Microsatellite Method Assessment Of The Effects Of Stock Enhancement Of Bighead Carp(Aristichthys Nobilis) In Jiangsu Reaches Of The Changjiang River

Bighead carp（Aristichthys nobilis）,an important cultured freshwater fish in China,is one of the four major Chinese carps.The natural habitation of bighead carp in the Changjiang River has been destroyed because of environmental water pollution and overfishing,among other factors.Stock enhancement of bighead carp has been conducted for years,being regarded as a positive fisheries management tool.Hatchery-reared bighead carp juveniles were released into the Changjiang River to supplement the wild population in 2016-2018.Tracing back to the source of stock enhancement,this research focuses on analyzing the status of the brood stocks germplasm resources,and evaluating the resources contribution rate of stock enhancement of bighead carp from main hatchery in Jiangsu province.Based on microsatellite technology,10 pairs of microsatellite fluorescently labeled primers were selected and combined with capillary electrophoresis detection methods to get genotyping data of brood strocks and recaptured samples of Changjiang River,in order to accurately evaluate the effects of storck enhancement,and standardize the production technology of released offspring.The main results are as follows:1. Current germplasm situation of bighead carp candidate parent and parent from hatchery in Jiangsu（1）A total of 638 individuals from 7 parent populations and 1 candidate parent population were assayed.The results showed the number of alleles of microsatellite markers was 368.The microsatellite loci genetic diversity parameters showed that the frequencies of invalid alleles were all less than 0.2 at 10 locus,the average of polymorphic information content（PIC）was 0.880,and the range of Shannon’s information index（I）was 1.860-2.355.F statistics showed that the average of inbreeding coefficient(F_IS)and genetic differentiation coefficient(F_ST)were 0.078 and0.050.The entire sample was in moderate genetic differentiation.Population genetic diversity results showed that the genetic diversity of eight populations was superior.The average number of alleles（N_a）was 14.83±1.45,the average values of observed heterozygosity（H_o）and expected heterozygosity（H_e）were 0.76±0.05 and 0.82±0.02,and the average Shannon’s information index（I）was 2.08±0.09.The level of genetic diversity was different among populations,but lack of heterozygotes.And the genetic diversity of the candidate parent population was the highest,nevertheless inbreeding risk existed within it.（2）The 638 samples belonged to two different clusters.The first Cluster was mainly composed of parental individuals,and the candidate parents were divided into the second cluster.The structure of populations were simple.The PCo A result was consistent with the Bayesian population genetic structure analysis map,and showed that the Nei’s genetic distance of the parent population was closer to the candidate parent after renewing.The range of genetic differentiation coefficient(F_ST)of each population was 0.009-0.18,and the genetic differentiation among parent populations had been no significant practically,in addition the genetic differentiation between parent population and candidate parent populations had been significant.AMOVA showed that the genetic variation in the samples mainly originated between individuals within the population（89%）,and a small part occurred between different populations（11%）,which was the same as the genetic variation results of wild populations.This study provided suggestions for the introduction and protection of wild stocks by revealing the genetic differences between their parents and candidate parents.It also provided theoretical support and data reference for building a healthy and responsible breeding system for stock enhancement.2. Contribution rate evaluation of bighead carp stock enhancement of main hatchery in Jiangsu section of the Changjiang River（1）System of contribution rate evaluationIn order to analyze the effect of stocking enhancement of bighead carp,a parentage test based on SSR markers were applied in this study.This study had established an evaluation system of“marking brood stocks-releasing offspring-recapturing-parentage assignment”to assess contribution rate for the large-scale stock enhancement.1250 fins of bighead carp with unknown sexes were sampled.10 SSR markers were modified using fluorescent dyes（FAM/HEX）.Genotype data of all samples were screened by using ABI Prism3 730 sequencer.The results showed the number of alleles of microsatellite markers ranged from 19 to 55,and the mean observed and expected heterozygosity was 0.748 and 0.882.The range of the polymorphism information content（PIC）was 0.797～0.924.The simulation of parentage assignment for the situation of unknown sexes showed the combined non-exclusion probability of 10 loci was 99.996%by using Cervus3.0.（2）Contribution rate evaluationGenetic information was obtained from 563 brood stocks of unknown sexes and687 recaptured individuals in Jiangsu province.By using pedigree identification software,it was efficient that the recapturing samples were assigned to the candidate parents as progeny.Through the comparison of genetic information between them,microsatellite markers can identify released individuals in recaptured individuals.The results showed that the 42 recaptured individuals were offspring of brood stocks.The study suggested that the total contribution rate of the brood stocks to the wild population was 6.11%in the Jiangsu reaches of the Changjiang River.The results indicate that stock enhancement has a good supplementary effect for the wild population,and provide reliable data support for the management of Changjiang River fishery resources.