100 million years ago,according to ancient fossil research.As a unique salmonid fish in China,the distribution range and evolutionary history of B.lenok tsinlingensis are of great value to the study of fish faunal composition,climate change,and geography.At the same time,B.lenok is an important fish component in China’s major rivers,and the conservation of its germplasm resources plays an important role in the stability of river biodiversity and the ecological protection of land and water areas.Based on years of research and experience accumulation,the study for the first time completed the key technologies of the whole process from artificial domestication to the second generation(F2)breeding and mastered the full artificial breeding technology of B.lenok tsinlingensis.Transcriptomics and genomics were used to study the response mechanism of heat stress and population genetics,and to systematically analyze the evolutionary history of B.lenok and the regions affected by domestication and selection in the genome.The relevant research results are as follows.1.From artificial domestication to second-generation breeding of B.lenok tsinlingensis,it involves multiple technical links such as parent domestication,cultivation,artificial propagation,First feeding domestication,summer,and winter cultivation.Based on the key technologies of each link,targeted research and experience summary have been carried out.Salmon gonadotropin-releasing hormone analogs were used for artificial oxytocin,and the injection dose was 0.5 m L/kg.Artificial extrusion method was used for artificial egg collection and insemination from48 hours to 72 hours after oxytocin.Water temperature 8℃～12℃,dissolved oxygen≥8.0 ㎎/L,daily water temperature difference≤2℃,dissolved oxygen saturation≥90%,p H7.8～8.3;In the early stage,the opening is mainly dominated by the yellow mealworm,and in the late stage,artificial compound feed should be added in time for transshipment domestication.Summer is mainly accomplished by adjusting and reducing feeding quantity and increasing oxygen supply,while overwintering is mainly accomplished by adjusting and increasing feeding frequency,reducing breeding density,and disease prevention and control.Disease prevention and control during summer and overwintering mainly relies on scientific and rigorous management and prevention to avoid group diseases.2.Through transcriptome sequencing of gill and liver tissues,385,314 transcripts were obtained for the first time,and 110,712 protein-coding transcripts were retained,of which N50 and average length were 1677 and 910 bp,respectively.A total of 90 individuals were randomly selected from three wild populations and 20 microsatellite markers with polymorphic loci were screened.These markers performed well in all three populations and may guide future monitoring and management of wild populations.In this study,only 192 SSR markers were tested,and 75,699 SSR markers were not utilized,which will be an important resource for developing high-quality SSR markers,and provide support for large-scale conservation research and breeding of endangered fish in the future.3.Transcriptomic studies on the body response mechanism of B.lenok tsinlingensis under heat stress showed showed that the transcriptome pattern was similar between the control group and the tolerant group under heat stress,and there were more differentially expressed genes in the sensitive group than in the tolerant group,suggesting that the gene expression in the tolerant group may return to the basic level with the increase of exposure time.Tissue differences were the main factors affecting gene expression and they also showed different physiological responses to heat stress.An interesting rule was found,that is,more DEGs up-regulation was found in gill tissue and more DEGs down-regulation was found in liver tissue.The differentially expressed transcriptome in gill tissue is mainly enriched in:(1)protein modification;(2)ion transport;(3)Cytoskeleton;(4)immune response;(5)antioxidant stress and(6)metabolic response.The main concentrations in the liver are:(1)metabolic changes;(2)immune response;(3)endocrine response;(4)cytoskeleton and(5)ion transport.The transcriptome showed tissue-specific response under heat stress.Heat stress often induces a stress-related inflammatory response that induces an immune response.Especially in the temperature resistant group,a range of immune systems was activated due to prolonged exposure to the thermal environment.Although this study assessing B.lenok tsinlingensis thermal safety mar(TSM)but based on our four years from 2017 to2020,the water temperature monitoring data analysis,The TSM for adult B.lenok tsinlingensis is about 3.5℃,and the TSM of embryos and sexually mature individuals will be very narrow,which may put them on the brink of extinction.4.The whole-genome resequencing of 24 individuals from 5 geographic groups of Taibaihe,Baiyunxia,Zhangjiachuan,Saihanba,and Yanbian by high-throughput sequencing technology,obtained 1.3Tb of data and detected 66.7 million SNPs site.Phylogenetic tree analysis and genetic structure analysis showed that 5 geographical populations were separated from each other and lacked gene exchange.According to the pedigree relationship,5 geographical populations came from 4 ancestors,among which Baiyunxia and Zhangjiachuan were the same ancestral population.The differentiation time calculated based on the evolution rate of single-copy homologous genes shows:Taibaihe population was the earliest to differentiate from other populations,indicating that Taibaihe population located in the tributary of Han River in the upper reaches of Yangtze River may be the oldest population,and B.lenok may have entered the southern part of the Qinling Mountains from the Atlantic Ocean and the Tethys Sea,and then were landbound with the uplift of The Qinghai-Tibet Plateau and the disappearance of the Tethys Sea.The isolation of Zhangjiachuan and Baiyunxia populations may be related to the uplift of the Qinling Mountains.The possible explanation for why The Baiyunxia population could not separate from Saihanba and Yanbian populations until16.49 Mya is that they had gene exchange between 24.91 Mya and 16.49 Mya,indicating that after no gene exchange between Baiyunxia population and Zhangjiachuan population,It was also able to communicate with Saihanba and Yanbian populations through rivers or oceans,and the only river supporting such communication was Weihe River,so Weihe river may have been open to the sea at that time.Population principal component analysis showed that the main influencing factors were related to the geographical location of the population,and different geographical environments made environmental selection and adaptation for different populations.In the historical analysis of population dynamics,Taibaihe,Baiyunxia,and Zhangjiachuan have similar population dynamics,while Yanbian and Saihanba have severe fluctuations,which may also be related to the small number of samples and unrepresentativeness.Linkage disequilibrium analysis showed that the probability of recombination within the same physical distance was Taibai River > Zhangjiachuan > Baiyunxia.Based on SNP data,selective clearance analysis of selected genes in each geographic population was carried out,and functional annotation and enrichment analysis of relevant databases were performed,which provided abundant reference data for further genetic breeding and related molecular markers.5.Based on the above research,it is urgent to strengthen the protection of Brachymystax lenok,especially B.lenok tsinlingensis in the Yangtze River and Yellow River basins where Taibai River,Baiyunxia,and Zhangjiachuan are located.Currently,the distribution in these regions is wide,small population,and has serious fragmentation,which requires active ecological protection and population restoration.Large-scale and continuous artificial breeding and proliferation and release activities since fully mature artificial breeding can play a positive role in the restoration of endangered natural populations.At the same time,the restoration methods and technologies can be adjusted and optimized through tracking and monitoring to establish a protection model of germplasm resources.Habitat expansion,elevation reduction,restoration of gene exchange between rivers,and population increase are expected in the future.In addition,based on the good natural population size,it will play a profound and important role in monitoring climate change and the ecological stability of large rivers. |
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