| Bighead carp?Hypophthamichthys nobilis?is a typical semi-migratory fish in large surface waters such as rivers,lakes and reservoirs in China.In recent years,the amount of natural resources and genetic diversity of bighead carp have declined due to overfishing and environmental damage.In addition,many breeding farms have problems such as effective small populations and inbreeding in the process of seed preservation and seed selection,which lead to degradation of growth and resistance of bighead carp in the breeding process.Therefore,the work of genetic improvement and breed selection of bighead carp can reduce the dependence on its natural germplasm resources and increase the coverage rate of improved varieties in breeding production,which is an important way to realize the effective protection of bighead carp natural resources and the sustainable development of fishery.This study collected nine populations,respectively from four national seed stock station populations?Shishou,Changsha,Ruichang and Hanjiang?,2 provincial seed multiplication farm parent populations?Caohu and Yulin?,three Reproduction and releasing populations?Zhenjiang,Zhangjiagang and Changshu?.In this study,molecular markers,population genetics,molecular genetics,quantitative genetics and transcriptome were used to carry out the research from the aspects of genetic variation analysis,pedigree construction,genetic parameter estimation and early development transcriptome analysis.The specific results are as follows:1.Analysis of genetic variation in bighead carp populationsGenetic variation of 10 bighead populations was analyzed by mt DNA d-loop region sequence.In a total of 401 d-loop sequences?939bp?,37 mutation sites?excluding insertion/deletion mutations?and 42 haplotypes were detected,and haplotype diversity?Hd?ranged from 0.665 to 0.905.The genetic distance between the two parameters of Kimura was between 0.0034-0.0068.Combined with the genetic clustering analysis,it was found that the genetic distance between the fishing population in the lower reaches of the Yangtze River and the original population in the middle and lower reaches was close,and that the genetic distance between the Shishou and American populations and other populations was far.Among them,the genetic distance between the Yulin population in the Pearl River basin and the American population and the middle reaches population in the Yangtze River basin?Changsha population?is relatively close.The analysis of molecular variance and genetic fixed index(FST)showed that genetic variation accounted for 6.58%of the total variation and the genetic difference was extremely significant?P<0.01?.The results showed that the germplasm resources of bighead carp in the Yangtze River basin showed high genetic variation,and the breeding and releasing populations in recent years maintained a high level of genetic diversity,which could provide a rich genetic material basis for genetic improvement and breed selection.15 microsatellite markers combined with fluorescence modification and capillary electrophoresis were used to detect bighead carp populations,the results showed that the number of alleles?Na?at each microsatellite locus was between 7 and 19?mean 13.53?,the number of effective alleles?Ne?was between 1.78 and 9.05?mean 4.54?,and the content of polymorphism information of each locus was between 0.40 and 0.88?mean0.70?.The comparison of genetic diversity parameters at population level was found that there was no significant difference in allele number and heterozygosity between the9 populations,the average observed heterozygosity?Ho?of the population was between0.69 and 0.75,and the expected heterozygosity?He?was between 0.70 and 0.74.According to the results of Nei's genetic distance and UPGMA dendrogram,the wild fishing population and the original population in the lower reaches of the Yangtze River were close and clustered first,and then clustered with the Pearl River population?Yulin?,the middle reaches of the Yangtze River?Changsha?and the upper reaches?Shishou?.According to the heat map analysis of genetic differentiation index and genetic distance between populations,the genetic differences between the upper and middle Yangtze River population,and the Yulin population of the Pearl River system and the downstream population were relatively greater.The analysis of individual genetic structure also found that the genetic structure of the upper Yangtze River population?Shishou?was relatively independent,which was quite different from that of the samples in other populations.The genetic structure of Yulin population was similar to that of Changsha and Ruichang.The results showed that the genetic resources of bighead carp in the Yangtze River basin presented high genetic variation,and the population of released in recent years maintained the high level of genetic diversity,which could provide abundant genetic material basis for genetic improvement and breeding.2.Analysis of genetic parameters of growth traits of bighead carp at the age of 30 days and 10 monthsThe aim of this study was to estimate the genetic improvement potential of early growth traits in bighead carp.The practices of mass spawning?group 1?and artificial insemination?group 2?were carried out using two populations of bighead carp,and 672fries at 30dph?days post-hatching?were collected from each group,that were used for the further analyses of early growth traits.Base on microsatellite markers,total of 628and 660 individuals from group 1 and group 2,respectively,were assigned with unambiguous pedigrees and reconstructed with population diallel cross combinations.Extremely significant unequal parental contributions were detected in both groups?P<0.01?.The extremely significant differences in BW?body weight?and SL?standard length?were detected among families in both groups?P<0.01?,and the significant differences in BW and SL were detected among diallel cross combinations in group 2?P<0.05?.Furthermore,the positive values in mid-parent heterosis?0.39–7.64%?and special combining ability?SCA??0.01–0.02?of two growth traits were detected in the hybridized combinations.Based on the animal model and restricted maximum likelihood?REML?algorithm,the heritability estimates of BW and SL in bighead carp?30dph?were calculated as 0.47 and 0.49,respectively?P<0.01?.The extremely significant genetic and phenotypic correlations were found between BW and SL,with0.89 and 0.83,respectively?P<0.01?.The results indicated that the early growth traits of bighead carp could be improved through family construction,cross breeding and selective breeding,but the unequal parental contribution should be taken into account in practical breeding programs.Two national populations?Shishou and Yangzhou?were selected for population breeding?group 1?and artificial insemination breeding?group 2?.When the offspring grew to the age of 10 months,1000 tails were randomly collected from group 1 and group 2,and paternity tests were carried out for paternity tests and main growth traits were measured for genetic parameters.Through the paternity test,group 1 and group 2identified 958 and 914,respectively.The body weight variation coefficients of group 1and group 2 were the largest?15.91%and 15.39%,respectively?,and the body length variation coefficients were the smallest?5.57%and 4.63%,respectively?.In group 1,different mating designs had extremely significant effects on 5 growth traits of body weight,body length,head length,body thickness and body height?P<0.01?;in group 2,different mating designs had extremely significant effects on 4 growth traits of body weight,body length,head length and body height?P<0.01?,but no significant effects on body thickness?P>0.05?.The mean weight,head length and body thickness of the children of the HJ×SS combination in group 1 and group 2 were the largest among the four combinations.All the 5 growth traits of the hybrid offspring in group 1 and group 2had a mesophile advantage,all the 5 growth traits of the HJ×SS hybrid offspring in group 2 had a hyperaffinity advantage,and the special combining ability of all the hybrid offspring was positive?0.01-0.02?.Based on the animal model and the restricted maximum likelihood method,the estimated heritability of bighead carp was 0.25,0.25,0.17,0.23 and 0.09,respectively?P<0.01?.The maximum genetic correlation between the two traits was body weight and body thickness?0.97?,and the minimum was body weight and head length?0.79?.The maximum phenotypic correlation between the two traits was body weight and body length?0.85?,and the minimum was body thickness and head length?0.42?.The results show that bighead carp seedlings with growth advantages can be obtained through pedigree construction and population hybridization.The growth characteristics of bighead carp at the age of 30 days and 10 months had high breeding potential,and the hybrid combination showed heterosis,which could be used as high quality breeding material for the genetic improvement of bighead carp.3.Comparison of growth performance between selected breeding lines and local bighead carpIn order to assess the growth performance of selected breeding lines of bighead carp,A batch of hybrid offspring of Shishou?Hubei?and Hanjiang?Jiangsu?populations were obtained by means of mass reproduction.A productive comparison experiment was carried out between selected breeding lines and local bighead carp in Guangxi aquatic breeding center experimental base.Growth traits of bighead carp were measured in September 2018,December 2018 and May 2019,respectively.The results showed that the individual growth performance of the bighead carp breeding line was better during the experiment.The average daily weight gain of the bighead breeding line and the local bighead was 0.908g/d and 0.858g/d,respectively.At the early stage of breeding,the coefficient variation of body weight of the bighead carp breeding lines was higher than that of the local.In the later two samples,the coefficient variation of body weight of the bighead carp breeding lines was significantly lower than that of the local bighead carp?P<0.01?,indicating that the individual size of the bighead carp breeding lines was more consistent than that of the local bighead carp during the breeding process.The correlation coefficient between body length,head length,body height,body thickness and body weight of bighead carp breeding line reached an extremely significant level?P<0.01?.The highest correlation coefficient between body weight and body length was 0.940,followed by 0.938 for head length and body length,and the lowest was 0.750 for body height and head length.4.Transcriptome analysis of early growth and development in bighead carpThe relationship between bighead carp transcriptome and the dynamic regulation of early development is unknown.In this study,transcriptome sequencing was performed at six early development stages of bighead carp to understand the dynamic molecular regulation of early growth and development of bighead carp.76,573reference transcripts,with an average length of 1768 bp,were received through the unattended transcription assembly.41742?54.54%?transcripts obtained valid functional annotations,and 59014 microsatellite?SSR?sites were identified.In addition,30199differentially expressed transcripts?DETs?were observed in the comparison of adjacent developmental stages,and 9 expression patterns were simulated by sequence clustering analysis of 6 growth and development stages.After DS1 stage?blastocyst stage?,the transcriptional expression level increased significantly,and the number of DETs decreased gradually in the subsequent development process.The transcriptome changes from DS1 to DS2?6 sarcomere phase?were the largest?up-regulated or down-regulated?,which enriched many biological processes and metabolic pathways related to maternal to zygotic transformation?MZT?.The protein-protein interaction?PPI?network of DETs during DS1 to DS2 is clearly mapped,and genes?or proteins?from the same pathway are integrated together and show significant co-regulatory patterns.In the sequence clustering analysis,a significant gene expression pattern?profile?48?was found,which may be related to the process of bigheys bigheys hatching out of the membrane,and from this expression pattern,the strict co-expression of an incubation enzyme gene?hce1?and 33 other annotated genes was determined. |
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