Study On The Parentage Identification Of Rhinogobio Ventralis Using Microsatellites

Rhinogobio ventralis(Sauvage et Dabry)belongs to Rhinogobio,Gobioninae,Cyprinidae,Cypriniformes,Osteichthyes,which is mainly distributed in Jinsha,Minjiang and Tuojiang River,and the lower reaches of Wujiang River,the area of middle and lower reach of Jialing River,the stem stream of the upper Yangtze River.It is a demersal and small fish,belonging to a typical species of rapids,which is habituated to live in the dark water environment of the bottom river of rapids and staggered rocks.Rhinogobio ventralis commonly known as rat fish which is unique in the upper Yangtze River and belongs to one of the 66 distinctive fishes in the protection list of International Protection Areas of Rare and Endemic Fish In the Upper of Yangtze River.In recent years,its natural resource show a sharp decline because of construction of large hydropower station and human overfishing and exploitation.From the species vale and threatened degreed evaluation,Rhinogobio ventralis has reached three-level desperate protection state and classified as low risk fish.In this regard,a number of domestic station to reproduce and put them into rivers has included Rhinogobio ventralis as a object.Studies on domestication and artificial reproduction technology of Rhinogobio ventralis have been paid great attention increasingly.In this study,we established microsatellite multiplex fluorescent PCR system by using polymorphic microsatellite loci which had been screened by us,assessed the genetic character of wild population of Rhinogobio ventralis,and established parentage identification technology based on microsatellite multiplex fluorescent PCR system.The research will provide theoretical basis and technical support for Rhinogobio ventralis artificial reproduction,family management and releasing effect evaluation.The main results of this papers as follows:(1)Establishment and optimization of microsatellite multiplex fluorescent PCR system in Rhinogobio ventralisWe obtained 70 microsatellite sequences of Rhinogobio ventralis from the NCBI library and selected 33 microsatellite loci with high polymorphism according to the reported papers.The 33 synthetic primers were amplified by PCR in 12 wild individuals of Rhinogobio ventralis,and the PCR products separated on 12% non-denaturing polyacrylamide gel and visualized by silver staining.The 15 microsatellite loci with high polymorphism and best amplification effects were selected for the development of multiplex PCR technique.We established five microsatellite multiplex fluorescent PCR panels(A?B?C?D and E groups),each group containing 3 microsatellite loci by optimizing the annealing temperatures,the primer ratio and the concentrations of reaction system.(2)Genetic diversity assessment of wild population of Rhinogobio ventralis from Luzhou using microsatellite markersAssess the genetic diversity and structure of 40 wild individuals collected from Luzhou in the upper Yangtze River using 15 polymorphic loci and microsatellite multiplex fluorescent PCR system.We found a total of 234 alleles in the Rhinogobio ventrali population,ranging from 5(REN02)to 23(REN17?RVE-13)per locus and the average number of alleles was 15.600.The polymorphism information content(PIC)varied from 0.729 to 0.940,with a mean of 0.872.The calculated expected heterozygosity(He)varied from 0.77825 to 0.95506,with a mean of 0.89600,whereas observed heterozygosity(Ho)varied from 0.55263 to 0.97500,with a mean of 0.82691.This results showed that the genetic diversity of this wild population of Rhinogobio ventrali from Luzhou was at a very high level.The inbreeding coefficient(FIS)ranged from-0.06283 to 0.29267,with a mean of 0.07961(P=0.00000±0.00000).There was no significant(P>0.05)linkage disequilibrium between any microsatellite loci.Except for REN16?RV-8?REN34?RVE-13 and REN02,the other microsatellite loci deviated significantly from Hardy-Weinberg equilibrium(P<0.05).The cluster analysis by the software of Structure showed that no obvious genetic differentiation occurred in this population.And the bottleneck test results indicated that the number of individuals of Rhinogobio ventrali declined and suffered severe bottleneck effect recently.(3)Establishment of parentage identification technique of Rhinogobio ventrali and genetic characteristics evaluation of first filial generation of breeding groupWe used 15 polymorphic loci and microsatellite multiplex fluorescent PCR system to construct the paternity test technology.Results showed that whether the parental genotypes were known the combined exclusion probability was more than 99.99%.When using multiplex PCR group A?B and C,the combined exclusion probability of the first parent was 99.83% and the combined exclusion probability of the second parent was 99.99%.All of offspring can correctly find their real parents.This results indicated that 15 polymorphic loci and microsatellite multiplex fluorescent PCR system were suitable for paternity test of Rhinogobio ventrali.The genetic diversity evaluation of artificial breeding F1 population showed that the number of microsatellite loci alleles was 98,ranging from 4 to 10 and the average number of alleles was 6.533.The PIC varied from 0.579 to 0.879,with a mean of 0.730.The He ranged from 0.606 to 0.894,average value was 0.766.The Ho ranged from 0.458 to 1.000,average value was 0.808.This results showed that the genetic diversity of offspring group was high.The total-population inbreeding coefficients of offspring F1 population(FIT)was 0.01469;the single population inbreeding coefficients(FIS)was-0.31731;the genetic differences between two families(FST)was 0.25203.This results indicated that there is a certain inbreeding phenomenon in the whole offspring F1 population,but no obvious in the single population.And there was different degrees of genetic differentiation occurred among three offspring F1 populations.Compared with the wild population,the genetic diversity of first filial generation of breeding group is relatively low.