Analysis Of Ginseng Genetic Diversity And Development And Verification Of SNP Locus Based On SLAF-seq

China is rich in ginseng germplasm resources,but due to the lack of sorting and identification of germplasm resources,the utilization efficiency of ginseng germplasm resources is low and breeding materials are scarce.At present,the genetic relationship of ginseng germplasm resources is not very clear,and there is a serious lack of germplasm identification methods.Molecular marker technology is a necessary technical method for the identification of germplasm resources.However,the number of molecular markers of ginseng is small,and the early-developed markers such as RAPD,RFLP and SSR are the main ones,which cannot achieve rapid and accurate identification of ginseng germplasm resources.In this study,based on the SLAF-seq technology,the genetic evolution analysis of ginseng populations was carried out,and the specific SNP sites of 21 ginseng resources were discovered.For the screened SNP flanking sequence information,primers were designed to amplify the genome fragments containing SNP sites,and the SNP sites were verified by the first-generation sequencing results.For successfully verified SNP sites,develop molecular markers based on conventional PCR and fluorescent quantitative PCR.It is expected to provide technical methods and theoretical references for the identification of ginseng germplasm resources.The main research results are as follows:1.A total of 745.17 Mb data were obtained from 98 individual samples.The average depth of this sequencing was 19.55 ×.A total of 1,160,215 SLAF tags were developed,including 356,324 polymorphic SLAF tags.The average Q30 value of sequencing was 93.19%,and the average GC was34.92%.A total of 1,766,585 population SNPs were obtained,and 17 population-specific SNPs were found.Among them,1 population-specific SNP was found in KTZ-01 ginseng,11 population SNPs were found in KTZ-02,and 2 specific SNPs were found in KTZ-04,2 specific SNPs were found in KTZ-05,and 1 specific SNP was found in KTZ-06.Through the analysis of population structure,PCA and phylogenetic tree,ginseng resources are divided into 8 subgroups.Among them,KTZ-01,KTZ-02,KTZ-04,KTZ-05 and KTZ-06 have relatively small internal genetic differentiation.However,ginseng resources present rich genetic diversity in general.2.34 pairs of universal primers are designed for the SNP flanking sequences of KTZ-01,KTZ-02,KTZ-04,KTZ-05 and KTZ-06.All primers can amplify the target band,but only 2 pairs of primers can amplify the target band.The amplified product is a single fragment,which is the primers of KTZ-01 and KTZ-02 specific SNPs.The first-generation sequencing results showed that the primer pair E-3F/R can amplify the 1235 bp target fragment.In the KTZ-01 sample,the base at 860 bp of the amplified sequence is T,and the base here in the amplified sequence of other samples is G;The primer pair M-11F/R can amplify a 1036 bp target fragment.In the KTZ-02 sample,the base of the amplified sequence at 644 bp is A,and the base of the amplified sequence of other samples is G here.3.Routine PCR results showed that the specific primer SEF/R only amplified a 279 bp band in the KTZ-01 sample,and no band was amplified in other samples;the specific primer SYF/R only amplified in the KTZ-02 sample A 250 bp band was amplified in the sample,and no target band was detected in other samples.The application of specific primers SEF/R and SYF/R can distinguish KTZ-01 and KTZ-02 from other ginseng varieties.4.RT-q PCR results showed that the specific primer SEF/R only produced an amplification curve in the KTZ-01 sample,and no amplification curve was generated in other samples;the specific primer SYF/R only produced an amplification curve in the KTZ-02 sample amplification curve,no amplification curve was detected in other samples,further verification of SNP sites was carried out.