Genotype classication and uorescence visual identication of the Rhizoma Paridis of Paris polyphylla var. yunnanensis based on the SNPs of ITS sequence

A uorescent visual identication system of Paris polyphylla var. yunnanensis was established based on internal transcribed spacer barcoding. It is proposed for the rst time that P. polyphylla var. yunnanensis should be divided into two types of genotypes: YN-I and YN-II according to single nucleotide polymorphism of internal transcribed spacer. In order to avoid false-negative results, two pairs of specic primers for YN-I and YN-II were designed, respectively, and specic visual uorescent identication systems was established by SYBR Green I uorescent dye was directly introduced into the PCR system which can be observed directly with the naked eye for the green uorescent color of PCR system. Therefrom, it has realized the rapid and directly visual identication of two genotypes of P. polyphylla var. yunnanensis from its common nine adulterants. This study proposed for the rst time the existence of different genotypes on the legal basis of Rhizoma Paridis, and provided a model for the accurate identication of different genotypes.

2019; Luo et al. 2017). Due to the lower natural propagation rate of PPY, the slow natural growth and the longer production cycle of Rhizoma Paridis, the annual demand increases at a rate of 20%, the wild PPY has been plundered for a long time and the resources are on the verge of exhaustion .
After the investigation of the arti cial planting base of Rhizoma Paridis in Sichuan and Yunnan province of China, it was found that the cultivation of Rhizoma Paridis was in the primary stage from wild breeding to arti cial cultivation, the production and trade of medical material were lack of an industrial standard. And the mixture of certi ed species recorded in China Pharmacopoeia(CP) and other varieties of Paris L. was common in the cultivation, production, circulation, and so on links because of economic interests. According to the national random inspection on the commodities in recent years, the pass rate of multiple batches of Rhizoma Paridis is less than 30% (Ju et al. 2019). It is di cult to distinguish PPY with the adulterants according to their phenotypic trait, microscopic characteristics, and chemical composition because the morphological characteristics of the rhizome of Paris species were similar, and the saponin types and contents of the species were widely overlapped. Also, the chemical constituents of the same species varied dynamically in different growth years and different parts of the rhizome (Wang et al. 2015;). This brings great hidden trouble to clinical medication safety. Therefore, it is of great signi cance for the quality control and drug safety to establish a rapid and accurate method for the identi cation of PPY with its adulterants in the production and trade of medicinal materials.
Internal transcribed spacer (ITS) is one of the core regions of plant DNA barcoding (Li et al. 2011). In recent years, it has been found that the ITS sequence can provide abundant mutation sites and information sites due to ITS rapid mutation and has become an important molecular marker in the phylogenetic and classi cation studies of angiosperms with lower taxonomic order (Liu et al. 2014) . Guo et, al. (2018) used ITS sequences to identify the origin of cultivated and wild samples of Paridis Rhizoma, and compared the differences between them. It was concluded that the diversity of the varieties in market circulation was caused by the confusion of the origin rather than the arti cial domestication. Fang et al. (2016) used ITS barcodes to identify the seeds and seedlings of o cial and un-o cial Paridis Rhizoma. Zhu et al. (2010) successfully ampli ed and sequenced the ITS2 sequences of 11 species of Paridis Rhizoma, and found that the identi cation success rate of ITS2 for the genus Paris was 100%, which was much higher than the other 5 chloroplast sequences. In the study, universal primers were used to amplify and sequence the ITS sequences of PPY and its common related species. By analyzing single nucleotide polymorphism (SNP) variation rule, it is found that PPY should be divided into two types of genotypes, YN-I and YN-II. When using ITS barcodes to identify PPY and its related species with only one pair of speci c primers, one genotype may be detected, but another may be missed. In order to avoid falsenegative results, two genotype-speci c primer pairs of PPY were designed and two genotype uorescence visualization identi cation methods were established respectively. The system through the macroscopic observation of uorescent color can realize the rapid and directly visual identi cation of PPY from its common adulterant products. It can be provided a new idea and way to solve the problem of authenticating the authenticity of the medicines of the same species and related species. Also, the exploration of common problems in the quality evaluation of Chinese medicine that the origin comes from multiple species of the same genus has a signi cant demonstration signi cance.

Plant Materials
One hundred and eighty-four samples of ten Paris species and subspecies in this experiments, including 34 samples of PPC; 65 samples of PPY; 17 samples of P. polyphylla;10 samples of P. stenophylla; 8 samples of P. veitnamensis; 5 samples of Paris axialis H. Li (P. axialis); 7 samples of P. vaniotii; 22 samples of Paris fargesii var. fargesii (P. fargesii); 13 samples of P. thibetica; 3 samples of Paris forrestii (Takht.) H. Li (P. forrestii) were collected from different localities in China (Table 1). All the samples were stored in the College of Ethnomedicine, Chengdu University of Traditional Chinese Medicine, China.

DNA extraction
The leaves of the Paris species were dried with allochroic silica gel, a 30 mg was weighed, and the total DNA of the samples was extracted according to the extraction steps of the plant group DNA isolation kit (Foregene Co., Ltd.). The rhizomes were ground into a powder, then was passed through No.3 sieve. In the study, the extraction steps of the DNA isolation kit in the plant group were improved, which a 20 mg sample of the material was suspended in cell lysis buffer (20µL 75% α-amylase with protease) and stored at -4℃.
DNA ampli cation and sequencing DNA extracts were ampli ed by ITS-4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS-L (5'-TCGTAACAAGGTTTCCGTAGGTC-3') primers of ITS sequence (Jiang et al. 2013). Te following polymerase chain reaction (PCR) steps were conducted: 5 μL of 10×EasyTaq buffer, 200 μM of deoxyribonucleoside triphosphate (dNTPs); 0.25μM of each primer, 2.5U of EasyTaq polymerase, 1μL (30ng) of template, and makes up 50μL with distilled water. The mixture was predenatured at 94℃ for 5min and was denatured at 94℃ for 1min, then it underwent 32 cycles of 1 min at 94°C, 1 min at 56.4°C, 1 min at 72°C, and then a nal extension for 10 min at 72°C. The PCR products (5μL) were detected by using the agarose gel (3%) electrophoresis method with Nanodrop for quality of DNA and were nally photographed under UV light exposure. The ampli ed products were directly methods Bidirectional DNA sequencing (Tsing Ke Biotechnology Co. Ltd., Chengdu, China).
The ITS sequences of PPY related cultivated species: P. polyphylla , P. stenophylla, P. vietnamensis, P. axialis, P. vaniotii, P. fargesii, P. thibetica, P. forrestii were compared using Megalign software in DNASTAR to nd out SNP sites with a difference of stability. Then designed speci c primers in the unique SNP site area of PPY by using Primer Premier 5.0 software and ampli ed the DNA template of Paris samples by PCR with the designed primers. The primer pairs were screened out which Tm difference value less than 5 and secondary structure and low mismatch rate (△G<7) (all primers in Table 2). Finally, the speci c primer pairs were screened by the above PCR reaction system, in which the objective band is clear and strong speci city and could effectively identify PPY and other common related species.
Establishment of uorescence visualization identi cation system Primer concentration, annealing temperature, deoxy-ribonucleoside triphosphate (dNTP) concentration, Taq enzyme dosage, cycle number, and template volume were optimized by the single factor method.In the 25 μL reaction system, the parameters of each component were set as follows: Speci c primer pairs ( 5U of EasyTaq polymerase, 1μL (30ng) of template, and makes up 25μL with distilled water. The mixture was predenatured at 94℃ for 5 min and was denatured at 94℃ for 30s, then it underwent 30 cycles of 30s at 64°C, 30s at 72°C, and then a nal extension for 5 min at 72°C.
The PCR products were stored at 12℃. 5µL of the PCR reaction product mixed with 6 × Loading buffer, then detected on a 3% agarose gel electrophoresis stained with Goldview I. and then performed electrophoresis at 220 V for 7 min and observed under the gel imaging system.20µL of the PCR reaction product mixed 1µL of 1000 × SYBR Green Type I uorescent dye and observed under ultraviolet light at 365 nm.

Methodological validation
A total of 25 samples as experimental materials including 10 batches of Paris samples from 7 traditional Chinese medicine (TCM) manufacturers with three parallel copies of each batch (except for individuals with less than 3 independent pieces in the batch). The universal DNA extraction method and ITS universal primers were used for DNA extraction and ITS ampli cation and sequencing, and the ITS sequences were compared using BLAST to determine the primitive plants of the experimental materials.

Results
Ampli cation and purity detection of ITS region A total of 184 plant samples were used for the ampli cation of ITS regional fragments. About 700 bp bands were obtained when the PCR products were detected by using the agarose gel electrophoresis method and successfully sequenced and the purity was 1.75-2.11 by Nanodrop test. SNP site analysis and primer screening of ITS sequence of PPY When analyzing the ITS sequences of 65 samples from PPY of different habitats, it was found that two genotypes could be classi ed according to the SNP sites. The diversity of the two genotypes in SNP sites was expressed as 40 sites, which were numbered as YN-I and YN-II respectively. The speci c classi cation criteria of genotypes were shown in Table 3. Among them, there were 35 samples of YN-I genotypes and 30 samples of YN-II genotypes. There were two speci c primer pairs that were designed for the two genotypes to avoid the omission of one of the genotypes and produced false-negative results of the PPY test. After sifting the primers in Table 2, YN-IF2/YN-IR2 and YN-IIF3/YN-IIR21 were determined to be the speci c primers pairs for the identi cation of PPY genotype I and II, respectively.
Construction and analysis of phylogenetic tree (N-J tree) The phylogenetic tree was shown in Figure 1. Phylogenetic tree results showed that the YN-II genotype of PPY was alone clustered into a large branch, indicating that the intraspeci c difference of the ITS sequence of YN-II genotype of PPY is smaller than the interspeci c differences, and it can be distinguished with its related species. The YN-I genotype of PPY was clustered into a small branch, and it was clustered into a large branch with P. forrestii and part of P. stenophylla and P. polyphylla, indicating that YN-I genotype of PPY has a close relationship with P. forrestii, P. stenophylla, and P. polyphylla. Also, it would provide more molecular evidence for PPY and P. stenophylla are classi ed as Paris polyphylla Smith. All PPC were clustered into one big branch, but some of them were interspersed with a few P. stenophylla and P. polyphylla, and P. stenophylla and P. polyphylla were clustered into several branches respectively, indicating that as the same subvarieties, P. stenophylla and P. polyphylla had a transitional period of gene differentiation and a large intrarespeci c genetic diversity. P. fargesii, P. thibetica, and P. vaniotii were clustered into one branch respectively, indicating that intraspeci c genetic distance of ITS of those 3 species sequences is less than interspeci c genetic distance and can be distinguished with other related species. P. axialis and P. vietnamensis were gathered together into a small branch, and then together into a large branch, indicating that the closer relationship between them.

Fluorescence visualization identi cation system
The optimized inspection results of the six parameters in the uorescence visualization identi cation system of two genotypes of PPY were shown in Fig According to the experimental results, the parameters were selected which the obvious speci c band brightness and the condition of avoiding nonspeci c band ampli cation and saving experimental materials were considered synthetically. The PCR reaction system and procedures for the two genotypes of PPY were nally determined as follows: The PCR steps for the identi cation of YN-genotype of PPY as follows: 2.5μL of 10×EasyTaq buffer, 280μM of dNTPs, 0.25μM of YN-IF2, 0.25μM of YN-IR2, 2.5U of EasyTaq polymerase, 1μL (30ng) of template, and makes up 25μL with distilled water. The mixture was predenatured at 94℃ for 5 min and was denatured at 94℃ for 30s, then it underwent 30 cycles of 30s at 64°C, 30s at 72°C, and then a nal extension for 5 min at 72°C.
The PCR steps for the identi cation of YN-II genotype of PPY follows 2.5μL of 10×EasyTaq buffer, 280μM of dNTPs, 0.2 μM of YN-IIF3; 0.2μM of YN-IIR21, 1.5U of EasyTaq polymerase, 1μL (30ng) of template, and make up 25μL with distilled water. The mixture was predenatured at 94℃ for 5 min and was denatured at 94℃ for 30s, then it underwent 30 cycles of 30s at 64°C, 30s at 72℃, and then a nal extension for 5 min at 72°C.

Fluorescent visualization identi cation of PPY with its common related species
The uorescence visualization identi cation results of PPY with its common related species were shown in Figure 14. In the site-speci c PCR identi cation system, there were only DNA ampli cation products of PPY with obvious bands in agarose gel, while the common related species had no bands. In the uorescence visualization identi cation system after the addition of uorescent substances, only the DNA ampli cation products of PPY could emit bright green uorescence, while other common related species could not emit uorescence. The results showed that the species-speci c primers and PCR ampli cation system designed for PPY had good speci city and could clearly distinguish PPY and its common relatives.

Results of method applicability veri cation
There are three samples (No. 10, 11, and 22) from 25 samples of Paris commercial crude drugs that failed to extract the DNA, and ITS bands still were not ampli ed after the reextraction. Therefore, the DNA of the three samples was not veri ed using the speci c PCR uorescence identi cation system. Analysis and comparison of 22 ITS sequences were shown that the main source of commercial medicinal materials was dried rhizomes of Paris and Trillium. There are 6 samples that were identi ed as PPY, including 2 samples of  and 4 samples of YN-II genotype (No. 1-4). The rest samples were identi ed as P. forrestii, P. polyphylla, and species of Trillium. The speci c information of commercial medicinal materials of Paris was shown in Table 4. As shown in Fig.15 Discussion DNA barcoding technology, as a new molecular identi cation technology that has been continuously developed in recent years. It has become an important supplement to traditional Chinese medicine identi cation technology because of its advantages of rapid, accurate, e cient, objective, and free from the change of individual morphological characteristics and the development of biological individual characteristics (Shi et al. 2016). It is widely used in the identi cation of authentic Chinese medicines, the identi cation of genuine products and substitutes, the identi cation of origin, the identi cation of multiorigin sources and genetic diversity, the identi cation of age, and so on. It plays an important role in ensuring the safety and effectiveness of traditional Chinese medicine, protecting the genetic diversity of medicinal plants, and nding or expanding new drug sources Srivastava et al. 2016). Among them, ITS (ITS1/ITS2) sequence was recommended to be included in the core barcode of seed plants by China Plant Barcode Of Life Group (China Plant BOL Group) in 2011. After a large number of studies, the ITS sequence had been proved to be able to accurately identify the origin of the bulk of Chinese herbal medicines such as Ginseng, Honeysuckle, Notopterygium incisum, Chinese wolfberry, and Rhizoma ligustici ).
Indeed, DNA barcode technology also has certain limitations. Standard DNA barcodes have better resolution e ciency between genus, but the resolution within the genus is lower, especially the identi cation of related species of the same genus in the large genera with high rates of adaptive radiation, which is not satisfactory. The identi cation e ciency of a single DNA barcode is often not ideal, and improperly designed primers can also lead to erroneous results (Hollingsworth et al. 2016;Ballin et al. 2019). For deeply processed proprietary Chinese medicines, the DNA is highly degraded and content is extremely low, so it is very di cult to extract enough DNA template (Xiong et al. 2015;Raclariu et al. 2018). Besides, for accurate molecular identi cation of species, it is necessary to establish a complete reference sequence database that can fully re ect the intra-species variation and inter-species differentiation, which is a huge workload and di cult for large genera ). In the face of the above problems, the application of DNA barcode technology in the identi cation of traditional Chinese medicine still needs to be continuously improved and developed.
There has been a great deal of controversy in the taxonomy of the Paris genus and its phylogenic relationship has been in a state of ambiguity. In addition to the continuous changes of global climate and geology since the middle ages, the continuous hybridization and geographical migration between related species of Paris genus have resulted in radial expansion of the genetic diversity of species of Paris, which is proved by the continuous discovery of new species in recent years (Ji et al. 2019;Huang et al. 2016;Xu et al. 2019). Rhizoma Paridis is a medicine material that the origin comes from multiple species of the same genus, the mixture of similar species of the same genus has always been an important factor affecting the quality of TCM. And the present situation has undoubtedly added great di culties to the identi cation of medicinal plants. With the unremitting efforts of predecessors such as CDDP marker , chloroplasts genome coding gene Song et al. (2017), ITS2 molecular regions coupled with high resolution melting analysis , new techniques and methods for the identi cation of Paris species have been developed continuously. However, current DNA barcoding methods generally require gel electrophoresis, or sequencing and homology analysis (constructed the N-J tree) to get results, which was not so intuitive in the actual test work. In this study, the site-speci c PCR identi cation system and uorescence visualization identi cation system of PPY were established for the rst time. This method signi cantly reduces the time and costs of electrophoretic detection and DNA introduction. Also, the intuitive results effectively improve the speed and ease of use of the method.
According to previous research such as Tang ling et al. (2013) analyzed 15 phenotypic traits of PPY from 20 populations, Chen Zhong-Su Zhi et al. (2017) used SSR molecular markers to analyze the genetic diversity of PPY in 5 different populations, the results showed that PPY is a complex because of phenotypic diversity. Its performance traits are affected by both environmental factors and genes. SNP refers to an intraspeci c or interspeci c variation of a single nucleotide base in a genome caused by mutations such as transformation, inversion, insertion, deletion, etc. It has become the third generation molecular marker and is widely used in variety identi cation and genetic diversity analysis, because of its wide distribution, strong genetic stability, high throughput, and fast detection (Mao et al. 2018). In this study, it was found that there are 40 SNP site variations in PPY from the different populations when sequencing the ITS sequences of 65 samples of PPY. This was consistent with Chen Shilin's statement that the comparison of four reference sequences of ITS2 of PPY with a length of 231 bp showed that there were 27 mutation sites (Chen, et al. 2011). Also, the level of genetic diversity of PPY was relatively high was further veri ed from SNP analysis. Therefore, this study proposed for the rst time that PPY should be divided into two genotypes: YN-I and YN-II according to SNP analysis. In order to give consideration to the identi cation of PPY with two genotypes and avoid false-negative results, speci c primers were designed and uorescence identi cation methods were established for PPY with two genotypes (Tang et al. 2012).
The e ciency of extracting DNA from commercial Chinese herbal medicines has always been a major problem in molecular identi cation. Thus, the methods of DNA extraction still need further study. At the same time, the Chinese herbal medicine industry chain was reminded to strengthen the management of bacteriostasis and the management of storage and logistics. (Wu 2016;Fang, et al. 2013;Mishra, et al. 2016). According to speci c uorescence detection and sequencing analysis of commercial medicinal materials of Rhizoma Paridis, the sources of Rhizoma Paridis on the market were complex, not only illegally related species of Paris but also other species of Trillium. It showed that the Trillium plants have ooded the medicinal material market as adulterant products of Rhizoma Paridis, which is a new phenomenon of adulteration of medicinal materials due to the shortage of resources and the rise of prices in recent years. The entry of a large number of adulterant products into the market will seriously affect the quality and clinical e cacy of Rhizoma Paridis. This result also shows the necessity of establishing an accurate, reliable, and practical authentication system for Rhizoma Paridis.

Acknowledgments
We sincerely appreciate Tsing Ke Biotechnology Co. Ltd., Chengdu, China for providing the service of DNA sequencing technology.

Competing interests
The authors declare that no one have competing interests.

Availability of data and materials
All data and conclusions are freely available to non-pro t colleges and research institutions.

Consent for publication
All authors consent for publication.   9  10  34  36  42  55  62  93  127  131  180   YN-I  T  G  C  T  C  T  A  C  G  A  T   YN-II  C  T  T  C  T  C  G  T  A  T  C   183  193  197  201  211  212  223  395  411  412 413   Figure 1 Phylogenetic tree of the Paris genusconstructed based on ITS.  Investigate into dNTP concentration of speci c PCR systemin YN-I. dNTP concentration was 80μM, 120μM, 200μM, and 280μM, respectively.
Page 29/34 Figure 6 Investigate into the number of cycles of speci c PCR systemin YN-I. The number of cycles was 26, 28, 30, 32, 34, respectively.

Figure 7
Investigate into the template quantities of speci c PCR systemin YN-I. The template quantities were 10ng, 30ng, 60ng, and 90ng, respectively.

Figure 12
Investigate into the number of cycles of speci c PCR systemin YN-II. The number of cycles was 26, 28, 30, 32, 34, respectively.

Figure 13
Investigate into the template quantities of speci c PCR systemin YN-II. The template quantities were 10 ng, 30 ng, 60 ng, and 90 ng, respectively.