Construction And Application Of Molecular Identification Methods For Atractylodis Macrocephalae Rhizoma,Atractylodis Rhizoma,and Its Hybrids

Atractylodis Rhizoma(AR),a traditional Chinese medicine,is derived from the dried rhizomes of Atractylodes lancea(Thunb.)DC.or Atractylodes chinensis(DC.)Koidz.,which has the effects of eliminating dampness and invigorating spleen,dispelling pathogenic wind and dispelling cold,and improving eyesight.And Atractylodis Macrocephalae Rhizoma(AMR)is derived from the dried rhizome of Atractylodes macrocephala Koidz,it can effectively invigorate the spleen and invigorate qi,eliminate dampness and diuresis,arrest sweating,and can also prevent miscarriage.Since the effects of AR and AMR are different,they should not be applied confusedly in clinic.In recent years,due to the scarce of wild resources of AR and AMR,their market demand has been increasing continuously,and the sources are dependent on artificial cultivation.Additionally,it has been reported that the hybrids of A.lancea and A.macrocephala exist in the cultivation and marketing of AR.As a result of the trait similarity of AR and its hybrids,it is difficult to distinguish them,which is one of the factors causing confusion and substandard quality of AR in the market.Therefore,it is of great significance to solve the problems of confusion between AR and AMR,and the adulteration from hybrids by accurate identification approach.In this work,stable SNP molecular markers were screened based on the differences in nuclear genome sequence of AR and AMR.Multiplex ligation-dependent probe amplification(MLPA),RNase H-dependent PCR(rh PCR),and Tetra-primer amplification refractory mutation system PCR were applied to identify AR,AMR,and hybrids,respectively.And the110 samples were used to verify the applicability and accuracy of the methods.The study contents and results were shown as follows:1.The bases at 152 bp of the ITS2 sequences of AR and AMR are "C" and "T",respectively,and the "C/T" heterozygous peaks were shown in hybrids.Based on this site,species-specific MLPA probes were designed and combined with the high-resolution melting curve analysis(HRM),an MLPA-HRM method was established for the simultaneous identification of AR and AMR,and hybrids.By pre-amplification and purification of DNA,hybridization of the probe with DNA,ligation,PCR amplification and melting curve analysis,single melting peaks with melting temperature(Tm)of 84.7±0.2℃ and 86.6±0.2℃ were generated for AMR and AR respectively,while double melting peaks were generated for hybrids.The method is highly specific and replicable,and nearly realizes accurate and efficient specific detection of AMR,AR,and hybrids.2.Based on the difference of base at 242 bp in the 18S-ETS sequence("G" for AR and "A" for AMR),rh PCR-HRM,a highly specific site-specific PCR method,was established for AR,AMR,and hybrids.Two highly specific amplification primers(B-Fr,C-Fr)containing RNA bases and closed groups and one common reverse primer(BC-Rr)were designed to construct duplex rh PCR-HRM reaction system.After optimizing the reaction conditions,the target fragments of 106 bp and 92 bp in length could be amplified in one-step PCR reaction from AR and AMR,and melting curve analysis yielded Tm values of 86.8±0.2°C and 84.7±0.2°C,respectively.PCR amplification and result analysis can be completed within 1 hour.The advantages of high specificity,ease of operation,and time saving making it suitable for the rapid identification of samples.3.Based on the 18S-ETS sequence,four amplification primers were designed(including two generic outer primers for Atractylodes,one AR-specific inner primer and one AMRspecific inner primer),and three specific fragments of 362 bp,263bp and 143 bp in length were amplified.The Tetra-primer ARMS-PCR method was established to identify AR,AMR,and hybrids by annealing temperature,number of amplification cycles and primer ratio.PCR amplificons could be analyzed by agarose gel electrophoresis.The method is simple,and the cost of amplification and detection is low,which feathers good application prospect for largescale sample identification.4.The three established molecular identification methods were used to identify 110 collected samples,and the same detection results were obtained: 89 batches of AR samples,7batches of AMR samples and 2 batches of hybrids samples were identified,and another 12 batches of AR samples were hybrids or adulterated with hybrids.The molecular identification methods constructed in this study are effective supplements for the Character Identification,macroscopical identification,and physical and chemical identification,and enhance the efficiency of the identification of AR,AMR,and hybrids.Meanwhile,it also provides technical support for the effective regulation of the herb market as well as a reference for the rapid identification research of other herbal medicines and processed products.