| Turbot(scophthalmus maximus,Linnaeus,1758),widely distributed along the Atlantic coast of Europe,is the most widely farmed commercial flatfish in the world.Since its introduction to China in 1992 by academician lei of the yellow sea fisheries research institute of the Chinese academy of fishery sciences,it has developed rapidly in China's bohai rim region through the cultivation of improved varieties and the promotion of varieties.Now it has become an important aquaculture species in China,and the scale of factory aquaculture is in a leading position in the world.However,the lack of reliable genealogical data and the risk of inbreeding decline limited the breeding of turbot.In order to distinguish the family lines,individual aquatic animals need to be marked.However,the traditional marking resource is expensive.Therefore,pedigree tracing based on microsatellite is of great significance in breeding.When using microsatellite molecular markers for genetic evaluation and pedigree tracing,people often pay more attention to data acquisition and processing analysis than to the accuracy and reliability of data results,and the final analysis results are often inconsistent with the expected and actual situation.Genetic evaluation using Simple Sequence Repeat(SSR)molecular markers can effectively improve the efficiency of breeding.Based on the selection and optimization of microsatellite marker,reconstruct genealogical using microsatellite markers and two methods based on spectrum reconstruction and molecular genetic correlation genetic parameter estimates early mixed genealogy turbot directly using microsatellite molecular marker genetic evaluation of turbot provide a basic reference standard varieties breeding work,on this basis,the comparison of the two methods of evaluation of accuracy.1.Screening and optimization of SSR molecular markersIn this paper,the key steps include selection of marker types,screening of microsatellite loci,template DNA quality control,PCR design and genotypic of the final product.Although these are routine operations to improve the accuracy of PCR,many relevant studies focus more on data collection and analysis at the later stage.The whole process of individual identification and pedigree tracing using molecular markers was analyzed,and the possible causes and solutions for various problems in PCR product genotypic were given,which can effectively reduce typing errors and provide more accurate reference for breeding.In this paper,the possible causes and solutions for the shadow peaks,plus "A" peaks,invalid alleles,and micro-mutations in genotypic process are given,and A set of strategies suitable for the processing of wrong results in our laboratory is established.2.Reconstruction of mixed pedigree of turbot by SSR markersIn this study,16 microsatellite loci with 3-4 bases of core repeat sequences were selected for turbot lineage traceability analysis.These microsatellite loci were optimized by experimental conditions and detected by experimental population,showing abundant polymorphism and high accuracy of genotypic(allele interpretation).The allele frequency,heterozygote,polymorphism information content and exclusion rate of microsatellite loci were calculated by Cervus 3.0,and the relationship between the number of microsatellite loci and the exclusion probability was simulated.According to the PIC value of each site,the cumulative exclusion rate of the site was calculated by increasing the number of bit points from high to low.It was found that when the number of microsatellite loci reached 6,the cumulative exclusion rate was above 0.999.In this paper,the family of turbot flounder was reconstructed by Colony1.0 and its accuracy was evaluated.The experiment analyzed 1110 individuals and 31 parents(some parents were lost),which were traced to 20 families,including 880 children and 27 parents(14 mother parents and 13 father parents).Four parents were not traced to their children,and 230 children were not traced to their parents.In this paper,a set of complete individual tracing standard procedure of SSR for turbot was established,which presents data support for later genetic evaluation.3.Estimation of genetic parameters of turbot in early mixed pedigrees based on two methods: pedigrees reconstruction and molecular genetic correlationIn this study,the genetic parameters of growth traits in turbot were assessed using pedigree reconstruction as the one generation pedigree data(PR)and genetic relationships between individuals calculated directly using microsatellite data(molecular correlation MR),respectively.The additive genetic matrix was constructed by molecular relationship and reconstructed pedigree,respectively,and applied to the same animal model to estimate genetic parameters and breeding values,and the results showed that the fixed effects of inbreeding coefficient and pool were not significant(p > 0.05).The additive genetic variances of body weight and body length obtained based on lineage reconstruction were 0.326 ± 0.172(BW)and 0.038 ± 0.046(BL),respectively,while the additive genetic variances estimated based on molecular genetic relationship were 0.139 ± 0.036(BW)and 0.031 ± 0.001(BL),respectively,and the maternal effects of body weight were 0.705 ± 0.303(PRBW),0.631 ± 0.251(MRBW),and maternal effects of body length were 0.106 ± 0.045(PRBL)and 0.126 ± 0.052(PRBL),respectively,and the phenotypic variances of body weight obtained by the two methods were also different,1.519 ± 0.298(PRBW)and 1.379 ± 0.253(MRBW),respectively.The breeding estimates obtained using pedigree information based on body mass length obtained by MR were positively correlated with the breeding values obtained by molecular kinship(P = 0.856).The mean Pearson correlation coefficients of observed and predicted phenotypic data obtained by the two methods after 500 10-fold crossvalidations were 0.692 ± 0.052(PRBW),0.615 ± 0.060(PRBL)and 0.717 ± 0.045(MRBW),0.629 ± 0.058(MRBL),respectively,and the results of cross-validation were used as genetic assessment accuracy,and the data showed that the accuracy of breeding value assessment based on MR(body length and body weight)was higher than that obtained using PR,and the cross-validation of breeding value data results of body weight showed its predictive ability relative to body length data,which was important for genetic assessment of future turbot larvae with unknown lineages. |
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