Genetic Diversity Research Of Scrophularia Ningpoensis From Different Origins

Objective: This study used the three techniques,named Near Infrared Spectroscopy?DNA barcoding and ISSR molecular markers,to analyze the chemical and genetic diversity of Scrophularia ningpoensis from different origins,in order to providing the foundation of the origins identification?excellent germplasms breeding?standard cultivation and sustainable development of Scrophularia ningpoensis.Methods:(1)Consulting the related literature in the early stage of the experiment to understand the biological characteristics?producing area distribution and market info of Scrophularia ningpoensis.The samples were collected after getting the preliminary knowledge of the basic situation of Scrophularia ningpoensis.(2)28 samples that concluded radix and decoction pieces of Scrophularia ningpoensis,which were collected from 14 different origins,needed to be drought ?smashed ?sieved and botted compactedly before setting parameters and scanning by NIR.(3)93 fresh leaf samples that collected from 22 different origins in 10 provinces are extracted DNA with the gene extraction kit.The samples total DNA were used to PCR amplify by the primers of ITS2 and psbA-trnH sequences,and the PCR products were sequenced.The sequences were matched by CodonCode Aligner and analyzed by MEGA6.06 and DnaSP.(4)The total DNA extraction and purity testing of the 33 samples from different origins and different haplotypes then the PCR amplification reaction process were ready for the following steps.The primers which could used to amplify the total DNA should be looked for from ISSR random primers,and every primer should screen their best annealing temperature.The concentration of every substrate in PCR amplification reaction system was screened by orthogonal experiment and tested by the methods of agarose gel electrophoresis.The sripe result was analyzed by statistic and clustering analysis.Results and conclusion:(1)The characteristic spectrum of Scrophularia ningpoensis from different origins was discovered,which could distinguish the origins on the south of Changjiang River from the north,showing that the internal composition and content of the samples were different between the north and south of Changjiang River.However,this conclusion needed to increase the number of samples to prove further.The NIR analysis technology showed a good prospect in the analysis and application of chemical diversity of Scrophularia ningpoensis.(2)There were 10 haplotypes in ITS2 sequences from 93 leaf samples of Scrophularia ningpoensis.The haplotype frequencies of A1 and A2 were 63.44% and 12.90% respectively,which was the highest frequencies of the two,representing the genetic characteristics in ITS2 sequence of Scrophularia ningpoensis from most origins.psbA-trnH sequence belonged to the maternal inheritance,which could reflect the maternal characteristics well.psbA-trnH sequences of all samples produced 4 haplotypes in total and among the haplotypes,C1 and C2 were similar.The haplotypes of sample came from Zhejiang were C1 and C2.This showed that samples came from most origins might be the same female parent with the samples came from Zhejiang which were regarded as geo-authentic crude drug.ITS2 sequences of all samples were analyzed by DnaSP software,the total coefficient of genetic differentiation(Gst)of all population was 0.70536,genetic flow(Nm)was 0.08;and psbA-trnH sequences result was: the total coefficient of genetic differentiation(Gst)of all population was 0.89007,genetic flow(Nm)was 0.06.The result showed that among the populations,the level of genetic exchange was very low and the phenonmenon of genetic differentiation was serious.(3)The experiment of ISSR molecular markers eventually adopted 13 primers and amplified 137 sites,including 109 polymorphism sites.The percentage of polymorphism site was 79.56% and the average number of every primer amplification sripes was 10.5.When the clustering figure distance was 21.5,all samples were divided into four parts and the Jaccard similarity coefficient among the samples from different origins was between 0.538 ~ 0.989.The clustering result and similarity coefficient of the samples from different origins showed that the genetic relationship of the samples had some associations with their geographical distribution distance,and the same population had higher similarity coefficient.The samples came from the three origins Qianxiang town in Zhejiang province ?Yuxi town in Sichuan province and Nanyang in Henan province could get together respectively in clustering figure and had longer distance from others.