Molecular Authentication Of Chinese Huangguo Ginseng Cultivar Based On Allele-specific PCR

Panax ginseng C.A.Meyer known as"the king of all herbs"is a high medical valuable traditional Chinese tonic herbal medicine.It plays a significant role in both traditional Chinese medicine and western medicine as a result of containing a large number of active ingredients.The mature fruits of general ginseng are red while that of Gumpoong and Huangguo is yellow.Huangguo with high content of ginsenoside and other active components is a new ginseng cultivar bred by Institute of Special Animal and Plant Sciences of Chinese Academy of Agriculture Sciences,which has added new valuable germplasm resource for the cultivation and breeding of ginseng.With the increasing development of ginseng breeding and production industry,problems including the cultivar confusion of ginseng,and degradation of seed quality had become increasingly outstanding,which had adversely affected the quality control of ginseng and germplasm purity.The variety identification of Chinese medicinal materials is the key of the quality control.For these reasons,it is of great significance to develop a simple and accurate authentication method of ginseng cultivar for the quality control of ginseng products and breeding of high-quality ginseng cultivar.DNA molecular markers can directly analyze the genetic material of different species at the molecular level compared with the traditional authentication method.So it can effectively eliminate the interference of different growth environmental factors and stage in growth and development.And it has the advantages of high polymorphism,accuracy and high repeatability,which provides an effective technology for the authentication of P.ginseng cultivars and breeding of high-quality ginseng.In this paper,SNP molecular markers were developed based on the previous analysis of ccs A gene in small single copy sequence and rpoC2 gene in long single copy sequence from chloroplast and bioinformatics analysis was carried out.And two specific primers were designed based on two"Huangguo"and"Hongguo"variety-specific SNPs to identify the cultivated ginseng Huangguo.Then the multiple allele-specific PCR method was established as well as real time allele-specific PCR and the method was validated and accurate to different Huangguo ginseng from Hongguo ginseng.In addition,another SNP marker was found by analyzing the sequencing results of the ccs A and 26S rDNA genes of Huangguo and Gumpoong ginseng cultivars.And in order to realize the identification of Gumpoong,a specific primer of Gumpoong was designed based on the SNP exploited from 26S rDNA gene and combined with multiplex PCR system to establish a molecular technology for identification of Gumpoong and Huangguo ginseng cultivars.The main results are as follows:1.The bioinformatics analysis was carried out according to the SNP sites of Huangguo ginseng cultivar found in previous studies.The result indicated that the two non-synonymous substitution of G for A and C for G were respectively detected at the635^thand 685^thnucleotide position of’Huangguo’variety.This transition,resulting in the modification of amino acid residue from glutamine（G）to arginine（A）and glycine（G）to setine（S）,not only changed the secondary structure but also modified the tertiary structure of ccs A and rpoC2 protein.Huangguo and Hongguo ginseng cultivars were accurately identified with the combination of ccs A and rpoC2 genes.In addition,Huangguo was C but Gumpoong was G at the 1737^thnucleotide position through the analysis of 26S rDNA gene sequence and combined with ccs A gene to distinguish Gumpoong from Huangguo.2.Based on the SNP sites developed,the site-specific primers of Huangguo and Gumpoong were designed by introducing a mismatch to ensure the specificity of the primers.The two pairs of allele-specific primers were designed based on the Huangguo species-specific SNP sites discovered in ccs A and rpoC2,respectively.Primes HgF（5′-CTATGCAGCTCTTCTATGTGG-3′）、HgR（5′-ATCCAACTGTGAATCAGCC-3′）and RNF（5′-GCGAAAGAATGAATCCTAAT-3′）、RNR（5′-CGGGGACTCACAGAAATA GC-3′）were designed for the identification of Huangguo.A specific primer was designed based on the Gumpoong species-specific SNP site discovered in 26S rDNA.Prime GPF（5′-CTAAGCCGGAGGTAGGATG-3′）and GPR（5′-TGACGAGGCATTTGGCTAC-3′）was designed for specific authentication of Gumpoong.3.Multiplex PCR system was established to authenticate Huangguo and Gumpoong ginseng cultivars.Huangguo variety generated 217 bp-specific bands with the usage of HgF and HgR,while Hongguo variety generated amplifications of 360 bp amplified by RNF and RNR.Meanwhile,a multiplex PCR system of discriminating Gumpoong was constructed by using specific primers"HgF/HgR and GPF/R".The results showed that both Huangguo and Gumpoong produced the bands of 217 bp and only Gumpoong ginseng amplified specific bands of 568 bp amplified by GPF and GPR.Therefore,Huangguo and Gumpoong could be accurately identified according to their separate brands.4.In the real time allele-specific PCR assay,samples of Huangguo showed intensive fluorescence signals owing to the binding of SYBR Green fluorescent dye to Huangguo variety specific amplicons.However,only weak fluorescence signals were detected for Hongguo variety due to failure in PCR amplification.Therefore,the rapid method of Huangguo ginseng cultivar could be easily established through Allele-Typing and the Endpoint analysis.The SNP molecular marker technology and multiplex PCR system established in this paper can accurately and effectively realize the identification of Chinese Huangguo and Korean Gumpoong ginseng,which provides a DNA molecular method for the authentication of Huangguo and Gumpoong and effective means for screening of high-quality cultivated ginseng Huangguo and ensuring the stability of its product quality.