Environmental DNA macro bar code sequencing method for evaluating influence of transgenic plant on farmland biodiversity

Abstract

The invention relates to an environmental DNA macro bar code sequencing method for evaluating the influence of a transgenic plant on farmland biodiversity, which comprises the following steps: collecting samples of root soil and leaf surfaces of the transgenic plant in the same period, and extracting the DNA of the samples; taking the extracted DNA as a template, and carrying out PCR (Polymerase Chain Reaction) amplification through an amplification primer; after a PCR amplification product is purified, high-throughput sequencing is carried out, an OTU sequence obtained in a result is compared with an nt public database of an NCBI website, arthropod species in a sample are obtained, and biodiversity related indexes are calculated, so that the influence of transgenic crops on farmland biodiversity is evaluated. According to the method, the professional requirements and workload of field investigation of biodiversity can be effectively reduced, the variety of identified species is more comprehensive, species classification can also be realized, and the time cost and the economic cost are relatively low.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular ecology, and specifically relates to an environmental DNA metabarcoding sequencing method for evaluating the impact of transgenic plants on farmland biodiversity. Background Art

[0002] The large-scale cultivation of transgenic crops is an important strategy for modern agriculture to address global food security challenges. Insect-resistant transgenic crops (such as Bt crops) indirectly protect biodiversity by reducing the amount of pesticides used. In addition, high-yield transgenic crops may slow down the encroachment of arable land expansion on natural ecosystems by increasing the yield per unit area. However, their ecological safety remains a major concern. Part of this controversy stems from the limitations of traditional ecological methods in biodiversity assessment. Therefore, there is an urgent need for a more accurate and efficient evaluation system.

[0003] Traditional biodiversity survey methods have significant limitations in the ecological impact assessment of transgenic crops. Species statistics rely on professional field collection and taxonomic identification by professionals, with limited spatio-temporal coverage and high costs. It is difficult to capture the dynamic changes of cryptic soil animals and small, highly mobile species, and it is impossible to accurately determine the species and quantity of animal communities. Although existing molecular techniques have improved efficiency, there are generally gene marker selection biases: the success rate of species-level identification of ribosomal RNA genes (such as 16S / 18S rRNA) in the animal kingdom is less than 40%, and most studies only focus on a single ecological niche (such as soil or air samples) to comprehensively evaluate the impact of transgenic crops on biodiversity. These deficiencies make it difficult for traditional methods to meet the needs of multi-interface and multi-taxa synchronous monitoring, and also impossible to distinguish the response differences of closely related species and cryptic species to transgenic crops. Summary of the Invention

[0004] The purpose of the present invention is to provide an environmental DNA metabarcoding sequencing method for evaluating the impact of transgenic plants on farmland biodiversity in order to solve the above problems.

[0005] The present invention achieves the above purpose through the following technical solutions:

[0006] An environmental DNA metabarcoding sequencing method for evaluating the impact of transgenic plants on farmland biodiversity, comprising the following steps:

[0007] Collect samples from the root zone soil and leaf surface of transgenic crops at the same time, and extract the DNA of the samples;

[0008] Using the extracted DNA as a template, perform PCR amplification with amplification primers;

[0009] After purifying the PCR amplification products, high-throughput sequencing was performed. The obtained OTU sequences in the results were compared with the nt public database on the NCBI website to obtain the arthropod species in the samples and calculate the biodiversity-related indices, so as to evaluate the impact of genetically modified crops on farmland biodiversity.

[0010] As a further optimization scheme of the present invention, the genetically modified crops are genetically modified corn or genetically modified soybeans.

[0011] As a further optimization scheme of the present invention, the genetically modified corn is DBN9229, and the genetically modified soybean is DBN9004×DBN8002×DBN8205.

[0012] As a further optimization scheme of the present invention, the soil samples of the genetically modified crops are collected as follows: after the genetically modified crops emerge, diagonal five-point sampling is used. At each sampling point, the floating matter on the soil surface layer is removed, and the rhizosphere soil samples with a depth of 2-15 cm are taken. The soil samples at the five sampling points are mixed and used for DNA extraction;

[0013] The leaf surface samples of the genetically modified crops are collected as follows: when taking the soil samples, one leaf from the upper, middle, and lower parts of the crop plants is taken. The leaves are thoroughly washed with ultrapure water, and vacuum filtration is carried out using a vacuum filter and a microporous filter membrane with a pore size of 0.22 μm. After the filter membrane is cut into pieces, it is used for DNA extraction.

[0014] As a further optimization scheme of the present invention, use Spin Kit for Soil kit to extract the DNA of the samples.

[0015] As a further optimization scheme of the present invention, the amplification primers are the upstream primer mlCOIintF and the downstream primer jgHCO2198R of the mitochondrial cytochrome oxidase COI gene, and the nucleotide sequences of the amplification primers are:

[0016] SEQ ID NO.1: Upstream primer mlCOIintF: GGWACWGGWTGAACWGTWT AYCCYCC;

[0017] SEQ ID NO.2: Downstream primer jgHCO2198R: TAAACTTCAGGGTGACCAA ARAAYCA.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1) The present invention collects samples of root zone soil and leaf surfaces of genetically modified crops at the same stage, extracts the DNA of the samples, performs PCR amplification on the samples using the mitochondrial cytochrome oxidase COI gene, purifies the amplification products, performs high-throughput sequencing, compares the obtained OTU sequences in the results with the nt public database on the NCBI website, obtains the types and relative abundances of animals in the sampled samples, calculates biodiversity-related indices, thereby evaluating the impact of genetically modified crops on farmland biodiversity, which can effectively reduce the professional requirements and workload of biodiversity field surveys, and can identify more species more comprehensively and specifically down to species classification, with lower time costs and economic costs.

[0020] 2) The present invention synchronously collects root zone soil samples and phyllosphere samples, combines the amplification of the 313bp mitochondrial COI hypervariable region and the combined annotation of multiple databases to achieve high-precision detection of multi-trophic level organisms such as soil springtails and phyllosphere pollinating insects. This technical system has created a new paradigm for the ecological risk assessment of genetically modified crops, can provide high-precision data support for the environmental release decision-making of genetically modified crops, has established a methodological benchmark for the sustainable management of agricultural ecosystems, and promoted the leap of biodiversity protection from macroscopic observation to molecular-scale detection. Description of the Drawings

[0021] Figure 1 It is a table showing the results of the investigation of the types of arthropods in the fields of the tested soybean and corn materials by the direct observation method.

[0022] Figure 2 It is a table showing the results of the investigation of the types of arthropods in the fields of the tested soybean and corn materials by the high-throughput sequencing method.

[0023] Figure 3 It is the clustering situation of all biological species in corn and soybean fields (high-throughput sequencing method).

[0024] Figure 4 It is the clustering analysis of corn and soybean sample levels (family level) (high-throughput sequencing method).

[0025] Figure 5 It is the arthropod diversity index in the fields of different corn materials (high-throughput sequencing method).

[0026] Figure 6 It is the arthropod diversity index in the fields of different soybean materials (high-throughput sequencing method).

[0027] Figure 7 It is the simpson index of arthropods in the fields of different corn materials (high-throughput sequencing method).

[0028] Figure 8 It is the simpson index of arthropods in the fields of different soybean materials (high-throughput sequencing method).

[0029] Figure 9 is the uniformity index of arthropods in the field of different maize materials (high-throughput sequencing method).

[0030] Figure 10 is the uniformity index of arthropods in the field of different soybean materials (high-throughput sequencing method). Detailed implementation manners

[0031] The present application will be further described in detail below with reference to the accompanying drawings. It is necessary to point out here that the following specific implementation manners are only used to further illustrate the present application and cannot be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0032] I. Materials

[0033] 1. The transgenic maize DBN9229 and the non-transgenic control maize DBN567 are both provided by Beijing Dabeinong Biotechnology Co., Ltd., and the quality of the above materials meets the requirements of not less than second-class maize seeds in GB 4404.1;

[0034] 2. The transgenic soybean DBN9004×DBN8002×DBN8205 and the non-transgenic receptor JACK are both provided by Beijing Dabeinong Biotechnology Co., Ltd.;

[0035] 3. Basta, with the active ingredient glufosinate-ammonium, a content of 18%, a dosage form of soluble concentrate, produced by Bayer CropScience, and the origin is Australia.

[0036] The methods used in this example are all conventional methods known to those skilled in the art unless otherwise specified. The reagents and other materials used are all commercially available products unless otherwise specified.

[0037] II. Methods

[0038] 2.1 Experimental design

[0039] Test time: from April 2024 to October 2024; Test location: The base of Beijing Dabeinong Biotechnology Co., Ltd., Sijian Village, Chengxi Town, Lvyuan District, Changchun City, Jilin Province (43°56′12″N, 125°15′07″E).

[0040] 2.1.1. Maize

[0041] The plots are arranged randomly, with a 1.0 m wide isolation belt between plots. The plot area is 150 m 2 (10×15 m) as one repetition, and each treatment has 3 repetitions. A total of 3 treatments are designed in the test:

[0042] Treatment 1: The transgenic line DBN9229 was sprayed with glufosinate-ammonium (600 g a.i. / hm 2 ), and the chemical used was Basta;

[0043] Treatment 2: The transgenic line DBN9229 was weeded manually;

[0044] Treatment 3: The non-transgenic maize variety DBN567 (the recipient variety) was weeded manually.

[0045] Sowing time, sowing method and seeding rate: Sown on April 27th by hill-drop seeding with the local conventional seeding rate, and managed in the conventional way;

[0046] Chemical application time: At the stage of 3 - 4 trifoliolate leaves of the transgenic maize, on June 3rd.

[0047] 2.1.2. Soybean

[0048] The plots were arranged randomly, with a 1.0 m wide isolation belt between plots. Each plot had an area of 150 m 2 (10×15 m) as one replicate, and each treatment had 3 replicates. A total of 3 treatments were designed for the experiment:

[0049] Treatment 1: The transgenic soybean DBN9004×DBN8002×DBN8205 was sprayed with glufosinate-ammonium (600 g a.i. / hm 2 ), and the chemical used was Basta;

[0050] Treatment 2: The transgenic soybean DBN9004×DBN8002×DBN8205 was weeded manually;

[0051] Treatment 3: The non-transgenic soybean variety Jack (the recipient variety) was weeded manually;

[0052] Sowing time, sowing method and seeding rate: Sown on May 8th by hill-drop seeding with the local conventional seeding rate, and managed in the conventional way;

[0053] Chemical application time: At the stage of 3 - 4 trifoliolate leaves of the transgenic soybean, on June 14th.

[0054] 2.2. Investigation of the impact on animal diversity

[0055] 2.2.1. Direct observation method

[0056] (1). Maize materials

[0057] The whole growth period of the transgenic lines and recipient maize varieties sprayed with glufosinate-ammonium and water in the three treatment groups in step 2.1.1 was investigated, using the diagonal 5-point sampling method:

[0058] From emergence to maturity, investigate once every 7 days. Fix 10 maize plants at each point, record the species and developmental stages of all arthropods on the whole maize plant (for aphids and spider mites, record the upper, middle, and lower 3 leaves), and record the quantity and species of the main arthropods including various insects and spiders during the investigation. Number the species that cannot be identified in the field and take them back to the laboratory for identification. When starting the investigation, first quickly observe the quantity of active and easily movable insects / spiders.

[0059] Use four indicators, namely the richness (number of species), diversity index, Simpson index, and evenness index of the arthropod community, to analyze and compare the occurrence and development trends of the insect communities, pest and natural enemy sub-communities in the fields of various maize materials, so as to determine whether the transformants and the spraying of glufosinate on the transformants have an impact on the diversity of arthropods in the field.

[0060] (2), Soybean materials

[0061] Investigate the transformants sprayed with glufosinate and the receptor soybean varieties sprayed with water in the three treatment groups in step 2.1.2 throughout the growth period. Use the diagonal 5-point sampling method:

[0062] From 10 days after thinning to maturity, investigate once every 10 days. Fix 10 soybean plants at each point, record the species and quantity of all arthropods on the whole soybean plant, record the quantity and species of the main arthropods including various insects and spiders during the investigation. Number the species that cannot be identified in the field and take them back to the laboratory for identification. When starting the investigation, first quickly observe the species and quantity of active insects.

[0063] Use four indicators, namely the richness (number of species), diversity index, Simpson index, and evenness index of the arthropod community, to analyze and compare the occurrence and development trends of the insect communities, pest and natural enemy sub-communities in the fields of various soybean materials, so as to determine whether the transgenic soybeans and the spraying of glufosinate on the transgenic soybeans have an impact on the diversity of arthropods in the field.

[0064] 2.2.2, High-throughput sequencing method

[0065] (1), Sample collection and processing

[0066] Sampling objects: Soils and leaves of all treatments and replicates for evaluating the impact on biodiversity in the 6 treatment groups in steps 2.1.1 and 2.2.2.

[0067] Sampling time: Sample once every 1 month starting from after crop emergence.

[0068] Sampling quantity: For each plot, 5 random sampling points are taken diagonally and mixed into 1 sample. When taking soil samples, remove the floating matter on the soil surface, use a soil sampler to take about 200 g of rhizosphere soil layer with a depth of 2 - 15 cm (about 1 kg in total for 5 points in each plot), place it in a sterile self-sealing bag, and mark the material name, number of replicates, and sampling time.

[0069] When taking soil samples, beside each sampling point, take 1 upper, middle, and lower leaf from 1 soybean plant (15 leaves in total for 5 points in each plot), and take 1 upper, middle, and lower leaf from 1 corn plant (15 leaves in total for 5 points in each plot), place them in a sterile self-sealing bag, and mark the information as shown in Table 1:

[0070] Table 1 Sample marking information table

[0071]

[0072]

[0073] Sample treatment: Pour 200 ml of ultrapure water into a sterile self-sealing bag for each replicate of leaf samples, wash the leaves thoroughly, use a vacuum filter (JOANLAB) and a microporous membrane with a pore size of 0.22 μm for suction filtration, and then cut the filter membrane into pieces for DNA extraction; mix the soil samples thoroughly for direct DNA extraction.

[0074] (2) Sample DNA extraction

[0075] Use Spin Kit for Soil kit to extract the DNA of the samples obtained in the above step (1) according to the instructions; use NanoDrop2000 to detect the purity and concentration of DNA; use 1% agarose gel electrophoresis with a voltage of 5 V / cm for 20 min to detect the integrity of DNA.

[0076] (3) PCR amplification and high-throughput sequencing

[0077] Using the DNA extracted in the above step (2) as a template, select the upstream primer mlCOIintF and downstream primer jgHCO2198R of the mitochondrial cytochrome oxidase COI gene for PCR amplification. The nucleotide sequences of the PCR amplification primers are:

[0078] SEQ ID NO.1: Upstream primer mlCOIintF: GGWACWGGWTGAACWGTWT AYCCYCC;

[0079] SEQ ID NO.2: Downstream primer jgHCO2198R: TAAACTTCAGGGTGACCAA ARAAYCA.

[0080] The PCR reaction system is as follows: 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 μM), 0.8 μL of downstream primer (5 μM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and made up to 20 μL.

[0081] The amplification procedure is as follows: pre-denaturation at 95°C for 3 min, 27 cycles (denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s), then stable extension at 72°C for 10 min, and finally stored at 4°C (PCR instrument: ABI Model 9700).

[0082] The PCR products were recovered using a 2% agarose gel, and the recovered products were purified using a DNA gel recovery and purification kit (PCR Clean-Up Kit, Yuhua, China), and the recovered products were detected and quantified using Qubit 4.0 (Thermo Fisher Scientific, USA).

[0083] The recovered products were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for high-throughput sequencing. Sequencing was performed using the Illumina PE300 / PE250 platform. The reads obtained from sequencing were assembled according to the overlap relationship, and the sequence quality was controlled and filtered; the nt public database on the NCBI website (ftp: / / ftp.ncbi.nlm.nih.gov / ) was used for OUT sequence alignment, and 97% sequence similarity was used as the species annotation standard; among the annotated OUT sequences, metazoans (Metazoa, the general term for all other animals except protozoa) were selected as the OUT set for subsequent biodiversity analysis.

[0084] 2.3. Results of investigation and statistical analysis

[0085] 2.3.1. Number of species

[0086] All data analyses were performed on the Majorbio Cloud Platform (https: / / cloud.majorbio.com). The statistical OUT annotation species data were statistically analyzed to analyze the community composition of each sample at each taxonomic level: class, order, family, and species. The Shannon diversity index, Simpson index, and Pielou_e community evenness index were calculated to analyze the biodiversity of each sample.

[0087] In this invention, the arthropod species in the fields of the tested soybean and corn materials during the growth period in 2024 were investigated by the direct observation method. The results showed that the arthropod populations on soybean and corn plants consisted of insects from 9 orders and 23 families (as shown in Figure 1 ). A total of 213 species from 132 families were detected by high-throughput sequencing (as shown in Figure 2 ).

[0088] As shown in Figure 3-4 , the number of species identified in soil samples was large but the difference was small. The number of species identified in leaf samples was relatively small, but the difference between different crops was significant. There were also species not identified in the soil. The sampling method had a greater impact on the identified species. The species identified in soil samples of different crops had little difference, while the species identified in leaf samples of different crops showed differentiation. Therefore, when using environmental DNA metabarcoding to detect the impact of transgenic technology on biodiversity, both soil and leaf sampling methods should be adopted simultaneously.

[0089] 2.3.2. Changes in arthropod community diversity (diversity index)

[0090] The diversity index reflects the occurrence of arthropods in the farmland system and also reflects the richness of arthropod species in this system. The larger the diversity index, the stronger the stability of the community. The diversity indices of arthropods in the receptor, transformant, and transformant sprayed with glufosinate in the field were calculated and analyzed using the diversity index calculation formula. The calculation results of the diversity index were retained to two decimal places. The diversity index calculation formula is as follows:

[0091]

[0092] In the formula: H - diversity index; Pi = Ni / N; Ni is the number of individuals of the i-th species; N is the total number of individuals. The calculation results of the survey data by the direct observation method are shown in Table 2, and the calculation results of the survey data by the high-throughput sequencing method are shown in Figure 5 :

[0093] Table 2 Diversity indices of arthropods in different corn materials in the field

[0094]

[0095]

[0096] The results showed that the diversity index of arthropods in the farmland system was relatively high in August. During some investigation periods, the diversity index of arthropods in the receptor was significantly higher than that in the transformant treated with herbicides. This was mainly due to the relatively small population of insects at the beginning or end of the investigation, and the damage caused by target Lepidoptera pests on the receptor. There were no significant differences in the arthropod diversity index between the receptor and the transformant during the remaining investigation times, indicating that compared with the receptor DBN567, the transformant and the transformant sprayed with glufosinate had no adverse effects on the changes in the diversity of field arthropods.

[0097] As Figure 5 shown, the results of high-throughput sequencing showed that there were no significant differences in the arthropod diversity index between the receptor and the transformant on June 24, July 29, and September 2. The results were consistent with those obtained by the direct observation method, indicating that high-throughput sequencing can be used to detect and calculate the animal diversity in crop fields.

[0098] By calculating and analyzing the survey data using the above diversity index calculation formula, the arthropod diversity indices in the fields of the receptor, transgenic soybean, and transgenic soybean sprayed with glufosinate were obtained. The calculation results of the direct observation method are shown in Table 3, and the calculation results of the survey data of the high-throughput sequencing method are as Figure 6 shown:

[0099] Table 3 Arthropod diversity indices in the fields of different soybean materials

[0100]

[0101] The results showed that the diversity index of arthropods in the farmland system was relatively high in late July. During some investigation periods, the diversity index of arthropods in the receptor was significantly higher than that in the two treatments of transgenic soybean. This was mainly due to the relatively small population of insects at the beginning of the investigation, and the damage caused by target Lepidoptera pests on the receptor. There were no significant differences in the arthropod diversity index between the receptor and the two treatments of transgenic soybean during the remaining investigation times, indicating that compared with the receptor Jack, transgenic soybean and transgenic soybean sprayed with glufosinate had no adverse effects on the changes in the diversity of field arthropods.

[0102] As Figure 6 shown: The results of high-throughput sequencing showed that there were no significant differences in the arthropod diversity index between the receptor and the transformant on July 5, August 4, and September 3. The results were consistent with those obtained by the direct observation method, indicating that high-throughput sequencing can be used to detect and calculate the animal diversity in soybean crop fields.

[0103] 2.3.3 Changes in the dominance concentration of the arthropod community (Simpson index)

[0104] The Simpson index (dominance index) reflects the dominance of arthropods in the entire community. The Simpson indices of arthropods in the receptor, transformant, and transformant sprayed with glufosinate in the field were calculated and analyzed from the survey data using the Simpson index calculation formula. The calculation results of the Simpson index were retained to two decimal places. The Simpson index calculation formula is as follows:

[0105]

[0106] In the formula: C is the Simpson index; Ni is the number of individuals of the i-th species; N is the total number of individuals. The calculation results of the direct observation method are shown in Table 4, and the calculation results of the survey data of the high-throughput sequencing method are as Figure 7 shown:

[0107] Table 4 Simpson indices of arthropods in the field of different maize materials

[0108]

[0109]

[0110] The results showed that there was no significant difference in the Simpson indices of arthropods between the receptor and transformant treatments at the same survey time. Compared with the receptor DBN567, the transformant and the transformant sprayed with glufosinate had no adverse effects on the change in the dominance concentration of arthropods in the field.

[0111] As Figure 7 shown: The high-throughput sequencing results showed that on June 24, July 29, and September 2, there was no significant difference in the Simpson indices of arthropods between the receptor and transformant treatments. The results were consistent with those obtained by the direct observation method, indicating that the high-throughput sequencing method can be used to detect and calculate the animal diversity in crop fields.

[0112] The Simpson indices of arthropods in the receptor, transgenic soybean, and transgenic soybean sprayed with glufosinate in the field were calculated and analyzed from the survey data using the above Simpson index calculation formula. The calculation results of the direct observation method are shown in Table 5, and the calculation results of the survey data of the high-throughput sequencing method are as Figure 8 shown:

[0113] Table 5 Simpson indices of arthropods in the field of different soybean materials

[0114]

[0115]

[0116] Results showed that there were no significant differences in the Simpson index of arthropods between the two treatments of the receptor and transgenic soybean at the same survey time. Compared with the receptor Jack, transgenic soybean and transgenic soybean sprayed with glufosinate ammonium had no adverse effects on the change of the dominance concentration of arthropods in the field.

[0117] As Figure 8 shown: The results of high-throughput sequencing showed that on July 5, August 4, and September 3, there were no significant differences in the Simpson index of arthropods between the two treatments of the receptor and the transformant. The results were consistent with those obtained by the direct observation method, indicating that high-throughput sequencing can be used to detect and calculate the animal diversity in crop fields.

[0118] 2.3.4. Changes in the evenness of the arthropod community (evenness index)

[0119] The evenness index reflects the uniformity of the number of each species in the arthropod population. The evenness index of arthropods in the fields of the receptor, the transformant, and the transformant sprayed with glufosinate ammonium was calculated and analyzed from the survey data using the evenness index calculation formula. The calculation results of the evenness index were retained to two decimal places. The evenness index calculation formula is as follows:

[0120] J = H / ln S

[0121] In the formula: J - evenness index; H - diversity index; S - number of species; The calculation results of the direct observation method are shown in Table 6, and the calculation results of the survey data of the high-throughput sequencing method are as Figure 9 shown:

[0122] Table 6 Evenness index of arthropods in the fields of different maize materials

[0123]

[0124] Results showed that there were no significant differences in the evenness index of arthropods between the two treatments of the receptor and the transformant at the same survey time. Compared with the receptor DBN567, the transformant and the transformant sprayed with glufosinate ammonium had no adverse effects on the change of the evenness of arthropods in the field.

[0125] As Figure 9 shown: The results of high-throughput sequencing showed that on June 24, July 29, and September 2, there were no significant differences in the evenness index of arthropods between the two treatments of the receptor and the transformant. The results were consistent with those obtained by the direct observation method, indicating that high-throughput sequencing can be used to detect and calculate the animal diversity in crop fields.

[0126] The evenness index of arthropods in the fields of the receptor, transgenic soybean, and transgenic soybean sprayed with glufosinate ammonium was calculated and analyzed from the survey data using the above evenness index calculation formula. The calculation results of the direct observation method are shown in Table 7, and the calculation results of the survey data of the high-throughput sequencing method are asFigure 10 As shown

[0127] Table 7 Uniformity Index of Arthropods in the Field of Different Soybean Materials

[0128]

[0129] The results showed that there was no significant difference in the uniformity index of arthropods between the two treatments of the recipient and transgenic soybeans at the same survey time, indicating that compared with the recipient Jack, transgenic soybeans and transgenic soybeans sprayed with glufosinate had no adverse effects on the change of the uniformity of arthropods in the field.

[0130] As Figure 10 shown: The results of high-throughput sequencing showed that there was no significant difference in the arthropod evenness index between the two treatments of the recipient and the transformant on July 5, August 4, and September 3. The results were consistent with those obtained by the direct observation method, indicating that high-throughput sequencing can be used to detect and calculate the animal diversity in crop fields.

[0131] The above embodiments only express several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Environmental DNA macrobarcode sequencing method for evaluating the impact of transgenic plants on farmland biodiversity, characterized by: The following steps are involved: Collect root soil and leaf surface samples of GM crops at the same time and extract DNA from the samples; The extracted DNA was used as a template and PCR amplification was performed using amplification primers; The PCR amplification products were purified and then subjected to high-throughput sequencing. The OTU sequences obtained in the results were compared with the nt public database on the NCBI website to obtain the arthropod species in the samples and calculate the biodiversity-related index to evaluate the impact of genetically modified crops on farmland biodiversity.

2. The environmental DNA macrobarcode sequencing method for evaluating the impact of transgenic plants on farmland biodiversity according to claim 1, characterized in that: The genetically modified crops are genetically modified corn or genetically modified soybeans.

3. The environmental DNA macrobarcode sequencing method for evaluating the impact of transgenic plants on farmland biodiversity according to claim 2, characterized in that: The transgenic corn is DBN9229, and the transgenic soybean is DBN9004×DBN8002×DBN8205.

4. The environmental DNA macrobarcode sequencing method for evaluating the impact of transgenic plants on farmland biodiversity according to claim 1, characterized in that: The soil samples of genetically modified crops were collected by sampling at 5 diagonal points after the emergence of genetically modified crops. The floating materials on the soil layer were removed from each sampling point, and the soil samples of the rhizosphere layer with a depth of 2-15 cm were taken. The soil samples from the 5 sampling points were mixed and used for DNA extraction. To collect leaf surface samples of genetically modified crops, when taking soil samples, take one leaf each from the upper, middle and lower parts of the crop plant, wash the leaves thoroughly with ultrapure water, and filter them using a vacuum filter and a microporous filter membrane with a pore size of 0.22μm. The filter membrane is cut into pieces and used to extract DNA.

5. The environmental DNA macrobarcode sequencing method for evaluating the impact of transgenic plants on farmland biodiversity according to claim 1, characterized in that: use The DNA of the samples was extracted using the Spin Kit for Soil.

6. The environmental DNA macrobarcode sequencing method for evaluating the impact of transgenic plants on farmland biodiversity according to claim 1, characterized in that: The amplification primers are the upstream primer mlCOIintF and the downstream primer jgHCO2198R of the mitochondrial cytochrome oxidase COI gene, and the nucleotide sequences of the amplification primers are: SEQ ID NO.1: Upstream primer mlCOIintF: GGWACWGGWTGAACWGTWT AYCCYCC; SEQ ID NO.2: Downstream primer jgHCO2198R: TAAACTTCAGGGTGACCAA ARAAYCA.

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