Effects Of Broodstock Enhancement On Fish Larval Resources And Genetic Diversity Of The Four Major Chinese Carps In The Middle Reaches Of The Yangtze River

The four major Chinese carps（black carp,grass carp,silver carp and bighead carp）are widely distributed in the major rivers and adjacent waters of China.The Yangtze River is an important habitat of their natural stock resources.In recent years,the monitoring results showed that the resources of the four major Chinese carps were in declining.In order to recruit the fish resources and restore the aquatic ecological system,many provinces and cities along the Yangtze River carried out the stock enhancement activities of four major Chinese carps near ten years.What is the effect of large-scale stock enhancement activities on the improvement of fish stock resources?Whether artificial releasing fish population has adverse effects on genetic diversity of natural population?In order to answer these questions scientifically,and investigate the influences of the artificial stock enhancement to restore resources on the natural fish stock resources of the Yangtze River,the present study used molecular markers of mitochondrial D-loop sequence and microsatellites of nuclear genomic DNA to analyze the impact on fish larval resources of the broodstock populations of the four major Chinese carps released in Hubei Province Jianli and Shishou sections of the middle Yangtze River from 2015 to 2017,the genetic differences between the progenies of stocking broodstocks and non-stocking parents,and the present genetic diversity of the catches of the four major Chinese carps in the middle Yangtze River.The above results could provide basic data for the protection of their stock resources,prevention and control the ecological risks,provide an important basis for draw a scientific and rational stock enhancement plan and make fish resources restoration strategy.The main research contents and results were as follows:1.Establishment and application in population genetics of microsatellite multiplex PCR reaction system for black carp and bighead carp.This study reported for the first time the application of multiplex fluorescent PCR system consisting of tetranucleotide repetitive microsatellite in the populations of black carp and bighead carp.Eleven tetranucleotide repetitive microsatellite markers were used to optimize the multiplex PCR system of black carp,and the 11 loci were divided into two quadruple systems and one triple system.Sixteen tetranucleotide repetitive microsatellite markers were used to optimize the multiplex PCR system of black carp,and the 16 loci were divided into four quadruple systems.The multiple systems of the two fish species all had high allele variability and high polymorphic information content（PIC）.In order to test the validity of bighead carp multiple systems for parentage assignment,39 offspring from two full-sibs families and their parents were genotyped.The combined exclusion probability（CEP）was as high as 99.99%,and 100%of offspring（39/39）were clearly assigned to their real parents.In addition,this study demonstrated that the multiple systems of two species of fish were successfully applied to the study of genetic characteristics of wild populations.Since no parent-offspring samples were collected,the multiplex PCR system of black carp was only applied in population genetics.2.Replenishment and construction of microsatellite gene bank of the four major Chinese carps broodstock populations released in the middle reaches of Yangtze River in the year of 2015-2017（bighead carp:2012-2017）.In this study,microsatellite markers were used to replenishment and construction of gene bank of the broodstock populations.The genetic diversity was further analyzed.The results showed that the effective allele number（Ne）was 2.93-9.95,the gene abundance（Ar）was 11.62-16.92,the expected heterozygosity（He）was 0.838-0.903,and the observed heterozygosity（Ho）was 0.765-0.900 in black carp broodstock populations.The Ne,Ar,He and Ho were9.56-10.57,18.27-19.45,0.872-0.885 and 0.953-0.999,respectively,in grass carp broodstock populations.The Ne,Ar,He and Ho were 7.48⁸.97,15.92¹7.62,0.824⁰.866 and 0.794⁰.901,respectively,in silver carp broodstock populations.The Ne,Ar,He and Ho were 2.88⁵.58,3.44⁴.03,0.725⁰.816 and 0.667⁰.760,respectively,in bighead carp broodstock populations.The results of molecular variance analysis（AMOVA）showed that the variation among individuals was the main source of the total variation,and the composition of variation showed that the genetic variation among populations was lower than that within populations.The F_ST of black carp broodstock populations,grass carp broodstock populations,silver carp broodstock populations and bighead carp broodstock populations was 0.03138,0.05675,0.01605and 0.0152 respectively.The genetic diversity of the broodstock populations of four species of fish was at a high level.There was no genetic differentiation in all the other three species except grass carp broodstock populations.The results showed that the broodstock populations of four major Chinese carps were suitable for release.3.Effects of the four major Chinese carps broodstock enhancement activity in the middle reaches of Yangtze River on fish larval resources.In this study,microsatellite multiplex fluorescent PCR systems of the four major Chinese carps established independent by our laboratory were used to analyze the parent-child relationship of broodstocks and fries collected during the fish larval resources survey in 2015-2017（Grass carp:2016-2017）.The results showed that 9 black carp fries were collected,but no fries had parent-offspring relationship with broodstocks were identified.A total of1122 grass carp fries were collected.Among them,61 fries were found to have parent-offspring relationship with broodstocks,including 7 fries whose parents were both broodstocks,and 54 fries produced by crossing broodstocks with un-broodstocks.A total of 1297 silver carp fries were collected.Among them,144 fries were found to have parent-offspring relationship with broodstocks,including 27 fries whose parents were both broodstocks,and 117 fries produced by crossing broodstocks with un-broodstocks.8 bighead carp fries were collected,but no fries had parent-offspring relationship with broodstocks were identified.In addition,the results showed that some of the broodstocks could also produce offspring in the years after release.In 2016 and2017,the annual contribution rates of grass carp broodstocks to the fries of fish larval resources in Jianli reach of the Yangtze River were 4.84%and 4.4%,respectively,and the total contribution rates were 4.84%and 7.46%,respectively.In 2015²017,the annual contribution rates of silver carp broodstocks to the fries of fish larval resources in Jianli reach of the Yangtze River were 3.74%,10.37%and 7.51%,respectively,and the total contribution rates were 3.74%,18.88%and 14%,respectively.4.Effects of grass carp and silver carp broodstock enhancement in the middle reaches of Yangtze River on genetic diversity of fish larval resources.Analysis of the genetic differences between the progenies with different parents.In this study,microsatellite DNA markers were used to analyze the genetic differences among the broodstock progenies,hybrid progenies and un-broodstock progenies of the four major Chinese carps in the middle reaches of the Yangtze River.Allele number（Na）,effective allele number（Ne）,gene abundance（Ar）,observed heterozygosity（Ho）and expected heterozygosity（He）were selected to reflect the level of genetic diversity.Among the progenies of silver carp,there were no significant differences in Ar,Ho and He,the Na and Ne of the broodstock progenies and hybrid progenies were lower than those of the un-broodstock progenies.Among the progenies of grass carp,there were no significant differences in Ho and He,the Na and Ne of the broodstock progenies and hybrid progenies were lower than those of the un-broodstock progenies,the Ar value of the broodstock progenies was significantly lower than that of the un-broodstock progenies,but there was no significant difference between the broodstock progenies and the hybrid progenies.Private alleles were all found in un-broodstock progenies only in two fish species.The frequencies of private alleles were very low.The highest frequencies were0.036 in grass carp progenies and 0.083 in silver carp progenies,which were far less than 0.5.Private alleles alone could not reflect differences in genetic relationships.Molecular variance analysis（AMOVA）,genetic differentiation index(F_ST)and discriminant analysis（DAPC）of the first and the second principal components of the population showed that no genetic differentiation occurred in progenies populations of grass carp and silver carp.Therefore,there was no significant difference in genetic diversity and structure among these progenies.5.The genetic diversity of the catches populations of four major Chinese carps in the middle reaches of the Yangtze River.Samples of four species of fish in this study were collected from fisherman’s catches at different locations in the middle reaches of the Yangtze River from June 2016 to September 2017.A total of 368 samples were collected,including 81 black carps,95 grass carps,108 silver carps and 84 bighead carps.Mitochondrial DNA control region（mtDNA D-loop）sequences were used to analyze the genetic diversity of the samples.The results showed that the sequence length of black carp was 927 bp and 30 haplotypes were defined.The haplotype diversity index（Hd）of each black carp population was 0.931-0.978,and the nucleotide diversity index（Pi）was 0.01323-0.01459;the sequence length of grass carp was 934 bp and 12 haplotypes were defined.The Hd of each grass carp population was 0.308⁰.569,and the Pi was 0.00090⁰.00240;the sequence length of silver carp was 890 bp and 35haplotypes were defined.The Hd of each silver carp population was 0.874⁰.906,and the Pi was 0.000717⁰.01081;the sequence length of bighead carp was 884 bp and 22haplotypes were defined.The Hd of each bighead carp population was 0.889⁰.942,and the Pi was 0.00448⁰.00479.The results of molecular variance analysis（AMOVA）showed that the variation among individuals was the main source of the total variation,and the composition of variation showed that the genetic variation among populations was lower than that within populations.The results of genetic differentiation index(F_ST)analysis and gene flow（Nm）analysis showed that the genetic differentiation degree of all the four species was lower(F_ST<0.05).Neutral test,mismatch distribution analysis and Bayesian skyline plot analysis（BSP）results showed that grass carp populations had experienced population expansion events in history,while the other three species have not.The results showed that the genetic diversity status of the four major Chinese carps populations in the middle Yangtze River was not different from the before fish artificial stock enhancement..In conclusion,the microsatellite multiplex PCR reaction systems of black carp and bighead carp could be effectively applied in these fish population genetics research.The2015-2017 microsatellite DNA data base of the four major Chinese carp broodstock populations（bighead carp:2012-2017）in the middle Yangtze River has been replenished and constructed.The population genetics analysis indicated that all broodstock populations were suitable for stock enhancement,but the management of grass carp broodstock populations should be strengthened.It has been confirmed that the broodstocks in the stock enhancement activity can produce offspring normally in the Yangtze River,which contributes to the larvae quantity of the four major Chinese carps in early resource.There was no significant difference in genetic diversity and structure between these offspring populations with broodstock parents and with un-broodstock parents.Analysis of mitoDNA D-loop sequence showed that the populations genetic diversity and genetic structure of the four major Chinese carps in the middle Yangtze River were not affected significantly by artificial stock enhancement population for the time being comparing with the before fish artificial stock enhancement.