Applications Of Microsatellite Loci In Population Genetics And Parentage Identification Of Cherax Quadricarinatus

Cherax quadricarinatus is a very important freshwater aquaculture economic species.The breeding of C.quadricarinatus is of great significance for the healthy development of the red crayfish aquaculture.In this study,microsatellite markers were developed based on whole-genome sequencing technology,and the genetic diversity of different populations was analyzed by microsatellite markers.At the same time,five microsatellite fluorescence multiplex PCR system for paternity identification of C.quadricarinatus was constructed.The main results are as follows:In order to understand the distribution characteristics of microsatellites in the genome of C.quadricarinatus,MISA software was used to search and analyze the 1-6bp repeat in the genome of C.quadricarinatus.A total of 32,35682 microsatellites were screened from the genome of C.quadricarinatus,and the sequence length accounted for 0.96 % of the genome.Among the six bases,dinucleotide has the largest number of microsatellites,followed by trinucleotide,mononucleotide,tetranucleotide pentanucleotide,and hexanucleotide.The dominant microsatellite types of six bases are A,AC,ACC,AAAT,AACCT and ACAGAG.Among the whole genome microsatellites of C.quadricarinatus,the first 10 dominant repeat types were AC,A,AG,ACC,ACT,AAT,C,AGC,AT,and AACCT.This study provided data for the whole genome microsatellite screening,further research of crayfish,molecular marker-assisted breeding,and genetic information evaluation of red claw crayfish.The genetic structure of 4 crayfish populations from Guangdong,Taiwan,Anhui,and Zhejiang was analyzed using 24 microsatellite markers developed in this study.The results showed that 221 alleles were detected in 4 populations.The number of alleles at24 loci ranged from 5 to 15,and Cqaa-195 had the highest number of alleles(Na = 15).However,primer Cqaa-124 had the lowest number of alleles(Na = 5).The average number of alleles in Guangdong,Taiwan,Anhui,and Zhejiang populations were 6,6,7,and 7,respectively.The average number of effective alleles was 3.189,3.243,3.344,and 3.696,respectively.The genetic differentiation coefficient(Fst)among the four populations was-0.00234-0.05009.Shannon diversity index(H)was greater than 1,and the Guangdong population was the lowest,while that of the Zhejiang population was the highest.The Shannon diversity index of the Zhejiang,Anhui,Taiwan,and Guangdong were 1.510,1.405,1.339,and 1.330 respectively,which was consistent with the average effective number of alleles in each population.The genetic diversity of Guangdong,Taiwan,Anhui,and Zhejiang populations is different.In this study,the obtained polymorphic tetranucleotide microsatellite genetic markers were combined with capillary electrophoresis,and five groups of stable multiplex PCR systems(including 20 loci)were obtained by continuously optimizing annealing temperature,primer concentration,and cycles.CERVUS 3.0 software was used to identify the genetic relationship of 12 C.quadricarinatus families.The results indicated that 112 alleles were amplified from these loci.The average number of alleles was 5.6.In addition,the polymorphism information content(PIC)was 0.375-0.788,the observed heterozygosity(Ho)was 0.458-0.917,and the expected heterozygosity(He)was 0.435-0.832.In the 20 microsatellite markers screened in this study,the average observed heterozygosity was 0.762 and the average expected heterozygosity was 0.683.The average polymorphism information content was 0.621,showing abundant polymorphism.When any four groups of microsatellite multiplex PCR systems were selected,the cumulative actual correct identification rate was greater than97.20 %;the cumulative correct identification rate of five multiplex PCR systems reached 100%.In addition,two other paternity analysis software COLONY 2.0 and PAPA 2.0 were used to verify the accuracy of paternity identification,and the identification results were completely consistent with pedigree records.Therefore,selecting these combinations can not only obtain accurate pedigree information but also reduce workload and cost.