Development And Application Of Microsatellite Markers Based On Next-generation Sequencing For Rana Dybowskii

Illumina Next Seq500 and Miseq sequenced and assembled were carried out on the transcriptome and genome of Rana dybowskii by using high-throughput sequencing technology,respectively.A large number of sequences containing microsatellite fragments were obtained and characterized.The transcriptome and genome sequencing results obtained 56668566 and 6108401 Raw Reads for subsequent bioinformatics analysis.After a series of treatments(including removal of low-quality fragments and de-redundancy filtering,etc.),56391524 and 2524353 Clean Reads were obtained.The transcriptome Clean Reads data de novo was assembled and de-redundant to obtain 116655 Unigenes.Microsatellite search and feature analysis were performed on Unigenes and Clean Reads of the two data sources,and two different strategies were used to develop microsatellite primers for the two data sources.Comparing the microsatellite abundance and repetition types of the two data sources,the frequency of microsatellites is similar(26.43%,24.38%),the transcriptome data shows that the proportions of 2mer,3mer,and 4mer are relatively average,32.25%,29.12%,and 27.91%,The genome has less 3mer and more 4mer(28.24%,22.07%,39.00%).The two sets of data show that the types of dinucleotide dominant repeat units are slightly different.AT / AT is the dominant repeat unit in the 2mer transcript group,a total of 6063,accounting for 61.0% of the total 2mer;the number of TC / AG repeats in the genome is the largest,accounting for 45.43% of all dinucleotide repeats,the CG / CG base pair content is extremely low,which is also reflected in the transcriptome data.The types of dominant repeat units in 3mer and 4mer are the same as AAT / ATT and AAAT / ATTT.A total of 15 microsatellite sites were developed by the two data sources,and the average polymorphic information content was similar,0.6189 and 0.6188,respectively.The average Ho and He of the transcriptome SSR are 0.3762 and 0.6484,while the genome date is 0.9086 and 0.6694.The genetic heterozygosity level of the genomic locus is slightly higher than that of the transcriptome,both of which show a higher level of genetic variation.Among the four populations of Rana dybowskii surveyed and evaluated,the average values of Shonnon?s Index(I),Observed Heterozygosity(Ho),Expected Heterozygosity(He),and Fixed Coefficient(F)were 1.08,0.528,0.557,0.038,respectively.It reflects that the population of Rana dybowskii has a high genetic diversity as a whole.The population of Qianshan Mountain and the population of northern Changbai Mountain have a moderate degree of genetic differentiation,and the Fst value is 0.08156.The genetic differentiation between populations in northern Changbai Mountain and southern Changbai Mountain and eastern Liaoning Mountain is very small.The degree of genetic differentiation between southern Changbai Mountain,eastern Liaoning Mountain and Qianshan Mountain is relatively small,0.02144,0.02807,0.04093,respectively.Through analysis of its molecular variance results,genetic variation mainly occurs among individuals,accounting for 98% of the total variation,and is the main source of genetic variation.Genetic variation among populations accounts for only 2% of the total share.