Analysis On Half-sib Families Of Procambrus Clarkii And The Parentage Assignment Using Microsatellite Markers

Ten microsatellite loci of procambrus clarkii were selected to analyze the half-sib families and the parentage assignment of procambrus clarkii.The polymorphism information of the 10 microsatellite loci for a procambrus clarkii group,containing 48 randomly selected individuals,was assessed firstly,the average observed heterozygosity of the group is 0.3455,the mean expected heterozygosity was 0.6224,the mean Shannon index was 1.1335,the mean polymorphism information content was 0.5656.According to the genetic information of ten microsatellite loci,the genetic distance among the 48 individuals was calculated and cluster analysis using the UPGMA method was performed.Results showed that the 10 microsatellite loci could apply for genetic diversity analysis of procambrus clarkii.Then,three microsatellite loci were selected to analyze half-sib families of procambrus clarkii.To obtain half-sibling family of procambrus clarkii,three randomly selected adult female and two male procambrus clarkii were put in the same tank to let them free mate.After three female shrimps brood,genetype information of three microsatellite loci for 14,14,15 children from the three half-sib families,respectively,and their parents were detected,genetic relationship for individuals was analyzed using GERUD2.0 software,Results showed that the offspring of the first half-sib family at least have two male parents,the offspring of the second half-sib family at least have three male parents,the offspring of the third half-sib family at least have four male parents,indicating before the trial,the female had mated with other males.This result suggests that the mating pattern of the procambrus clarkii is one female to many males.In the paternity test experiment,the same experiment materials as in half-sib families of procambrus clarkii analysis were selected.As one female procambrus clarkii can mate with several male procambrus clarkii,half-sib family can be made based on female parent.No.1 and No.2 individuals are suspicious male parents,No.3 individual is the first female parent,From No.4 to No.17 individuals are No.3’s offspring,No.18 is the second female parent,from No.19 to No.32 are No.18’s offspring,No.33 is the third female parent,from No.34 to No.48 are No.33’s offspring.The observed heterozygosity is in the range of 0.354 to 0.896 based on genetic information of the 10 microsatellite loci using CERVUS3.0 software,the expected heterozygosity is in the range of 0.588 to 0.807,the mean expected heterozygosity is 0.7193,the polymorphism information content is in the range of 0.521 to 0.767,the average polymorphism information content is 0.6662,and each gene locus has an average of 5.6 alleles.The parent-child ruled out rates based on the 10 microsatellite loci genetic informations were obtained:in the case of both two parental genotypes were unknown,the range of parent rule out rate of each site(E-1P)was measured from 0.177 to 0.417;in the case of one parent genotype known,the range of rule out rate of another parent(E-2P)range was measured from 0.325 to 0.596;the range of rule out rate of both parents for one parental pair rate(E-PP)was measured from 0.487 to 0.794,so the combined exclusion probability(first parent)was 0.97616685,the combined exclusion probability(second parent)was 0.99869019,the combined exclusion probability(parent pair)was 0.99998479.For the simulation analysis,in the case of the gender of both parents known,simulate children were chose 10000,candidate female parents were 3,candidate male parents were 2,the success rate for simulation pairing between parent and offspring reached 100%.in the case of the gender of both parents unknown,with the 100%identification rate and under the 95%confidence level,based on the genetic data of 10 microsatellite loci,paternity test can be fulfilled at most 45 candidate parents,which has met test requirements for the most of pond breeding.In parental authority appraisal,the results show that two suspicious male parents were not real male parents of these 43 children,while their definite female parents of 43 children were indentified successfully:the female parent of the 14 children which from No.4 to No.17 was identified as No.3,the female parent of the 14 children which from No.19 to No.32 was identified as No.18,and the female parent of the 14 children which from No.34 to No.48 was identified as No.33.The result of UPGMA clustering analysis according to the genetic distance of these 48 procambrus clarkii individuals also proved that the two suspicious male parents were not their real father,which was consistent with the conclusion of parental authority appraisal,show that the 10 micro satellite loci can be used for crayfish paternity tests.Finally,a method to effective identify different family of procambrus clarkii was established based on the genetic information of these three half-sib families.In the previous experiment,it had been proved that they had no relationship among the three half-sib families,the genetic distances among the individuals from three family were obtained using Population software and evolutionary tree was built using UPGMA method,the genetic distance among individuals in the half-sib family or the full-sib family was less than 0.579289.If the genetic distance between the two individuals is less than 0.579289,it can be considered the two individuals are full sibling relationship or half sibling relationship,which can be used to avoid inbreeding among individuals.From the UPGMA evolutionary tree,individuals from different family were clustered in different clades.we believe that this effective method for distinguishing different family of individual will be good for breeding and prevent inbreeding of procambrus clarkii.