Advances in Molecular Identification Of Cistanche

Abstract: Cistanches Herba, a rare and valuable medicinal material in China, has high medicinal value and ecological value. Amid the advancement of molecular biology techniques, a variety of DNA-based molecular identification techniques have been gradually improved and major headway has been made in the research onCistanches Herba. This paper reviews the DNA-based molecular identification techniques for Cistanche and discusses the limitations and application prospects, which is expected to serve as a reference for accurate identification and quality evaluation of the Cistanches Herba, the protection and rational utilization of the resources, and variety of breeding. 

Keywords: Cistanche; molecular identification; molecular marker technique; the identification and quality; resource conservation

Click here to know more details about the components of Cistanche

Cistanche is the dry scale-leaved fleshy stem of Cistanche deserticola Y. C. Ma and Cistanche tubulosa (Schenk) Wight, a plant of the genus Cistanche in the family Cistanche. Jiangyun, Cunyun, Cistanche, and Chagangaoya (Mongolian language) are known as "desert ginseng" [2]. In recent years, as the resources of wild Cistanche are on the verge of depletion and the domestic demand is increasing day by day, a large number of counterfeit products of Cistanche have flowed into the market of Chinese herbal medicines, causing huge fluctuations in market prices, and the quality of medicinal materials cannot be guaranteed, which seriously endangers the safety of clinical medication. sex [3]. Therefore, the preservation, research and rational development and utilization of Cistanche plant germplasm resources are imminent, and the accurate identification of Cistanche plant germplasm resources is especially important.

as important. The 2020 edition of the "Pharmacopoeia of the People's Republic of China" (hereinafter referred to as "Chinese Pharmacopoeia") only records that the dried scaly fleshy stems of Cistanche deserticola are used as genuine medicines. From the perspective of the situation, in addition to the plants Cistanche and Cistanche tubehua recorded in the 2020 edition of the "Chinese Pharmacopoeia", there are also Cistanche sinensis G. Beck, C. salsa (C. A. Mey.) G. Beck, Lanzhou Cistanche C. lanzhouensis Z. Y. Zhang, etc. [4], there are also a large number of counterfeit doping phenomenon. With the development and continuous improvement of molecular biology technology, molecular identification technology has the advantages of less sample consumption, high speed and high accuracy, and has been widely used in the species identification of animals and plants. The development of resource mining research is also relatively rapid [5]. At present, there has been some progress in the identification of Cistanche medicinal materials by molecular identification technology. According to the classification of molecular marker technology required for molecular identification [6], this paper reviews the germplasm identification of Cistanche deserticola and other aspects, and discusses the existence of germplasm identification of Cistanche deserticola. Analyze the problems and put forward corresponding solutions, aiming to provide reference for the protection, rational utilization and cultivation of new varieties of Cistanche plants.

1 Application of DNA barcoding technology in the identification of Cistanche plants

1.1 DNA barcoding technology

In 2003, Professor Paul Hebert of the University of Guelph in Canada introduced barcode technology into the biological world and first proposed the concept of "DNA barcode" [7]. DNA barcode technology is an effective method for identifying traditional Chinese medicine and multi-based raw materials. For the respective DNA, the candidate fragments were amplified by the general primer polymerase chain reaction (PCR), the PCR amplification products were purified, sequenced and analyzed, the target DNA barcode sequence was searched, and a DNA barcode recognition system was constructed [8]. In conclusion, DNA barcode identification is a biomolecular identification method that utilizes one or a few relatively short, standard DNA fragments for species identification [9].

In recent years, through the combination of high-throughput sequencing technology and DNA barcode identification technology, a new technology that can detect barcode sequences of multiple species in mixed samples at the same time has been developed——DNA metabarcode, the basic principle of which is to apply high-throughput sequencing The technology obtains the amplified sequence of the mixed barcode, and identifies the species composition in the mixed sample by means of bioinformatics analysis[10].

1.2 Selection of DNA barcode sequences

The barcode sequences that can be used in DNA barcoding technology include mitochondrial coenzyme Ⅰ (CO Ⅰ) DNA, 12S rRNA, 16S rRNA sequences and ribosomal 18S rDNA for animal species identification[11]; ribosomal 16S rDNA for bacterial identification[12] ], ribosomal internal transcribed spacer (ITS) gene-specific fragments and CO I sequences for fungal identification[13]; due to the slow evolution rate of mitochondrial genomes in plants, barcode fragments are mainly selected on the chloroplast genome The proposed gene fragments mainly include rpoB, rpoC1, matK, rbcL, and UPA, and the non-coding region fragments include atpF-atpH, trnH-psbA, psbK-psbI, and

Nucleated gene ITS[14]. In 2006, Chen Shilin's research group tested the discrimination ability of ITS2 on more than 6,600 plant samples, and found that the identification efficiency of ITS2 at the species level was as high as 92.7%, indicating that the ITS2 sequence can identify standard DNA barcodes of medicinal plants and closely related species. ITS2 was used as a new type of universal DNA barcode for medicinal plants [15], and was recognized by international peer experts [16].

In 2013, the National Pharmacopoeia Committee discussed and approved the inclusion of the guidelines for the molecular identification of DNA barcodes for Chinese medicinal materials in the supplementary edition of the "Chinese Pharmacopoeia". ITS2 is the core DNA barcode identification system for plant medicinal materials [17].

At present, many scholars have carried out molecular identification research on Cistanche plants. According to the research of Chen Shilin et al. [18], ITS2 is suitable as a standard barcode sequence for identification of medicinal plants. Sun Zhiying et al [19] found that the ITS2 sequence can be used as the basis for effectively identifying the Chinese herbal medicine Cistanche deserticola and its counterfeit products in DNA barcodes. Wang Xiaoyue et al[20] used ITS2 barcodes to identify 4 common obfuscated products of Cynomorium, Cistanche, Liedang and Cistanche, and successfully established the "molecular identity card" of Cistanche's obfuscated products. The method of identifying medicinal plants through ITS2 sequences is relatively mature, and has the advantages of rapidity, accuracy, and efficiency. Therefore, using the ITS2 sequence to identify Cistanche plants has become the most commonly used method.

1.3 Workflow of DNA barcoding

The workflow of DNA barcoding is similar to the operation of molecular phylogenetic research, and the main steps are shown in Figure 1. Gu Xiuyan [21] obtained the base sequence of ITS and analyzed the differences between species, and found that Cistanche is closely related to Cistanche saline, and Cistanche in Lanzhou is closely related to Cistanche, which also provides a basis for the development of new drug sources of Cistanche. basis. Li Zhenhua et al[22] carried out DNA molecular identification research on Cynomorium, Cistanche and Huanghua Liedang, and realized rapid and accurate identification of Cistanche and counterfeit Cynomorium, Cistanche and Huanghualiedang by site-specific PCR.

In short, there is already a relatively complete process for identifying plant species using DNA barcodes. Identifying Cistanche plants by analyzing DNA sequences and establishing a related database can provide a more basis for the identification and classification of Cistanche plants in the future.

1.4 Data analysis of DNA barcoding

Processing and analyzing the obtained data is a very important task[18]. After the sequencing is completed, sequence comparison and manual correction are performed to remove low-quality sequences and primer regions. Commonly used software includes Chromas, CExpress[23], etc.; The genetic distance analysis of the final sequence is generally carried out by MEGA software [24] to analyze the genetic distance between samples of different plant species, and the K2P model [25-26] is used to calculate the intraspecific distance between species; then construct the neighbor-joining (NJ) phylogeny Tree, using the iTol online website [27] to improve and beautify the developmental tree (https://itol.embl.de/), and check the support rate of each branch according to the bootstrap (1000 repetitions).

The BLAST method is a search algorithm based on BLAST. It is necessary to establish or download a reference sequence database for species identification on the GenBank database (https://www.ncbi.nlm.nih.gov/) for subsequent analysis of gene fragments and species identification work [28]. Xu Danyun et al[29] used 3 pairs of DNA barcode universal primers to identify 22 species of Lauraceae plants, and successfully identified 20 species of plants. Adolfo et al[30] successfully identified 3 species of Pueraria plants using ITS2 and matK barcodes. The above studies demonstrate that by obtaining and analyzing the DNA sequences of medicinal plants, plant species can be quickly and efficiently identified.

2 Application of other molecular marker techniques in the identification of Cistanche plants

For organisms, their traits above the molecular level are ultimately determined by molecular traits. Compared with morphological analysis [31] and chromosome analysis [32], molecular markers can reveal the true face of biological genetic diversity. Sarwat et al[33] used amplified fragment length polymorphism (AFLP), selectively amplified polymorphic microsatellite locus technology (SAMPL), simple intersequence amplification of repeats (ISSR), random amplified polymorphic DNA ( RAPD) and other molecular marker techniques detected the genetic diversity of Tribulus terrestris samples collected from different places in India, and the results showed that these four molecular marker techniques can obtain different DNA fingerprints unique to each geographic region. The International Union for the Protection of Plant Variety Rights (UPOV) also uses DNA molecular marker identification as an auxiliary means for DUS (distinctness uniformity and stability) testing of crop varieties [34]. At present, molecular marker technologies such as AFLP, RAPD, and ISSR are relatively mature and widely used in the identification of Cistanche plants (Table 1).

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