| Siniperca chuatsi belongs to the order perciforme,family hippidae,subfamily Siniperca and genus Siniperca.Since ancient times,S.chuatsi has been regarded as a treasure on the table because of its delicious meat.In the 1970 s,China broke through the problem of artificial propagation of S.chuatsi and soon carried out breeding in many provinces and cities.In 2010,China's annual aquaculture output exceeded 250,000 tons,and in 2018,it exceeded 310,000 tons,with an output value of more than 20 billion yuan.However,due to the neglect of parent selection in long-term artificial propagation,the degradation of germplasm,decline of growth rate,early sexual maturity and poor resistance to disease,which seriously affect the breeding efficiency and product quality and safety.So it is of great significance for the healthy and sustainable development of the culture industry of S.chuatsi to carry out the breeding of excellent varieties of S.chuatsi.For this reason,we selected the growth rate and survival rate as the target characters,adopted the techniques of family selection,population selection and fish parent-child identification,and bred a new mandarin fish strain Guangqing No.1 with excellent growth performance through five generations of breeding.In this study,we evaluated the genetic parameters of growth traits in different generations,compared their growth performance,and analyzed the genetic diversity of different generations to evaluate the impact of five generations of breeding on their genetic diversity.In order to explore the possibility of polyploid breeding,the experiment of polyploid preparation of S.chuatsi was also carried out.Finally,the first high-density genetic linkage map of S.chuatsi was constructed,and QTL mapping of its growth and gender was carried out,which laid a foundation for the next research of molecular marker assisted breeding and monoculture of S.chuatsi.The main results are as follows:1.Evaluation of genetic parameters of growth characters in different generations of S.chuatsi.Based on the analysis of SSR genetic diversity of several cultured and wild populations in the early stage,three populations of S.chuatsi from different sources,Anhui wild population,Hunan wild population and Guangdong breeding population,were selected as the breeding base population.Through complete diallel crossing and pairing combination,the families were constructed,and the offspring of each family were cultured in the same pond after marking.At the end of the breeding cycle,the growth data were collected and the breeding value was evaluated,then the breeding parents and pairing scheme of the next year were determined,and the breeding of five generations in succession was completed.In this paper,the narrow sense heritability of growth traits of S.chuatsi was evaluated by using the growth data of F1-F3 generations(body weight and daily gain)based on the growth data of five generations of lineage and population selection.At the same time,the weight data of F1-F5 generation and each generation of control group were used to evaluate the actual heritability,selection response and genetic gain of body weight.The results were as follows: the narrow sense heritability of body weight of F1-F3 generation of S.chuatsi was 0.26,0.42 and 0.23,respectively,showing medium and high heritability.The narrow sense heritability of daily gain was 0.26,0.16 and 0.11,respectively,which showed that the growth traits of S.chuatsi still had some space for genetic improvement.The actual heritability of F2-F5 generation of S.chuatsi was 0.14,0.30,0.35 and 0.24 respectively,the selection response was 0.28,0.54,0.62 and 0.42 respectively,the genetic gain was 10.02%,12.84%,13.25% and 9.38%respectively,and the average genetic gain was 11.37%.The coefficient of variation of harvest weight of F1-F5 decreased by generations,which were 18.68,15.32,13.16,10.10 and 9.16,respectively.The results showed that the degree of variation of family weight gradually decreased,the heredity tended to be stable,and the improvement effect was obvious.The above results showed that the growth characters of S.chuatsi were greatly improved.The selection effect was good,and the growth rate doubled in F4 generation.The genetic progress was great.A new strain Guangqing No.1 of S.chuatsi was developed by four generations of breeding,with growth rate and survival rate as the target characters,adopting the techniques of family selection,population selection and fish parent-child identification.According to the data statistics of small-scale test,mediumsized test and farmhouse pond head harvest,"Guangqing No.1" showed obvious growth advantage and strong resistance,and its growth speed,survival rate and evenness were significantly improved.The growth rate and survival rate of Guangqing No.1 increased by 16.3% and 12.6% respectively.The popularization and application of S.chuatsi "Guangqing No.1" will play a positive role in promoting the healthy and sustainable development of mandarin fish industry in China.2.Genetic diversity analysis of different generations of S.chuatsi.In this study,microsatellite SSR markers were used to analyze and compare the genetic diversity among generations of S.chuatsi.50 pairs of microsatellite primers were selected from the transcriptome database and the simplified genome database of S.chuatsi,which were constructed by Gen Bank,literature and the previous research institute of the team.Nine pairs of primers with good specificity and polymorphism were screened out.The fluorescent primers with Tamra,hex and fam were synthesized.The amplified products of the fluorescent primers were used for STR genotyping.In this study,32 samples from6 selected generations(F0 to F5)of S.chuatsi were STR typed to analyze their genetic diversity.The results showed that 8 of the 9 microsatellite loci were highly polymorphic and 1 was moderately polymorphic.The genetic diversity of F0 generation of Siniperca chuatsi was the highest(He = 0.7433).The genetic diversity of F4 was the lowest(he =0.6561),and the genetic diversity of F5 increased slightly.According to the analysis of Fst and AMOVA,the level of genetic differentiation between populations is low(Fst <0.15),15.98% of genetic variation comes from between populations,84.02% from within populations.The UPGMA phylogenetic tree based on genetic distance showed that six generations of S.chuatsi were divided into two groups,F0,F1 and F2 were one group,F3,F4 and F5 were the other group,which indicated that there was a process of transformation and improvement from F3.Although the results showed that the genetic diversity of six generations of S.chuatsi decreased after five generations of breeding,but they all maintained a high level of genetic diversity(He > 0.65).The genetic diversity of the selected population of S.chuatsi is maintained at a relatively high level,which suggests that it has good breeding potential and resistance to stress.3.Preliminary study on polyploid breeding of S.chuatsi.In this study,the polyploidy of S.chuatsi was studied.Heat shock and cold shock methods were used to induce tetraploid and triploid,flow cytometry was used to detect the ploidy,and primary culture growth was observed.Through the comparison and optimization of various conditions,the best induction condition of tetraploid of S.chuatsi was confirmed: when the fertilized eggs were treated with heat shock at 42? for 2 minutes at 50 minutes of fertilization,and tetraploid was obtained by inhibiting the first cleavage and doubling the number of chromosomes.More than 50% tetraploid was obtained.While the best inducement condition for triploid of S.chuatsi was that the fertilized eggs were treated with cold shock at 4? for 20 minutes at 8 minutes of fertilization,and the second polar body could be inhibited to get triploid.The triploid rate obtained by this method is more than 40%.The results showed that the body weight of triploid fish and diploid fish were not significantly different(105.1g and 102.4g,respectively),and there was no significant growth advantage.However,the standard deviation of triploid fish population is larger than that of diploid fish population,indicating that the body weight fluctuation range of triploid fish is relatively large,and the upper limit of body weight is relatively high,suggesting that the weight growth space of some triploid fish individuals is larger than that of diploid fish.In this study,only the growth performance of triploid fish prepared by cold shock method was observed.Although the triploid S.chuatsi produced by this method did not show obvious growth advantage over the diploid S.chuatsi,how the triploid fish produced by crossing the tetraploid mandarin fish with the diploid mandarin fish needs further observation.The sterility of triploid fish has a good application value for the culture of S.chuatsi.The energy saved during gonad development and reproduction can be used for individual growth.Therefore,the comparison of its preparation method and culture performance is worth further study.4.Construction of a high-density linkage map and mapping of sex-determined and growth-related loci in the mandarin fish(S.chuatsi).Here,we constructed a high-density linkage map for mandarin fish based on the dd RAD-Seq.The linkage map contained 3283 dimorphic single nucleotide polymorphism markers and 24 linkage groups(LGs).The total map length was 1972.01 c M,with an average interlocus distance of 0.61 c M.Five significant quantitative trait loci(QTLs)for sex-determined traits were detected on LG23.They were clustered together between the positions of 60.27 and 68.71 c M on LG23.The highest logarithm of odds value(17.73)was located at 60.27 c M near marker r1?73194,which accounted for 53.3% of the phenotypic variance.Genotypes of all the male fish on r1?33008 were homozygous,while those for females were heterozygous.Thus,LG23 was considered a sex-related linkage group.Nineteen significant QTLs for three growth traits at two growth stages were found to distribute on seven different LGs,and their contributions to phenotypic variation were quite low(11.1–17.2%),suggesting that multiple genes affected the growth traits.This high-resolution genetic map provides a valuable resource for fine mapping of important traits,and identification of sex-related markers will facilitate single-female fish breeding and sex determination mechanism studies.In this study,Guangqing No.1,a fine strain of S.chuatsi,was bred by means of family selection and population selection.In this study,the genetic parameters of growth characters in different generations were evaluated.It was found that the growth rate and survival rate of S.chuatsi were significantly improved after generations of breeding.The coefficient of variation of body weight decreased year by year,which showed that Guangqing No.1 tended to be stable genetically.The growth rate and genetic gain of S.chuatsi were also significantly increased,which indicated that the selection effect of S.chuatsi was better.Further genetic diversity analysis showed that the genetic diversity of each selected generation of S.chuatsi decreased slightly,but maintained at a high level.At the same time,the prospects of triploid and tetraploid species of S.chuatsi were discussed.Finally,the first high-density genetic linkage map of Siniperca chuatsi was constructed,and QTLs related to sex and growth were obtained.This study enriches the methods and strategies of mandarin fish breeding in China,provides good materials and new strains for S.chuatsi breeding.It provides useful gene resources for the analysis of complex characters and genetic breeding of S.chuatsi.It will greatly promote the healthy and sustainable development of S.chuatsi industry in China. |
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