| Macrobrachium nipponense,an important freshwater aquaculture species in China,was bred in large quantities due to its fast growth speed,short reproductive cycle,and strong stress resistance.With the rapid development of aquaculture,the coexistence of multiple generations caused by precocious puberty,growth inhibition caused by high-density aquaculture,and degradation of germplasm resources have seriously hindered the development of the shrimp aquaculture industry.In this paper,the F3 generation hybridized between the Yangtze River（CJ,E119.56,N32.24）and Dongting Lake（DT,E112.99,N29.30）populations were chosen as the male parent,as well as the population in the Dianshan Lake（DS,E120.97,N31.13）were chosen as the female parent.Hence,two three-line hybrid populations were obtained including SCD（DS♀ × CD♂）and SDC（DS♀ × DC♂）.Employing the parental population（DS,CJ,and DT）and hybrid population（SCD and SDC）as research objects,the analysis of SNP genetic molecular markers and morphological indexes were proceeded to discuss the differences in morphological characteristics and genetic diversity between them.The growth performance and biochemical components of these five populations were compared and analyzed from the perspective of growth speed and nutrient accumulation.On this basis,a comparative study of the parental population（DS）and hybrid population（SCD）based on transcriptome and proteome analysis was conducted to further explore the differences between them at the gene and protein levels.Finally,ammonia nitrogen exposure experiments were carried out on DS and SCD.The differences in stress resistance were comprehensively compared from the aspects of antioxidant immunity,energy metabolism,and detoxification ability.This study will lay a theoretical foundation for the hybrid breeding of M.nipponense.The main findings and conclusions of the study are listed below:1 Morphological analysis and comparison of SNP genetic molecular markers between hybrid population of M.nipponense and their parentsIn this study,the research objects were three parent populations of M.nipponense from CJ,DT,DS and two hybrid populations SCD and SDC.The F1 generation of five populations was taken as the experimental object for a three-month breeding experiment.The dominant characteristics that showed significant differences between hybrid and parental populations were obtained by measuring 19 morphological indexes and using statistical analysis methods.At the same time,SNP genetic molecular markers were analyzed in them.The results show that:（1）SCD and SDC gather into one branch,while DT and CJ gather into another.（2）The traits that have a significant influence on body mass in hybrid populations include both genetic traits and traits that distinguish them from their parents.The genetic ones include the fantail width（FW）and overall length（OL）.And the distinguished ones include the second periopod length（P2L）and carapace width（CW）.（3）The hybrid population has higher nucleotide diversity and haplotype diversity than the parent population.2 Comparison of growth performance and biochemical components between hybrid and parents of M.nipponenseThree-month breeding experiments were carried out on the parental populations（DS,DT,and CJ）and hybrid populations（SCD and SDC）of M.nipponense.And their growth performance-related indicators and biochemical components were measured.The results obtained are as follows:（1）The survival rate of the parental population is significantly lower than that of the hybrid population.（2）The hybrid population’s biochemical indexes,which include triglyceride,total cholesterol,and glucose in muscle and hepatopancreas,were significantly higher than the corresponding biochemical indexes in the parent population.Compared with the parent population,the activity of digestive enzymes in the hepatopancreas of the hybrid population was significantly higher,while the difference in the activity of digestive enzymes in the muscle was not significant.（3）The content of polyunsaturated fatty acids in the hepatopancreas and the content of essential amino acids in the muscle were significantly different between the two groups,and the content of these was more in the hybrid group.（4）The expression level of some growth-related genes such as CHS,Ec R,RXR,CDA1,LGR2,and Ca MKI in the muscle of the parental population exhibited significantly lower than that of the hybrid population.3 Comparative analysis of omics between hybrid and parents of M.nipponenseBased on the transcriptomic and proteomic analysis,the parental population（DS）and hybrid population（SCD）after three months of breeding were used for comparative study.Transcriptome sequencing showed that 259 unigenes were upregulated and 555 unigenes were down-regulated,which is a total of 814 differentially expressed unigenes.The KEGG pathway enrichment includes oxidative phosphorylation,apoptosis,and MAPK signaling pathways.The results of proteome sequencing revealed 57 differential proteins,including 35 up-regulated proteins and22 down-regulated proteins.The KEGG pathway enrichment mainly includes arginine synthesis,glutamate,and alanine metabolism signaling pathways.Fluorescence quantitative PCR was used to verify and analyze the gene sequences corresponding to15 differential genes randomly selected in the transcriptome and 14 differential proteins in the proteome.Both transcriptome and proteome results showed that the hybrid population had certain advantages in growth and immunity compared with the parental population.4 Comparative analysis of stress resistance between hybrid and parent populations of M.nipponense based on ammonia nitrogen exposureTo further verify the results of the transcriptome and proteome,a 96-hour acute ammonia nitrogen exposure experiment and a 28-day chronic ammonia nitrogen exposure experiment were performed on the parent population（DS）and the hybrid population（SCD）.The results obtained show that the hybrid population is more tolerant to ammonia nitrogen than the parent population.With the increase in ammonia nitrogen concentration,the number of apoptotic cells in the hepatopancreas showed an upward trend.There was a significant difference in the apoptosis rate of the two populations under the same ammonia nitrogen concentration,and the SCD population was lower.Under the same ammonia nitrogen concentration,the antioxidant and immune enzyme activities of the SCD population were higher than those of the DS population.After chronic 28-day ammonia nitrogen exposure,the survival rate of the SCD population was higher,but the weight gain and growth rate were not significantly different from the parent population.The histological structure of hepatopancreas became abnormal,and the number of vacuoles and lipid droplets increased when ammonia nitrogen concentration increased.Under the same ammonia nitrogen concentration exposure,there were significant differences in lipid substances,lipid metabolism-related enzyme activities,and gene expression between the two populations.In high ammonia concentration group,the number of apoptotic cells in gills increased significantly.With the increase of ammonia nitrogen exposure concentration,urea cycle metabolism-related substances,glutamine synthesis-related enzyme activities,and nitrogen metabolism-related gene expression were upregulated,while respiratory metabolism-related gene expression was inhibited.In conclusion,the degree of genetic differentiation,growth performance,and nutrient accumulation of the hybrid population,which has the advantage of heterozygous selection,were significantly better than those of the parent population.The hybrid population showed stronger stress resistance under the same ammonia nitrogen exposure.The above results provide a theoretical reference for the building of germplasm resources and subsequent crossbreeding. |
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