Study On The Genetic Relationship And Molecular Marker Of Murraya Exotica And M.paniculata

According to the 2020 edition of the Chinese Pharmacopoeia,Murrayae folium et cacumen(MFC)originates from Murraya exotica or Murraya paniculata,which can promote qi circulation,relieve pain and invigorate the blood circulation of scattered stasis.MFC is used in the treatment of stomachache,rheumatism,arthralgia,toothache.It is also one of the main medicines of the Chinese patent medicine Sanjiu Weitai,which is used in large quantities.However,the classification status of M.exotica and M.paniculata is controversial and has been changed many times.Because of the existence of morphologic intermediates,most foreign scholars agreed that the two species should be combined as one.At present,it is generally supported that M.exotica and M.paniculata are two separate and distinct species in the domestic environment.Studies have shown that M.exotica and M.paniculata have great differences in chemical composition.The main constituents of M.exotica leaves are coumarins,while those of M.leaves are polymethoxylated flavones.In the current market,the main resource of MFC is M.paniculata,while M.exotica is used as a green plant in southern China.Thus,it is necessary to clarify the genetic relationship between M.exotica and M.paniculata and develop molecular identification markers for them for the safety and efficacy of MFC in clinical trials.In this study,69 samples of M.exotitca and M.paniculata from different districts and counties were numerically classified based on leaf traits.And the chloroplast genome comparison of 8 species of the genus Murraya was performed.The DNA Signature Sequence(DSS)was developed for the identification of M.exotica and M.paniculata based on the chloroplast genomes of the genus Murraya.Specific primers were designed according to single nucleotide polymorphism(SNP)on the DSS,and a multiplex allele-specific PCR identification method was establised for the identification of M.exotica and M.paniculata.The main contents and results of this study are as follows:1.Based on the differences in traits between M.exotica and M.paniculata,8 leaf traits were selected for a numerical taxonomic study of 69 samples,including widest point,phylliform,leaf apex,tip micro concave,leaf margin,number of lobules,lobule length,and lobule width.The R-type clustering results showed that lobule length and lobule width were highly correlated.The results of principal component analysis showed that the widest point,phylliform,leaf apex and lobule width had a great influence on the classification of M.exotica and M.paniculata,and could be used as an important indicator for the classification of M.exotica and M.paniculata.The Q-type cluster tree showed that the total samples could be divided into two groups,M.exotica and M.paniculata,indicating that they have significant differences in leaf traits.2.The chloroplast genomes of M.exotica,M.paniculata,M.alata,M.euchrestifolia,M.kwangsiensis,M.kwangsiensis var.macrophylla,and M.microphylla were sequenced and assembled.And the chloroplast genome of M.koenigii was downloaded from GenBank 33 chloroplast genomes of 8 species were annotated.The results showed that the chloroplast genome of species in the genus Murraya was relatively conserved,which had a classical tetrad structure,consisting of two single copy regions and two reverse repeat regions.Chloroplast genome length ranged from 15 8,573～160,817 bp with an average of 160,169 bp;and GC content ranged from 38.54%to 38.65%with an average of 38.62%.The chloroplast genomes of the 8 species contained 112 non-repeating genes,including 78 protein-coding genes,30 tRNA genes,and 4 rRNA genes.33 chloroplast genomes contained 1233 repeated sequences and 7924 simple repeat sequences(SSR).3.The results of IR boundary analysis showed that there was expansion and contraction of the IR region in the chloroplast genomes of 8 species.In some samples of M.exotica,expansion was observed in the IR region,while contraction was observed in M.kwangsiensis var.macrophylla.The results of comparative analysis and collinearity analysis showed that the chloroplast genome structure of 8 species was conserved,and the sequences were highly similar without obvious structural rearrangement.Phylogenetic relationships of the genus Murraya were inferred using maximum likelihood(ML)and Bayesian inference(BI)methods.Three data sets were generated for phylogenetic analyses,as follows:(1)the whole chloroplast genome;(2)a concatenated data set consisting of coding sequence(CDS);and(3)the non-coding sequence(NCS).The six phylogenetic trees had the same topological structure,and each evolutionary branch had high support.M.exotica,M.paniculata,and M.alata of Sect.Murraya were clustered into a monophyletic group,and M.exotica was more closely related to M.alata.The chloroplast genome phylogenetic analysis supported the classification of M.exotica and M.paniculata as two separate species.4.The chloroplast genome of the genus Murraya was analyzed using IdenDSS software.DSS molecular markers were screened and verified to identify M exotica and M.paniculata.On this basis,SNPs of M.exotica and M.paniculata were screened out and species-specific identification primers were designed.The multiplex allele-specific PCR identification method was established for the identification of MFC,while the PCR method was explored and verified in terms of tolerance and feasibility.In this method,about 330 and 230 bp of specific fragments were amplified from DNA templates of M.exotica and M.paniculata,respectively.The identification can be done in a single identification assay.