Method for investigating regional medicinal plant diversity based on atmospheric eDNA macro-barcoding technology

Abstract

The application discloses a regional medicinal plant diversity investigation method based on atmospheric eDNA macro-barcode technology. rbcL The primers of the gene and the ITS2 sequence are subjected to regional specific base optimization, and two-segment sequence primers of the intron are combined to construct four DNA barcode independent amplification with high adaptability, so that the regional dominant medicinal plant diversity investigation is realized at low cost, non-destructive and in a whole-year and whole-cycle dynamic monitoring mode. trnL The defects of traditional methods, such as large consumption of manpower and material resources, low investigation efficiency and inability of dynamic monitoring, are effectively solved, and a standardized technical scheme and scientific basis are provided for long-term dynamic monitoring, protection and utilization of medicinal plant resources.

Description

Technical Field

[0001] This invention belongs to the field of medicinal plant resource survey and environmental DNA molecular ecology technology. Specifically, it relates to a method for regional medicinal plant diversity survey based on atmospheric EDNA macrobarcoding technology. Background Technology

[0002] Medicinal plants are an important natural source for drug development, and their unique secondary metabolites provide irreplaceable molecular frameworks and bioactive substances for new drug discovery and lead compound screening. As a vital strategic biological resource in my country, medicinal plants hold a crucial strategic position in the traditional Chinese medicine industry, the inheritance of ethnic medicine, and national biosecurity. Conducting systematic surveys of medicinal plant diversity in specific regions can provide fundamental data and decision-making basis for the development, protection, and sustainable utilization of medicinal plant resources, serving as a key link between resource reserves and industrial needs.

[0003] Currently, methods for investigating regional diversity of medicinal plants mainly include field quadrat surveys, field surveys and specimen collection, remote sensing image surveys, and methods based on traditional species monographs or catalogues. Among these, field quadrat surveys and traditional species monographs and catalogues are the mainstream methods. However, these methods have significant drawbacks: field surveys and specimen collection are inefficient, require a large number of professionals for long-term fieldwork, and are limited by terrain, season, and transportation conditions, making it difficult to achieve large-scale, high-frequency continuous monitoring; at the same time, medicinal plants that are elusive, inaccessible to humans, or lack distinctive features outside their flowering period are often overlooked; and they are somewhat destructive to habitats and species, which is detrimental to species conservation. Remote sensing image surveys are mainly used to obtain general information on vegetation distribution at a certain scale, involving a limited number of species, and their resolution is highly dependent on professional experience. Traditional species monographs or catalogues cannot reflect the constantly updated dynamic patterns of species diversity. Therefore, developing a new survey method that is low-cost, non-destructive, suitable for long-term dynamic monitoring, and can reflect the dominant regional medicinal plant groups is of positive significance for the development, protection, and sustainable utilization of medicinal plant resources.

[0004] eDNA (environmental DNA) macrobarcoding technology extracts biological DNA fragments from environmental media and combines them with high-throughput sequencing and species comparison to achieve rapid, sensitive, and non-destructive detection of biological species. It has been successfully applied in fields such as freshwater and marine aquatic biodiversity monitoring and soil microbial community surveys. However, there are few reports on the use of this technology in systematic and standardized surveys of regional medicinal plant diversity.

[0005] In eDNA macrobarcoding technology, the screening of DNA barcodes and the design of specific primers are crucial for accurate species identification. Because different plant groups or plants from different geographical regions differ in their genetic composition, the discriminative power and species specificity of the same set of barcodes and primers often vary. Therefore, it is essential to screen suitable barcodes and design or optimize corresponding primer sequences for the target group in the target region. Currently, the detection of eDNA macrobarcodes for microorganisms (bacteria, fungi, etc.) in the atmospheric environment is relatively mature, often using 16S rRNA or ITS fragments. In contrast, the detection of DNA barcodes for atmospheric plants still faces significant challenges. Furthermore, the atmospheric plant DNA barcoding primers reported in existing literature are all designed for wide-area use and have poor adaptability to the characteristics of regional flora components. They generally have two major technical problems: (1) Low amplification efficiency: There are base mismatches between the universal primers and the template DNA binding sites of regional plants and medicinal plants, resulting in low amplification success rate and some medicinal plant DNA cannot be effectively amplified; (2) Serious non-specific amplification: Universal primers are prone to amplifying the DNA of non-target organisms such as fungi and bacteria in the atmosphere, resulting in a low proportion of effective sequences and further reducing the detection sensitivity of medicinal plants. Summary of the Invention

[0006] To overcome the problems existing in the background technology, this invention provides a regional medicinal plant diversity survey method based on atmospheric eDNA macrobarcoding technology. This method, based on atmospheric eDNA macrobarcoding technology, establishes a low-cost, non-destructive, year-round, full-cycle dynamic monitoring method for medicinal plant diversity survey through regionally adapted primer optimization and standardized operating procedures. It solves the problems of high cost, low efficiency, strong destructiveness, inability to conduct long-term dynamic monitoring, and lack of relevant standardized medicinal plant survey procedures in traditional medicinal plant survey methods. This provides a standardized technical solution and scientific basis for the long-term dynamic monitoring, protection, and utilization of medicinal plant resources.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] The method for surveying regional medicinal plant diversity based on atmospheric eDNA macrobarcoding technology includes the following steps:

[0009] S1 Atmospheric plant tissue collection: Sampling points are set up in the target survey area to collect plant tissue samples from the atmosphere;

[0010] S2 Total DNA Extraction: Genomic DNA was extracted from the collected atmospheric plant tissue samples. The integrity and purity of the DNA were tested, and the samples were kept for use after passing quality control.

[0011] S3 Region Adaptive Primer Combination PCR Amplification: Using the total DNA extracted from the sample in step S2 as a template, the four DNA barcodes rbcL, ITS2, trnL (c / h) and trnL (g / h) were independently amplified by PCR. After the amplification products were purified and accurately quantified, they were mixed in equimolar proportions to construct a sequencing library.

[0012] Among them, the rbcL primer pair has the forward primer sequence shown in SEQ ID NO.1 and the reverse primer sequence shown in SEQ ID NO.2; the ITS2 primer pair has the forward primer sequence shown in SEQ ID NO.3 and the reverse primer sequence shown in SEQ ID NO.4.

[0013] S4 High-throughput sequencing and sequence preprocessing: Perform paired-end high-throughput sequencing on the sequencing library obtained in step S3 to obtain the raw sequencing sequence; perform data quality control and splicing, sequence denoising and dechimerism processing on the raw sequence to obtain high-quality effective sequence;

[0014] S5 Species Classification and Annotation: The high-quality valid sequences obtained in step S4 are clustered by OTU with a similarity of 97%. The representative sequences obtained from the clustering are compared with the local plant reference database to complete the species annotation. After species screening, the list of valid seed plant species in the atmosphere of the survey area and the detection information are obtained.

[0015] Furthermore, the survey method also includes the following steps:

[0016] S6 Medicinal Plant Dataset Construction: Combining botanical classics and professional databases, the plant species list obtained in step S5 was checked one by one, and species with reported medicinal value were selected to construct a medicinal plant dataset for the survey area.

[0017] S7 Medicinal Plant Diversity System Analysis: Based on the medicinal plant dataset from step S6, conduct multi-dimensional diversity characteristic analysis to complete the regional medicinal plant diversity survey.

[0018] Furthermore, in step S1, an active sampling method using a pollen trap and volumetric sampling is employed to collect plant tissue samples from the atmosphere; a Burkard pollen trap is used to actively draw in air to collect bioaerosols and plant tissue fragments from the atmosphere; centrifuge tubes containing bioaerosols are periodically retrieved from the sampler and replaced with new tubes in a timely manner to achieve continuous sampling.

[0019] Furthermore, in step S2, total DNA is extracted from the sample using the DP302-02 plant genome extraction kit, and the integrity and purity of the DNA are detected by 2% agarose gel electrophoresis to ensure that the DNA quality meets the requirements for subsequent PCR amplification.

[0020] Furthermore, in step S3,

[0021] The trnL (c / h) primer pair has the forward primer sequence shown in SEQ ID NO.5 and the reverse primer sequence shown in SEQ ID NO.6; the trnL (g / h) primer pair has the forward primer sequence shown in SEQ ID NO.7 and the reverse primer sequence shown in SEQ ID NO.6.

[0022] Furthermore, in step S3, the PCR amplification reaction system is as follows: 4 μL of 5×FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL each of 5 μM forward and reverse primers, 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and sterile ddH2O to a final volume of 20 μL.

[0023] The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 45 s; 35 cycles were set for the rbcL and trnL (c / h) primer pairs, and 30 cycles were set for the ITS2 and trnL (g / h) primer pairs; after the cycles, a final extension was set for 10 min at 72℃, followed by incubation at 4℃; three technical replicates were set for each sample; the amplification products were detected by 2% agarose gel electrophoresis, and the gels were extracted and recovered using the AxyPrep DNA gel extraction kit. After precise quantification using a QuantiFluor™-ST fluorometer, the products were mixed in equimolar proportions.

[0024] Furthermore, in step S4, high-throughput sequencing was performed using the Illumina NovaSeq sequencing platform, and the specific steps for sequence preprocessing were as follows:

[0025] (1) Based on the barcode, identify the sample, perform quality filtering on the original sequence to remove low-quality bases and short sequences, and splice the sequences based on the overlapping regions between PE reads to obtain high-quality sequences of a single sample;

[0026] (2) The sequence was corrected using Usearch software. By comparing it with the Gold database, chimeric sequences were removed by combining de novo and reference, and finally high-quality effective sequences were obtained.

[0027] Furthermore, in step S5, the OTU clustering is to perform operational classification unit clustering on high-quality sequences with 97% similarity to obtain OTU representative sequences and OTU tables; the species classification annotation adopts the uclust algorithm and blastn alignment method, and the alignment databases include UNITE, SILVA, PR2 and NT databases;

[0028] The species screening process is as follows: First, species annotated by OTUs with fewer than 10 reads in each month are removed. Second, based on the iplant plant intelligence database and the updated versions of botanical monographs and databases such as the "List of Seed Plants in Central Yunnan", the coverage area of ​​various plant tissue fragments scattered in the atmosphere is delineated with the sampling point as the center and a radius of 200km. Seed plants that are naturally distributed in this area and are the main source of medicinal plants are selected as effective species.

[0029] The principle for integrating species detection information is that if a species is detected by at least one of the four DNA barcodes of the same sample from the same sampling month, it is considered a valid species detected at that sampling point in that month.

[0030] Furthermore, in step S6, the medicinal plant classics and professional databases include the Pharmacopoeia of the People's Republic of China, Chinese Materia Medica, Yunnan Materia Medica, Flora of China, Flora of China, China Pharmaceutical Information Query Platform, and Plant Intelligence Medicinal Plant Database; the screening criterion is that species whose medicinal efficacy and medicinal value are clearly recorded in the above classics and databases are all included in the medicinal plant dataset.

[0031] Furthermore, in step S7, the multidimensional diversity feature analysis includes species composition feature analysis, pharmacognosy feature analysis, and seasonal dynamic feature analysis.

[0032] The beneficial effects of this invention are:

[0033] (1) This invention uses atmospheric eDNA macro barcoding technology to conduct diversity surveys of regional medicinal plants. By constructing four highly adaptable DNA barcodes and independently PCR amplifying the four DNA barcodes, the invention achieves effective detection of regional dominant medicinal plants using eDNA macro barcoding technology.

[0034] (2) This invention significantly improves the amplification efficiency, detection stability and detection sensitivity of regional plants in Kunming City by optimizing the regional adaptability of four DNA barcode primers, and achieves accurate identification of species. Through year-round monitoring at only two sampling points, 302 species of medicinal plants can be stably detected, covering the core medicinal groups such as Asteraceae, Fabaceae and Amaranthaceae in the region. It also effectively makes up for the shortcomings of traditional surveys in detecting species that are not in bloom or grow in seclusion. It can reflect the composition characteristics of regional dominant medicinal plant resources more stably.

[0035] (3) This invention constructs a standardized method for the entire process from sample collection, eDNA extraction, amplification and sequencing to data analysis. It is not limited by the terrain in the field or the non-flowering period of plants. It can accurately analyze the seasonal dynamic changes of regional dominant medicinal plant species through continuous monthly sampling. The entire process is standardized and can realize dynamic monitoring throughout the year and the entire cycle. It makes up for the shortcomings of traditional surveys that can only realize single or low-frequency static surveys. It provides a replicable and scalable technical solution for long-term dynamic monitoring and trend analysis of regional medicinal plant resources.

[0036] (4) This invention can not only clarify the species composition of regional dominant medicinal plants, but also systematically analyze their efficacy categories, medicinal parts, life forms and other core pharmacognosy characteristics. The survey results can provide basic data and scientific basis for the protection planning, sustainable utilization and germplasm resource monitoring of regional medicinal plant resources. At the same time, it can provide basic data on medicinal plant groups for air pollen allergy risk assessment.

[0037] (5) This invention completes the survey of medicinal plant diversity by collecting plant tissue samples in the atmosphere. It does not require field sampling and plant destruction, avoids the interference of traditional surveys on wild plant populations and native habitats, saves manpower and material costs, avoids environmentally destructive surveys, and realizes environmentally friendly monitoring of wild medicinal plant resources. Attached Figure Description

[0038] Figure 1 This is a flowchart illustrating the regional medicinal plant diversity survey method based on atmospheric eDNA macrobarcoding technology of the present invention.

[0039] Figure 2 Venn diagram showing the shared medicinal plants at the Kunming Chenggong sampling site (CG) and the Pingzheng sampling site (PZ) in the example;

[0040] Figure 3 This is a bar chart comparing the distribution of dominant medicinal plant families in CG and PZ regions in the example.

[0041] Figure 4 This is a comparative diagram of the distribution of medicinal plant efficacy categories in CG and PZ regions, as shown in the example.

[0042] Figure 5 This is a comparison diagram of the distribution of medicinal parts of medicinal plants in CG and PZ regions in the example;

[0043] Figure 6 This is a comparison diagram of the distribution of medicinal plant life forms in CG and PZ locations in the example;

[0044] Figure 7 The example shows a seasonal dynamic line graph of the number of medicinal plant species detected monthly in pollen from CG and PZ locations. Detailed Implementation

[0045] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0046] To illustrate the present invention more clearly, the following embodiments will be described in detail.

[0047] Example 1

[0048] This embodiment uses Kunming City, Yunnan Province as the target survey area, and sets up two sampling points: ① Kunming Medical University Chenggong Campus (CG, E 102°49'41'', N24°50'48'', a new urban area, representing a low-to-medium intensity urbanization environment); ② Pingzheng Campus (PZ, E 102°42'36'', N25°3'7'', a core urban area, representing a high-intensity urbanization environment). A systematic survey of medicinal plant diversity in Kunming City is conducted based on the method described in this invention. The specific implementation steps are as follows:

[0049] 1. Atmospheric plant tissue collection

[0050] Both sampling sites avoided industrial pollution sources and areas with dense vegetation. A Burkard pollen trap, a high-flow-rate airborne particulate matter sampler, was placed on the rooftop with a sampling flow rate of 16.5 L / min. Every 30 days, 1.5 mL centrifuge tubes collecting bioaerosols from the sampler were collected and replaced with new tubes to ensure continuous sampling. Three biological replicates were set up for each sampling site. The sampling period was from December 2023 to November 2024, covering the entire calendar year. Sampling was conducted on the 30th of each month. In case of extreme weather such as heavy rain or strong winds, the sampling time was postponed by 1 to 2 days to ensure the consistency of the sampling environment. After sampling, the centrifuge tubes were immediately placed in a -80°C ultra-low temperature freezer and stored in the dark until eDNA extraction to prevent nucleic acid degradation.

[0051] 2. Extraction of total DNA from pollen

[0052] All operations were performed in a sterile laminar flow hood, with blank centrifuge tubes used as negative controls throughout the process to eliminate external environmental contamination.

[0053] Genomic DNA was extracted from samples stored at -80℃ using the DP302-02 Plant Genome Extraction Kit. After extraction, the integrity and purity of the DNA were assessed using 2% agarose gel electrophoresis to ensure that the DNA quality met the requirements for subsequent PCR amplification.

[0054] 3. Optimization of DNA barcoding primers adapted to the Kunming region and PCR amplification

[0055] rbcL, ITS2, trnL(c / h), and trnL(g / h) were used as marker genes, and PCR amplification was performed to construct a comparison database.

[0056] The four DNA barcodes selected in this invention include: chloroplast coding gene rbcL, ribosomal nuclear gene ITS2, and chloroplast non-coding regions trnL (c / h) and trnL (g / h). Among them, rbcL and ITS2 are internationally recognized plant DNA barcodes with high species resolution, but they are also fragments with low primer specificity and amplification rate for plants in the Kunming region, thus serving as core optimization targets; trnL (c / h) and trnL (g / h) are conserved non-coding fragments with good stability for amplifying degraded biological (pollen) aerosol DNA.

[0057] In this embodiment, four DNA barcoding primer pairs optimized for plants in the Kunming region according to the present invention were used for independent PCR amplification. The primer sequences correspond to SEQ ID NO.1-7, as follows:

[0058]

[0059] The rbcL forward primer inserts the base CA, resulting in high binding efficiency with plant templates from the Kunming region, effectively improving the binding specificity and amplification efficiency of the primer with Kunming plants. The ITS2 reverse primer inserts the base A, reducing primer mismatch rate and improving the amplification success rate and coverage of species from the Kunming region.

[0060] All primers had adapter sequences adapted for the Illumina sequencing platform and sample-specific barcode sequences added to their 5' ends. PCR reactions were performed using TransStart Fastpfu DNA Polymerase on an ABI GeneAmp® 9700 PCR instrument, with three technical replicates per sample.

[0061] PCR amplification was performed using a standardized 20 μL reaction system: 4 μL of 5×FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL each of 5 μM forward and reverse primers, 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and sterile ddH2O to a final volume of 20 μL.

[0062] PCR amplification reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 45 s; rbcL and trnL (c / h) primer pairs were set for 35 cycles, and ITS2 and trnL (g / h) primer pairs were set for 30 cycles; after the cycles, the final extension was set at 72℃ for 10 min, and then incubated at 4℃.

[0063] Three technical replicates were set up for each sample. After the PCR products of the three technical replicates of the same sample were thoroughly mixed, the amplification effect was detected by 2% agarose gel electrophoresis. The target fragment was recovered using the AxyPrep DNA gel recovery kit. The recovered products were accurately quantified using a QuantiFluor™-ST fluorometer and mixed in equimolar proportions for library construction.

[0064] The main steps for constructing a PE300 sequencing library using the above-mentioned mixed PCR products include: Y-shaped adapter ligation, magnetic bead screening to remove adapter self-ligation fragments, PCR amplification to enrich the library template, and sodium hydroxide denaturation to generate single-stranded DNA.

[0065] 4. High-throughput sequencing and sequence preprocessing

[0066] The final constructed library was sequenced using the Illumina NovaSeq sequencing platform with paired-end (PE) sequencing to obtain the raw sequencing sequences. The raw sequences obtained from sequencing were then normalized, specifically through the following steps:

[0067] (1) Data quality control and splicing: Based on the barcode identification sample, the original sequence is filtered for quality, low-quality bases and short sequences are filtered out, and splicing is performed based on the overlapping area between PE reads to obtain a high-quality sequence of a single sample;

[0068] (2) Sequence denoising and chimera removal: The sequences were corrected using Usearch software and chimera sequences were removed by combining denovo and reference methods by comparing with the gold database to obtain high-quality effective sequences;

[0069] (3) OTU clustering and generation: high-quality sequences are clustered into operational classification units with a similarity of 97% to obtain OTU representative sequences, and all sequences are mapped to the representative sequences to generate OTU tables.

[0070] (4) Species annotation: Using the uclust algorithm and blastn alignment method, the OTU representative sequence was compared with databases such as UNITE, SILVA, PR2 and NT to annotate the pollen source plants at multiple taxonomic levels.

[0071] 5. Species Classification Notes

[0072] To ensure the accuracy of the annotation results, the initial annotation results were screened in two steps: First, species annotated by OTUs with fewer than 10 reads in each month were removed to reduce interference from low-abundance and random sequences. Second, based on the iPlant plant intelligence database and botanical monographs, such as "A Checklist of Seed Plants in Central Yunnan" (edited by Wang Huanchong, Yang Feng, and Zhang Rongzhen, Beijing: Science Press, June 2022, ISBN 978-7-03-058236-2), the natural distribution of candidate species was verified. The study area was delineated with Kunming City as the center and a radius of 200 km, and finally, seed plants naturally distributed within this area were selected as valid species.

[0073] The principle for integrating species detection information is as follows: for the same sample from the same sampling month, a species detected by at least one of the four DNA barcodes is considered a valid detected species from that sampling point in that month. A total of 517 species of higher plants were detected.

[0074] 6. Construction of medicinal plant dataset

[0075] Based on the "Pharmacopoeia of the People's Republic of China (2020 Edition, Part I)," "Chinese Materia Medica," "Yunnan Materia Medica," "Flora of China," the China Pharmaceutical Information Query Platform, and the Plant Intelligence Medicinal Plant Database, the medicinal properties of the 517 plants detected were verified one by one. All species whose medicinal efficacy and medicinal value are clearly recorded in the above authoritative classics and databases were included in the medicinal plant dataset.

[0076] The final Kunming medicinal plant dataset includes 302 species belonging to 99 families and 231 genera. Of these, 249 species were detected at the CG (Cumulative Graphite) point and 230 at the PZ (Polygonal Graphite) point, totaling 177 species, accounting for 58.6% of the total. 72 species are unique to the CG point, and 53 species are unique to the PZ point. Furthermore, the medicinal efficacy, medicinal parts, and life forms of all included species were standardized and organized to form a complete dataset of medicinal plant attributes.

[0077] 7. Systematic analysis of medicinal plant diversity

[0078] Based on the constructed medicinal plant dataset, a multi-dimensional diversity feature analysis was conducted to complete the medicinal plant diversity survey in Kunming City. The specific analysis results are as follows:

[0079] (1) Analysis of species composition characteristics

[0080] The common and unique distribution of medicinal plants in the two regions are as follows: Figure 2As shown in the figure, the medicinal plant resources within the study area exhibit high levels of sharing, but also some regional differences. The distribution of families and genera of medicinal plants detected in the atmosphere of Kunming is highly concentrated. The top 10 dominant families contain 204 species of medicinal plants, accounting for 67.5% of the total species. The distribution of dominant families is shown in the figure below. Figure 3 As shown; among them, the Asteraceae family is the absolute dominant family shared by both places, with a total of 56 species detected in both places, followed by the Fabaceae family (28 species in total) and the Amaranthaceae family (25 species in total). These families are not only the dominant families of the airborne plant community in Kunming, but also the core enrichment groups of regional medicinal plants as verified by monographs and data.

[0081] (2) Analysis of pharmacognosy characteristics

[0082] According to the classification system of Chinese medicine efficacy, medicinal plants are categorized by efficacy, including heat-clearing drugs, diuretic and dampness-removing drugs, wind-dampness-dispelling drugs, and blood-activating and stasis-removing drugs. Based on traditional Chinese medicine usage habits, the medicinal parts of medicinal plants are classified and statistically analyzed, including roots and rhizomes, fruits and seeds, whole herbs, leaves, stems and woody parts, flowers, and barks. Simultaneously, the life-form composition of medicinal plants is statistically analyzed, including four categories: herbs, shrubs, trees, and vines, comprehensively interpreting the pharmacognosy characteristics of medicinal plants.

[0083] The distribution of medicinal plant efficacy categories in the two regions is as follows: Figure 4 As shown in the figure, it can be seen that the distribution of medicinal plant efficacy in the two regions is highly consistent, with heat-clearing drugs being the absolute dominant type (69 species at CG point and 68 species at PZ point), followed by diuretics and rheumatoid drugs. This survey result is consistent with the survey results of medicinal seed plants in Kunming by Yang Guansong et al. (Yang Guansong, Liu Xinran, Yang Chuangfeng. Study on medicinal seed plant resources and diversity in Kunming, Yunnan Province [J]. Anhui Agricultural Sciences, 2021, 49(14): 162-165.), revealing that this distribution pattern is highly consistent with the overall distribution trend of medicinal plant efficacy in Yunnan Province as investigated through field surveys or monograph cataloging.

[0084] The distribution of medicinal parts of medicinal plants in the two regions is as follows: Figure 5 As shown in the figure, the medicinal parts of the plants used in the two regions are mainly whole herbs, fruits and seeds, and roots and rhizomes, accounting for more than 70% in total, covering the main types of medicinal parts in traditional Chinese medicine.

[0085] Distribution of life forms of medicinal plants in the two regions as follows Figure 6As shown in the figure, it can be seen that the medicinal plants in both regions are mainly herbaceous plants, accounting for more than 50%, followed by shrubs and trees, with vines accounting for the lowest proportion. This is consistent with the distribution characteristics of medicinal plant life forms reported in Kunming (Yang Guansong et al. Study on medicinal seed plant resources and diversity in Kunming, Yunnan Province [J]. Anhui Agricultural Sciences, 2021, 49(14): 162-165.).

[0086] (3) Analysis of seasonal dynamic characteristics

[0087] Based on monthly sampling data throughout the year, the number of medicinal plant species detected in different months was counted, the seasonal variation pattern of species richness was analyzed, and the annual occurrence characteristics of medicinal plant tissues in the atmosphere were clarified, providing data support for regional dynamic monitoring of flowering period of medicinal plants and resource assessment.

[0088] Seasonal dynamics of the number of medicinal plant species detected monthly in the two locations, as follows: Figure 7 As shown in the figure, it can be clearly seen that the number of medicinal plant species detected in the two regions exhibits a significant seasonal fluctuation pattern: the number of species remains at a high level in winter (December to February of the following year), reaches its peak in spring (March to May), falls back to a low to medium level in summer, drops to its lowest level in autumn (September), and slowly recovers in October and November, thus clarifying the annual occurrence characteristics of medicinal plant pollen in the Kunming area.

[0089] This embodiment verifies the feasibility and effectiveness of the method of the present invention from multiple dimensions. By combining atmospheric eDNA macrobarcoding technology with four DNA barcodes specific to the Kunming region, a non-destructive dynamic survey of the diversity of medicinal plants in Kunming City was achieved. The species composition, pharmacognosy characteristics and seasonal dynamics of regional dominant medicinal plants were obtained, providing comprehensive basic data and scientific support for the protection, monitoring and sustainable utilization of important medicinal plant resources in the Kunming area.

[0090] This invention focuses on achieving seasonal dynamic tracking of regional dominant medicinal plant groups, rather than pursuing complete coverage of all species (such as rare species). It makes up for the shortcomings of traditional surveys in terms of temporal continuity, cost controllability, and monitoring repeatability, and provides technical support for long-term dynamic monitoring, protection decision-making, and sustainable utilization of regional medicinal plant resources.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for surveying regional medicinal plant diversity based on atmospheric eDNA macrobarcoding technology, characterized in that, Includes the following steps, S1 Atmospheric plant tissue collection: Sampling points are set up in the target survey area to collect plant tissue samples from the atmosphere; S2 Total DNA Extraction: Genomic DNA is extracted from collected atmospheric plant tissue samples. The integrity and purity of the DNA are tested, and the samples are kept for use after passing quality control. S3 region-adaptive primer combination PCR amplification: using the total DNA extracted from the sample in step S2 as a template, the four DNA barcodes rbcL, ITS2, trnL (c / h) and trnL (g / h) were independently amplified by PCR. After purification and precise quantification of the amplification products, they were mixed in equimolar proportions to construct a sequencing library. Among them, the rbcL primer pair has the forward primer sequence shown in SEQ ID NO.1 and the reverse primer sequence shown in SEQ ID NO.2; the ITS2 primer pair has the forward primer sequence shown in SEQ ID NO.3 and the reverse primer sequence shown in SEQ ID NO.

4. S4 High-throughput sequencing and sequence preprocessing: The sequencing library obtained in step S3 is subjected to paired-end high-throughput sequencing to obtain the raw sequencing sequence; the raw sequence is subjected to data quality control and splicing, sequence denoising and dechimerism processing to obtain high-quality effective sequences. S5 Species classification and annotation: The high-quality valid sequences obtained in step S4 are clustered by OTU with a similarity of 97%. The representative sequences obtained from the clustering are compared with the local plant reference database to complete the species annotation. After species screening, the list of valid seed plant species in the atmosphere of the survey area and the detection information are obtained.

2. The survey method according to claim 1, characterized in that, It also includes the following steps, S6 Medicinal Plant Dataset Construction: Combining botanical classics and professional databases, the plant species list obtained in step S5 was checked one by one, and species with reported medicinal value were selected to construct a medicinal plant dataset for the survey area. S7 Medicinal Plant Diversity System Analysis: Based on the medicinal plant dataset from step S6, conduct multi-dimensional diversity characteristic analysis to complete the regional medicinal plant diversity survey.

3. The survey method according to claim 1 or 2, characterized in that, In step S1, an active sampling method using a pollen trap and volumetric sampling is employed to collect plant tissue samples from the atmosphere. A Burkard pollen trap is used to actively draw in air to collect bioaerosols and plant tissue fragments from the atmosphere. Centrifuge tubes containing bioaerosols are periodically retrieved from the sampler and replaced with new tubes in a timely manner to achieve continuous sampling.

4. The survey method according to claim 1 or 2, characterized in that, In step S2, total DNA was extracted from the sample using the DP302-02 plant genome extraction kit, and the integrity and purity of the DNA were detected by 2% agarose gel electrophoresis to ensure that the DNA quality met the requirements for subsequent PCR amplification.

5. The survey method according to claim 1 or 2, characterized in that, In step S3, The trnL (c / h) primer pair has the forward primer sequence shown in SEQ ID NO.5 and the reverse primer sequence shown in SEQ ID NO.

6. The trnL (g / h) primer pair has the forward primer sequence shown in SEQ ID NO.7 and the reverse primer sequence shown in SEQ ID NO.

6.

6. The survey method according to claim 5, characterized in that, In step S3, the PCR amplification reaction system is as follows: 4 μL of 5×FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL each of 5 μM forward and reverse primers, 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and sterile ddH2O to a final volume of 20 μL. The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 45 s; 35 cycles were set for the rbcL and trnL (c / h) primer pairs, and 30 cycles were set for the ITS2 and trnL (g / h) primer pairs; after the cycles, a final extension was set for 10 min at 72℃, followed by incubation at 4℃; three technical replicates were set for each sample; the amplification products were detected by 2% agarose gel electrophoresis, and the gels were extracted and recovered using the AxyPrep DNA gel extraction kit. After precise quantification using a QuantiFluor™-ST fluorometer, the products were mixed in equimolar proportions.

7. The survey method according to claim 1 or 2, characterized in that, In step S4, high-throughput sequencing was performed using the Illumina NovaSeq sequencing platform. The specific steps for sequence preprocessing were as follows: (1) Based on the barcode, identify the sample, perform quality filtering on the original sequence to remove low-quality bases and short sequences, and splice the sequences based on the overlapping regions between PE reads to obtain high-quality sequences of a single sample; (2) The sequence was corrected using Usearch software. By comparing it with the Gold database, chimeric sequences were removed by combining de novo and reference, and finally high-quality effective sequences were obtained.

8. The survey method according to claim 1 or 2, characterized in that, In step S5, the OTU clustering is to cluster high-quality sequences into operational taxonomic units based on 97% similarity to obtain OTU representative sequences and OTU tables; the species classification annotation uses the uclust algorithm and blastn alignment method, and the alignment databases include UNITE, SILVA, PR2 and NT databases; The species screening process is as follows: First, species annotated by OTUs with fewer than 10 reads in each month are removed. Second, based on the iplant plant intelligence database and the updated versions of botanical monographs and databases such as the "List of Seed Plants in Central Yunnan", the coverage area of ​​various plant tissue fragments scattered in the atmosphere is delineated with the sampling point as the center and a radius of 200km. Seed plants that are naturally distributed in this area and are the main source of medicinal plants are selected as effective species. The principle for integrating species detection information is that if a species is detected by at least one of the four DNA barcodes of the same sample from the same sampling month, it is considered a valid species detected at that sampling point in that month.

9. The survey method according to claim 2, characterized in that, In step S6, the medicinal plant classics and professional databases include the Pharmacopoeia of the People's Republic of China, Chinese Materia Medica, Yunnan Materia Medica, Flora of China, Flora of China, China Pharmaceutical Information Query Platform, and Plant Intelligence Medicinal Plant Database; the screening criterion is that species whose medicinal efficacy and medicinal value are clearly recorded in the above classics and databases are all included in the medicinal plant dataset.

10. The survey method according to claim 9, characterized in that, In step S7, the multidimensional diversity feature analysis includes species composition feature analysis, pharmacognosy feature analysis, and seasonal dynamic feature analysis.

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