Applied Research Of Molecular Markers In Quality Control Of Fritillaria Cirrhosa D.Don

Objective Fritillaria cirrhosa D.Don is a kind of medicine and food homologous plant with high price and high medicinal value,and it has many kinds,its medicinal value varies with different producing areas.Due to the great differences in resources and prices of Fritillaria,the phenomenon of confusing sale of Fritillaria often occurs.SSR molecular markers have the advantages of good repeatability and high polymorphism.Using molecular markers to study the quality control of Fritillaria cirrhosa D.Don at the genetic level will help to improve the accuracy of genetic diversity analysis and provide more ways to identify adulterated Fritillaria cirrhosa D.Don.Methods The transcriptome information of Fritillaria cirrhosa D.Don was obtained by Illumina double-terminal sequencing technique,and the SSR loci were screened and analyzed by MISA software.20 pairs of SSR primers were synthesized,the PCR system was optimized,and the SSR primers for follow-up experiments were screened.Taking Fritillaria cirrhosa D.Don from different habitats as the research object,the polymorphism of Fritillaria cirrhosa D.Don was analyzed by SSR molecular markers and the genetic map was constructed.The genetic distance was calculated and the genetic relationship was analyzed by cluster analysis.Through the comparison of amplified bands,the differential SSR molecular marker bands were recovered,cloned,sequenced and SCAR primers were designed,which were applied to the identification of different varieties of Fritillaria cirrhosa D.Don,Fritillaria thunbergii and Fritillaria hupehensis Hsiao et K.C.Hsia.Results The transcriptome information of Fritillaria cirrhosa D.Don was obtained and the correlation of SSR loci was analyzed.In this study,20 pairs of SSR primers were randomLy synthesized according to nucleotide repeat types,including the existence of 5 repeat types of SSR from dinucleotide to hexanucleotide.Through the optimization experiment,the best reaction system of SSR primer amplification was determined.The amplification products of 7 pairs of SSR primers were selected,which were NO.4,NO.7,NO.8,NO.12,NO.17,NO.18 and NO.20,respectively,without trailing,and the background was clean without clutter.In the genetic diversity analysis experiment of Fritillaria cirrhosa D.Don,7 pairs of SSR primers were used to amplify 5 species of Fritillaria cirrhosa D.Don,and their polymorphisms were analyzed.After calculating the PIC value,the genetic map of Fritillaria cirrhosa D.Don was constructed.According to the genetic distance of Fritillaria cirrhosa D.Don,the genetic relationship of Fritillaria taipaiensis P.Y.Li is the farthest.Based on the cluster analysis of five varieties of Fritillaria cirrhosa D.Don,Fritillaria taipaiensis P.Y.Li was divided into one group at 0.49,which indicated that there was a great genetic difference between Fritillaria taipaiensis P.Y.Li and other varieties of Fritillaria cirrhosa D.Don.Through the amplification of band comparison,the differential bands of Fritillaria cirrhosa D.Don,Fritillaria thunbergii and Fritillaria hupehensis Hsiao et K.C.Hsia were found by SSR primers NO.20 and NO.4,respectively,and on this basis,SCAR primers 20-1 and 4-1 were designed,which can be used for the adulteration identification of Fritillaria thunbergii and Fritillaria hupehensis Hsiao et K.C.Hsia mixed with Fritillaria cirrhosa D.Don.Conclusion The SSR molecular markers developed based on the transcriptome of Fritillaria cirrhosa D.Don can be used in the study of heritage diversity,variety identification,genetic relationship identification and genetic map construction of Fritillaria thunbergii,which provides a theoretical basis for source protection and quality control of Fritillaria cirrhosa D.Don.