Isolation Of Microsatellite Markers For Urechis Unicinctus Based On High-Throughput Sequencing And Genetic Structure Analysis Of Five Populations Using Microsatellite Markers

Urechis unicinctus is a new type of mariculture species in northern China,it is delicious taste,rich nutrition,and has great economic value.But with overfishing and environmental damage,the resources of U.unicinctus have declined,while market prices are rising.So it is imperative to implement artificial enhancement and releasing.Although in the Shandong area of artificial breeding has been a breakthrough,the study on geographic population genetic analysis and genetic structure difference has not been reported,then the rapid development of microsatellite marker assisted breeding system provides a feasible and effective tool for the artificial breeding and rapid propagation.We got a lot of microsatellite sequences from U.unicinctus by RAD — seq high throughput sequencing.After screening,22 pairs of highly polymorphic microsatellite markers were developed.Furthermore we used these SSRs to analysis and evaluation of population genetic structure of 5 wild geographic populations in the Bohai Sea(Shandong,Yantai,YT;Shandong,Weifang,WF;Shandong,Qingdao,QD;Hebei,Qinhuangdao,QHD,Liaoning,Dalian,DL).1.The isolation and characterization of 22 microsatellite lociFirst of all,we used the modified phenol chloroform isoamyl alcohol extraction method to extract high quality DNA from U.unicinctus.Moreover we obtained the library of RAD—seq by Illumina HiSeq2500,then we got the clean data and analyzed.The sequence produced a total of Raw data 2.113 G,filtered Clean data 2.109 G,sequencing quality at Q20 > = 96.76%,at Q30 > = 92.22%,GC content is 40.06%,and the assembled GC content is 39.98%,the difference is small.A large number of heterozygous single nucleotide polymorphisms(SNPs)and a small number of homozygous SNPs were detected in the sequence fragments,and the heterozygosity wasabout 96.5%.Based on the above results,we can conclude that the results of RAD sequencing are accurate and reliable.The sequencing of a total of 1287 SSRs were detected,of which the number of the three base repeat microsatellite sequence is 818,the largest number of all,accounting for 63.56%.of the total number.The primers were designed for the sequence of microsatellite length above 18 bp,and 50 sequences of microsatellite length above 24 bp were selected.And through a Qinhuangdao from U.unicinctus wild populations the 50 pairs of primers were screened.Among them,38 pairs of clear target bands could be amplified stably,22 of which were polymorphic.All of the 22 microsatellite sequences were Perfect,of which 5 were identified to be Dinucleotide microsatellite,Trinucleotide microsatellite were 14,2 Tetranucleotide microsatellite and 1 Five nucleotide microsatellite,the percentage is 22.73%,63.64%,9.09% and 4.55%,respectively.In the QHD population of 30 individuals DNA as template were detected up to 234 alleles per microsatellite locus,allele number(Na)between 7 to 16,the average number of alleles(Na)was 10.6364;the effective number of alleles(Ne)between 4.369 to 13.534,the average effective allele was 8.096;the average observed heterozygosity(Ho)and the average expected heterozygosity(He)values were 0.790 and 0.883.The average polymorphism information content(PIC)was 0.854,and the polymorphism information content(PIC)of the UuS34 locus was the lowest,which was the highest in UuS9,and all of the 22 microsatellite markers were highly polymorphic markers.2.Analysis of genetic structure of 5 wild U.unicinctus populationsThe genetic structure of 5 wild U.unicinctus populations in Bohai Sea was analyzed by using the high polymorphic microsatellite markers developed in this paper.The results showed that 22 microsatellite loci in 5 populations showed high polymorphism,each marker to detect the number of alleles(Na)ranged from 24 to 44.The polymorphism information content(PIC)ranged from 0.921 to 0.967.The total average effective number of alleles(Ne)in 5 groups ranged from 6.629 to 8.850,the average heterozygosity(Ho)and the average expected heterozygosity(He)range was0.790 to 0.912,0.851 to 0.896,DL population had maximum heterozygosity(He=0.8962),WF population(He =0.8510)was minimum.The average FST showed that the population differentiation level was in the moderate differentiation level,the FST value was 0.0880 to 0.1136,the WF group had the smallest differentiation,but the DL group had the most obvious differentiation.This shows that the 5 geographic groups of U.unicinctus had abundant diversity,the genetic diversity of DL population in the 5 groups was the highest,and WF group was the lowest;9 of 22 microsatellite markers in different populations showed significant deviation from Hardy—Weinberg equilibrium;From the Nei's standard genetic distance,the distance between YT and DL group was the furthest,as the genetic relationship of these two groups was the farthest.However,YT group was closest to QD group.DL group had a far genetic distance with other groups.The UPGMA phylogenetic tree showed that YT population and QD population were the closest,and they gathered together to a genetic branch at the beginning,then QHD population gathered together,while WF population and DL population were opposite.IBD analysis indicated there is no significant linear relationship between geographical and genetic distance in U.unicinctus populations.The above results suggest that U.unicinctus from the Yellow Sea and Bohai Sea had rich genetic diversity,while no significant difference observed between the populations.The largest genetic distance was YT with DL group,this phenomenon can be explained by ocean current blocking gene exchange.Microsatellite markers developed in this study not only can be used to study microsatellite polymorphism,genetic diversity,genetic analysis and population evolution,but also to strengthen germplasm resources protection and management of U.unicinctus,and then provide guidance for artificial breeding in the future.