Development Of Indel Markers For Panax Ginseng And Panax Quinquefolius And Their Application In Origin Authentication Of Ginseng Products

Panax ginseng and Panax quinquefolius are traditional Chinese herbal medicines with similar morphology,they have been used for supplementing Qi and nourishing body.The chemical compositions and pharmacological effects of P.ginseng and P.quinquefolius are similar,but traditional Chinese medicine theory and modern medical experiments have been proved that the medicinal properties and efficacy of them are very different.P.ginseng is warm,sweet or slightly bitter in flavor,it has been used to supplement Qi and nourish spleen and kidney.P.quinquefolius is cool,sweet or slightly bitter in flavor,it has been used to tonify lung and reduce internal heat,supplement Qi and nourishi Yin.Therefore,Panax ginseng and Panax quinquefolius can not be confused.Ginsenosides are the constituents responsible for the bioactivities of P.ginseng and P.quinquefolius.The total ginsenosides content in P.quinquefolius is higher than that in P.ginseng,which is the evaluation criterion of ginseng products.The phenomenon of the intentional or misidentificated adulteration of P.quinquefolius into P.ginseng often occurs,owing to the similarity in appearance from each other.Therefore,accurate identification of P.ginseng and P.quinquefolius is very important for ensuring pharmaceutical efficacy and food safety.Many traditional methods have been used for discrimination of P.ginseng and P.quinquefolius,these methods are vnlnerable to environment condition,subjective factor and machining process.Therefore,it is necessary to develop a rapid and effective method to realize the identification of P.ginseng and P.quinquefolius and their products.Based on the fact that intron position in orthologous genes is highly conserved across plant species,intron length polymorphisms were exploited from unigenes of P.ginseng and P.quinquefolius in this study.In the genomic sequence of Cytochrome P450,compared with P.quinquefolius,P.ginseng has a 365 bp insertion sequence and a 29 bp deletion sequence.In the GAPDH genomic sequence,compared with P.quinquefolius,P.ginseng has a 5 bp insertion sequence and a 20 bp deletion sequence.Specific primers PgF and PgR were designed according to the 365 bp-insertion sequence in Cytochrome P450 gene of P.ginseng,specific primers PqF and PqR weredesigned according to the 20 bp-insertion sequence in GAPDH gene of P.quinquefolius.Molecular authentication of P.ginseng and P.quinquefolius was performed by multiplex PCR system with two specific primer pairs.The P.ginseng samples generated their unique bands of 440 bp,whereas P.quinquefolius samples yielded their specific amplicons with size of 692 bp.And the developed multiplex PCR system was effective for the detection of as low as 0.01 ng of total DNA.As a result of the mixture DNA of P.ginseng and P.quinquefolius by multiplex PCR system,they generated two specific bands of 440 bp and 692 bp.And the detection of intentional adulteration of P.quinquefolius into P.ginseng down to 1% level,that is to say,detection limit is 0.01 ng.The assay showed absolute species-specificity and high sensitivity,and therefore,could be used for molecular authentication of P.ginseng and P.quinquefolius.This study developed the insertion or deletion sequence of P.ginseng and P.quinquefolius from the cox? gene.Amplification of the cox? gene of P.ginseng and P.quinquefolius,according to the sequence alignment,was found that there is an 8 bp insertion sequence and a 7 bp deletion sequence in P.ginseng,on the contrary,there is a 7 bp insertion sequence and an 8 bp deletion sequence in P.quinquefolius.Specific primers PF and PR were designed according to the 8 bp-insertion sequence in cox?gene of P.ginseng,specific primers QF and QR were designed according to the 7bp-insertion sequence in cox? gene of P.quinquefolius.These two specific primer pairs were together used to construct a multiplex PCR system for authentication of P.ginseng and P.quinquefolius.The P.ginseng samples generated their unique bands of404 bp,P.quinquefolius samples yielded their specific amplicons with size of 138 bp,whereas the mixture of P.ginseng and P.quinquefolius generated two specific bands of 404 bp and 138 bp.Under the multiplex PCR system,P.ginseng and P.quinquefolius products also generated specific bands of 404 bp and 138 bp,respectively.The developed authentication system was proved to be effective not only for detection of intentional adulteration of P.quinquefolius into P.ginseng down to0.1% level,but also for detection of intentional adulteration of P.ginseng into P.quinquefolius down to 0.1% level,detection limit is as low as 0.001 ng.The developed indel molecular markers of mitochondrion in cox? gene could be used formolecular authentication of P.ginseng and P.quinquefolius and application in authentication of their products,and this method showed absolute species-specificity and high sensitivity.In this study,the assay from Cytochrome P450 gene,GAPDH gene and cox?gene exploited the insertion or deletion sequences of P.ginseng and P.quinquefolius,and the identification of P.ginseng and P.quinquefolius as well as their products can be realized by indel molecular markers.Compared to other DNA molecular markers,indel molecular markers do not require stringent reaction conditions and optimization of reaction system,and it is efficient,simple and sensitive.Therefore,this study provides an objective criterion for botanical origin authentication of ginseng products,and can be employed in origin authentication of other herbal preparations.