A method for identifying pseudohybrids of Zhengai No. 2 based on SCoT molecular markers

Abstract

This application belongs to the field of seed variety identification technology, specifically relating to a method for identifying Zheng Ai No. 2 based on SCoT molecular markers. The method includes steps such as: extracting the genome of the sample to be identified, PCR amplification, and identification based on electrophoresis results. In this application, addressing the identification needs of Zheng Ai No. 2, the inventors, through polymorphic screening of SCoT primers and optimization and design of related reaction systems, provide a relatively accurate and rapid method for identifying the Zheng Ai No. 2 variety. This method has significant technical implications for the promotion and cultivation of Zheng Ai No. 2 and the sustainable development of the Artemisia argyi industry.

Description

Technical Field

[0001] This application belongs to the field of seed variety identification technology, specifically involving a method for identifying false hybrids of Zhengai No. 2 based on SCoT molecular markers. Background Technology

[0002] Ai ( Artemisia Artemisia argyi (Level. et. Vant.) is a perennial herb belonging to the genus Artemisia in the family Compositae. It is also a major traditional Chinese medicine used in medicine, food, and health products. Modern research shows that Artemisia argyi leaves are rich in minerals, flavonoids, and volatile oils, possessing properties such as warming the meridians and promoting blood circulation, antibacterial, anticancer, anti-inflammatory, immunomodulatory, and anticoagulant effects. Therefore, with the rapid development of the traditional Chinese medicine health service industry, the market demand for Artemisia argyi is increasing daily. Thus, the cultivation of new varieties of Artemisia argyi is of significant technical importance for the rapid development of the Artemisia argyi industry.

[0003] "Zheng Ai No. 2" is a new Artemisia argyi variety developed by the Sesame Research Center of the Henan Academy of Agricultural Sciences. It was bred using a systematic selection method, starting with a variant strain from a local Nanyang farm, focusing on high yield, stable yield, high quality, disease resistance, and wide adaptability. After years of selection and screening, it was approved for cultivation in 2022 (Identification No.: 2021014). Related analysis results show that its acid-insoluble ash, eucalyptus oil, and borneol content are 1.74%, 0.960%, and 0.066%, respectively, all meeting the limits set in the 2020 edition of the *Pharmacopoeia of the People's Republic of China*. Due to its high quality, high and stable yield, strong resistance to adverse conditions, wide adaptability, and simple cultivation management, Zheng Ai No. 2 has good prospects for widespread application as a new variety of Chinese medicinal herb.

[0004] In species (variety) identification, identification based on differences in the genome sequence of different species (variety) is a relatively rapid and accurate method. Among these, start codon targeted polymorphism (SCoT) is a novel marker system developed based on the short conserved regions flanking the ATG start codon in plant genes. This technology boasts advantages such as high amplification efficiency, rich detection of polymorphisms, good repeatability, strong primer universality, acquisition of abundant genetic information, identification of subtle differences between different genotypes, and accurate determination of phylogenetic relationships. Furthermore, it can be detected by agarose gel electrophoresis after the PCR reaction, making it simple and low-cost. Therefore, it has been widely adopted in the identification of some species (variety). However, based on the principle of this technology, it is clear that identification is mainly based on differences in the start codon sequence. Therefore, whether this method can be applied to the identification of Artemisia argyi-related varieties, especially whether it can distinguish the Zheng'ai 2 variety (which is planned for widespread cultivation) from other Artemisia argyi varieties, still requires further exploration and experimentation to determine. Summary of the Invention

[0005] Based on the technical need for identification of Zheng Ai No. 2, the purpose of this application is to provide a set of SCoT molecular markers and a method for identifying Zheng Ai No. 2 based on SCoT molecular markers, thereby laying a certain technical foundation for the promotion of Zheng Ai No. 2 and the development of the Artemisia argyi industry.

[0006] The technical solution adopted in this application is described in detail below.

[0007] A method for identifying pseudohybrids of Zhengai No. 2 based on SCoT molecular markers includes the following steps:

[0008] (1) Extracting the genome from the sample to be identified

[0009] Based on existing technology, the DNA genome of the Artemisia argyi sample to be tested is routinely extracted for later use;

[0010] Meanwhile, the DNA genome of leaf samples from Zhengai No. 2 was used as a control;

[0011] (2) PCR amplification

[0012] PCR amplification was performed using any of the following primer sequences.

[0013] SCoT7: 5'-CAACAATGGCTACCACGG-3';

[0014] SCoT8: 5'-CAACAATGGCTACCACGT-3';

[0015] SCoT10: 5'-CAACAATGGCTACCAGCC-3';

[0016] SCoT25: 5'-ACCATGGCTACCACCGGG-3';

[0017] SCoT26: 5'-ACCATGGCTACCACCGTC-3';

[0018] SCoT27: 5'-ACCATGGCTACCACCGTG-3';

[0019] SCoT76: 5'-CCATGGCTACCACTACCG-3';

[0020] The 15μL reaction system is designed as follows:

[0021] Primers, 1.0 μL;

[0022] 2x Tag Master Mix, 9μL;

[0023] DNA template, 3 μL (50 ng / µl);

[0024] The remaining portion was supplemented with ddH2O to a final volume of 15 μL.

[0025] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 90 s, 50℃ annealing for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 8 min.

[0026] The reaction products were then analyzed by 1.2% agarose gel electrophoresis (electrophoresis time was 30 min for all samples).

[0027] (3) Identification

[0028] First, based on phenotypic differences, the phenotypes of the Artemisia argyi to be tested were compared with those of Zheng Ai No. 2, so as to make a preliminary determination as to whether the Artemisia argyi to be tested belongs to "false hybrid".

[0029] The main phenotypic identification criteria for Zhengai No. 2 are as follows:

[0030] The stem is bluish-purple with 8 longitudinal ridges; the upper surface of the leaves is green with purple veins and the leaves are flat; the lower surface of the leaves is green with dense white spiderweb-like hairs, and the new leaves at the top are yellowish-white.

[0031] Subsequently, the results of the electrophoresis bands of the Artemisia argyi sample to be tested were compared with those of Zheng Ai No. 2. If the band results were inconsistent, the Artemisia argyi sample to be tested was determined to be a "false hybrid". If the band results were consistent, the Artemisia argyi sample to be tested was determined to be Zheng Ai No. 2.

[0032] In this application, in response to the identification needs of Zheng Ai No. 2, the inventors, by combining SCoT-PCR technology, screened the polymorphism of SCoT primers and optimized and designed the relevant reaction system, providing a relatively accurate and rapid method for identifying false hybrids of Zheng Ai No. 2. This method has significant technical implications for the promotion and planting of Zheng Ai No. 2 and the sustainable development of the Artemisia argyi industry. Attached Figure Description

[0033] Figure 1 The results of agarose gel electrophoresis of the extracted Artemisia argyi genomic DNA are shown in the figure; A1-A10 represent the results of different Artemisia argyi samples.

[0034] Figure 2 PCR amplification diagrams showing different amounts of template DNA used; from left to right, the amounts of DNA template used are 2, 2.5, 3, and 4 µl.

[0035] Figure 3 Electrophoresis images of PCR amplification with different primer amounts; from left to right, the primer amounts are 0.7, 0.9, 1.0, and 1.2 µl.

[0036] Figure 4 Electrophoresis images of PCR amplification with different amounts of 2×tagMaster mix; the amounts of 2×tagMaster mix from left to right in the image are 6, 7, 8, and 9 µl respectively.

[0037] Figure 5 Electrophoresis images of PCR amplification at different annealing temperatures; the annealing temperatures from left to right in the image are: 40, 45, 50, and 55℃.

[0038] Figure 6 Electrophoresis images of PCR amplification products at different agarose concentrations; the agarose concentrations from left to right in the images are: 1, 1.2, 1.5, and 2%.

[0039] Figure 7 Electrophoresis diagram for screening polymorphic primers;

[0040] Figure 8 Electrophoresis image of SCoT7 molecular marker on a single plant of "Zheng Ai 2"; In the figure: M is the marker; A1-A10 are the results of different Artemisia argyi samples;

[0041] Figure 9 Electrophoresis image of SCoT8 molecular marker on a single plant of "Zheng Ai 2"; In the figure: M is the marker; A1-A10 are the results of different Artemisia argyi samples;

[0042] Figure 10Electrophoresis image of SCoT10 molecular marker on a single plant of "Zheng Ai 2"; In the figure: M is the marker; A1-A10 are the results of different Artemisia argyi samples;

[0043] Figure 11 Electrophoresis image of SCoT25 molecular marker on a single plant of "Zheng Ai 2"; In the figure: M is the marker; A1-A10 are the results of different Artemisia argyi samples;

[0044] Figure 12 Electrophoresis image of SCoT26 molecular marker on a single plant of "Zheng Ai 2"; In the figure: M is the marker; A1-A10 are the results of different Artemisia argyi samples;

[0045] Figure 13 Electrophoresis image of SCoT27 molecular marker on a single plant of "Zheng Ai 2"; In the figure: M is the marker; A1-A10 are the results of different Artemisia argyi samples;

[0046] Figure 14 Electrophoresis image of SCoT76 molecular marker on a single plant of "Zheng Ai No. 2"; in the figure: M is the marker; A1-A10 are the results of different Artemisia argyi samples. Detailed Implementation

[0047] The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the experimental background of some embodiments is briefly described below.

[0048] Biomaterials:

[0049] Zheng Ai No. 2: The applicant participated in the cultivation of a new variety of Artemisia argyi, and therefore has a copy of the material (this variety has been approved by the Henan Provincial Committee for the Identification of Chinese Herbal Medicine Varieties and can be obtained through public channels).

[0050] Nanyang Dengzhou Artemisia, from Dengzhou, Nanyang, is a common variety of Artemisia argyi cultivated in China and can be obtained through public channels.

[0051] Main reagent kits and experimental instruments:

[0052] The novel plant genomic DNA extraction kit (Plant Genomic DNA Extraction Kit 36302-01) was purchased from Shanghai Tulugang Biotechnology Co., Ltd.

[0053] Electrophoresis apparatus, Beijing Liuyi DYY-3C dual-stability electrophoresis apparatus;

[0054] Gel imaging system, Tanon-2500.

[0055] Example 1

[0056] The identification method for Zheng Ai No. 2 based on SCoT molecular markers provided in this application includes conventional PCR amplification, band identification and other operations. The relevant operation process is briefly introduced below.

[0057] (a) Extracting the genome from the sample

[0058] When extracting genomic DNA from a sample, standard procedures can be used. Specific procedures are as follows:

[0059] Take 0.2g of Artemisia argyi leaves during the vegetative growth period, place them in a 2 mL round-bottom centrifuge tube, add two steel balls (0.5 cm in diameter), and grind them with a DNA extraction grinder (Heros-Mole TL2010S high-throughput tissue grinder, Dinghaoyuan Technology). Shake for 40-50 seconds, and then extract the DNA from the leaves according to the instructions of the DNA extraction kit.

[0060] Subsequently, the concentration of the obtained DNA solution was measured and diluted (ddH2O to 50 ng / µl), aliquoted, and stored at -20℃ for later use.

[0061] The extracted genomic DNA was analyzed by agarose gel electrophoresis (1.2% agarose content), and the results are as follows: Figure 1 As shown, the DNA bands are clear and undegraded, indicating good extraction quality. Further analysis using an ultra-micro spectrophotometer revealed that the A260 / A280 ratio was between 1.74 and 1.89, indicating that the extracted Artemisia argyi genomic DNA had high purity and met the requirements for subsequent experiments.

[0062] It should be noted that the relevant electrophoresis detection methods and specific procedures are as follows:

[0063] Add 1.2 g agarose to 100 mL of 1×TAE, heat in a microwave oven to dissolve, cool slightly, add 10 μL of Goldview Type I nucleic acid staining agent (MonTrack Monad), then pour into a gel casting plate to make an agarose gel and place it in an electrophoresis tank;

[0064] After electrophoresis at 120 V and 100 mA for 30 min, the gel was imaged on a GelDocXR+ gel imaging system (Tanon-2500).

[0065] (II) Design of primer sequences for PCR amplification

[0066] Based on the existing Artemisia argyi gene sequence, 80 universal dominant SCoT primers were designed. Due to the large number of related primer sequences, only the 7 SCoT primer sequences for which protection is sought in this application are described below:

[0067] SCoT7: 5'-CAACAATGGCTACCACGG-3';

[0068] SCoT8: 5'-CAACAATGGCTACCACGT-3';

[0069] SCoT10: 5'-CAACAATGGCTACCAGCC-3';

[0070] SCoT25: 5'-ACCATGGCTACCACCGGG-3';

[0071] SCoT26: 5'-ACCATGGCTACCACCGTC-3';

[0072] SCoT27: 5'-ACCATGGCTACCACCGTG-3';

[0073] SCoT76: 5'-CCATGGCTACCACTACCG-3'.

[0074] (III) Design and optimization of the reaction system

[0075] Based on the primers designed in step (II), in order to ensure that the technical indicators such as band clarity and distinguishability in the final electrophoresis results are good, we combined the L16 (43) orthogonal experiment and single-factor experiment to further explore several factors affecting the PCR system (template DNA amount, primer concentration, 2×Taq PCR premixed reagent II amount, etc.). The specific experimental situation is briefly described below.

[0076] (1) Amount of template DNA

[0077] The PCR reaction system was designed to be 15 μL:

[0078] Primer (primer SCoT26), 1 μL (5.7 nmol);

[0079] 2x Tag Master Mix, 7.5μL;

[0080] At a DNA concentration of 50 ng / µl, the dosages were 2 μL, 2.5 μL, 3 μL, and 4 μL, respectively.

[0081] The remaining portion was supplemented with ddH2O to a final volume of 15 μL.

[0082] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 90 s, 50℃ annealing for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 8 min; after the PCR reaction, the reaction products were subjected to 1.2% agarose electrophoresis (electrophoresis time was 30 min).

[0083] The results are as follows Figure 2 As shown, it can be seen that under the condition of DNA concentration of 50 ng / µl, 2~4 μL (i.e., template DNA amount of 100~200 ng) can amplify clear bands.

[0084] (2) Primer dosage

[0085] Based on the aforementioned results, the PCR reaction system was still designed to be 15 μL, but the amount of different primers was adjusted, specifically:

[0086] Primers (primer SCoT7, 5.6 nmol) were designed in solutions of 0.7 μL, 0.9 μL, 1.0 μL, and 1.2 μL, respectively.

[0087] 2x Tag Master Mix, 7.5μL;

[0088] DNA template, 3 μL (50 ng / µl);

[0089] The remaining portion was supplemented with ddH2O to a final volume of 15 μL.

[0090] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 90 s, 50℃ annealing for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 8 min; after the PCR reaction, the reaction products were subjected to 1.2% agarose electrophoresis (electrophoresis time was 30 min).

[0091] The results are as follows Figure 3 As shown, it can be seen that when the primer dosage is in the range of 1~1.2μL, the amplified bands are well distinguished and clear.

[0092] (3) 2×tag Master mix dosage

[0093] Based on the aforementioned results, the PCR reaction system was still designed to be 15 μL, but the amount of different 2x Tag Master Mix was adjusted, specifically:

[0094] Primer (primer SCoT8, 5.8 nmol), 1.0 μL;

[0095] 2x Tag Master Mix, in the following quantities: 6μL, 7μL, 8μL, and 9μL respectively;

[0096] DNA template, 3 μL (50 ng / µl);

[0097] The remaining portion was supplemented with ddH2O to a final volume of 15 μL.

[0098] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 90 s, 50℃ annealing for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 8 min; after the PCR reaction, the reaction products were subjected to 1.2% agarose electrophoresis (electrophoresis time was 30 min).

[0099] The results are as follows Figure 4 As shown, when the amount of 2x Tag Master Mix is ​​9μL, the amplified bands are clearer and have higher discrimination.

[0100] (4) Annealing temperature

[0101] Based on the aforementioned results, the PCR reaction system was still designed to be 15 μL, but different annealing temperatures were adjusted. Specifically:

[0102] Primers (primer SCoT7, 5.6 nmol), 1.0 μL;

[0103] 2x Tag Master Mix, 7.5μL;

[0104] DNA template, 3 μL (50 ng / µl);

[0105] The remaining portion was supplemented with ddH2O to a final volume of 15 μL.

[0106] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 90 s, annealing (annealing temperatures were designed to be 40℃, 45℃, 50℃, and 60℃) for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 8 min; after the PCR reaction, the reaction products were subjected to 1.2% agarose electrophoresis (electrophoresis time was 30 min).

[0107] The results are as follows Figure 5 As shown in the figure, annealing temperatures of 45℃ to 50℃ can clearly amplify bands with good differentiation.

[0108] (5) Agarose gel concentration during electrophoresis

[0109] Based on the aforementioned results, the PCR reaction system was still designed to be 15 μL, but the agarose concentration for electrophoresis was adjusted. Specifically:

[0110] Primers (primer SCoT7, 5.6 nmol), 1.0 μL;

[0111] 2x Tag Master Mix, 7.5μL;

[0112] DNA template, 3 μL (50 ng / µl);

[0113] The remaining portion was supplemented with ddH2O to a final volume of 15 μL.

[0114] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 90 s, 50℃ annealing for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 8 min; after the PCR reaction, the reaction products were subjected to 1%, 1.2%, 1.5%, and 2% agarose gel electrophoresis (electrophoresis time was 30 min for each).

[0115] The results are as follows Figure 6 As shown, agarose concentrations of 1–1.5% can effectively ensure clear bands during amplification.

[0116] Based on the above experimental results, the design of different reaction systems for different primer sequences can be summarized in Table 1 below.

[0117] Table 1. Reaction system design for different SCoT primer sequences:

[0118] .

[0119] (6) Screening and determination of SCoT primers

[0120] Based on the aforementioned experimental system, the optimal reaction system design is further identified as follows:

[0121] Primers, 1.0 μL;

[0122] 2x Tag Master Mix, 9μL;

[0123] DNA template, 3 μL (50 ng / µl);

[0124] The remaining portion was supplemented with ddH2O to a final volume of 15 μL.

[0125] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 90 s, 50℃ annealing for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 8 min; after the PCR reaction, the reaction products were subjected to 1%, 1.2%, 1.5%, and 2% agarose gel electrophoresis (electrophoresis time was 30 min for each).

[0126] Using the clear and distinct electrophoretic bands as the technical standard, the 80 designed SCoT primers were specifically screened and distinguished (partial results are shown in Figure 7). It can be seen that the 7 primers for which protection is sought in this application have good application potential. Specifically, the 7 primers for which protection is sought in this application are: SCoT7, SCoT8, SCoT10, SCoT25, SCoT26, SCoT27, and SCoT76. Their sequences are shown in SEQ ID Nos. 1-7, as follows:

[0127] SCoT7: 5'-CAACAATGGCTACCACGG-3';

[0128] SCoT8: 5'-CAACAATGGCTACCACGT-3';

[0129] SCoT10: 5'-CAACAATGGCTACCAGCC-3';

[0130] SCoT25: 5'-ACCATGGCTACCACCGGG-3';

[0131] SCoT26: 5'-ACCATGGCTACCACCGTC-3';

[0132] SCoT27: 5'-ACCATGGCTACCACCGTG-3';

[0133] SCoT76: 5'-CCATGGCTACCACTACCG-3'.

[0134] Example 2

[0135] Based on the reaction system and procedure specified in Example 1, ten individual samples of *Artemisia argyi* var. *zhengai* and *Artemisia argyi* var. *dengzhouensis* from Nanyang were randomly selected (samples randomly numbered: A01, A02, A03, A04, A05, A06, A07, A08, A09, A10). These samples were then tested and identified using the seven primers (SCoT7, SCoT8, SCoT10, SCoT25, SCoT26, SCoT27, and SCoT76) determined during screening. Specifically:

[0136] The 15μL reaction system is designed as follows:

[0137] Primers, 1.0 μL;

[0138] 2x Tag Master Mix, 9μL;

[0139] DNA template, 3 μL (50 ng / µl);

[0140] The remaining portion was supplemented with ddH2O to a final volume of 15 μL.

[0141] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 90 s, 50℃ annealing for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 8 min; after the PCR reaction, the reaction products were subjected to 1%, 1.2%, 1.5%, and 2% agarose gel electrophoresis (electrophoresis time was 30 min for each).

[0142] Electrophoresis results as follows Figure 8 , Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 , Figure 14 As shown. By comparing the electrophoretic bands, it can be clearly seen that A5 and A8 are different varieties using SCoT7, SCoT8, SCoT10, SCoT25, SCoT26, SCoT27, and SCoT76. Furthermore, combined with the relevant phenotypic identification results, it can be clearly determined that A5 and A8 are hybrid plants of Artemisia argyi from Dengzhou, Nanyang.

[0143] It should be explained that in actual production applications, when identifying Artemisia argyi varieties, it is generally necessary to conduct preliminary screening and identification based on relevant phenotypic differences, and then further accurately determine the variety by comparing the results of PCR amplification and electrophoresis as described above. In production, the main phenotypic identification criteria for Zheng Ai No. 2 are as follows:

[0144] The stem is bluish-purple with 8 longitudinal ridges. The upper surface of the leaves is green with purple veins and a flat shape. The lower surface of the leaves is green with dense white spiderweb-like hairs, and the new leaves at the top are yellowish-white.

Claims

1. A method for identifying false hybrids of Zhengai No. 2 based on SCoT molecular markers, characterized in that, The method includes the following steps: (1) Extracting the genome from the sample to be identified Extract the DNA genome from the Artemisia argyi sample to be tested, for later use; Meanwhile, the DNA genome of leaf samples from Zhengai No. 2 was used as a control; (2) PCR amplification PCR amplification was performed using primer sequences from SEQ ID Nos. 1 to 7; the specific primer sequences are as follows: SCoT7: 5'-CAACAATGGCTACCACGG-3'; SCoT8: 5'-CAACAATGGCTACCACGT-3'; SCoT10: 5'-CAACAATGGCTACCAGCC-3'; SCoT25: 5'-ACCATGGCTACCACCGGG-3'; SCoT26: 5'-ACCATGGCTACCACCGTC-3'; SCoT27: 5'-ACCATGGCTACCACCGTG-3'; SCoT76: 5'-CCATGGCTACCACTACCG-3'; (3) Identification First, based on phenotypic differences, the phenotypes of the Artemisia argyi to be tested were compared with those of Zheng Ai No. 2, so as to make a preliminary determination as to whether the Artemisia argyi to be tested belongs to "false hybrid". The phenotypic characteristics of Zhengai No. 2 are as follows: the stem is bluish-purple with 8 longitudinal ridges, the upper surface of the leaves is green with purple veins and the leaf shape is flat; the lower surface of the leaves is green with dense white spiderweb-like hairs, and the new leaves at the top are yellowish-white. Subsequently, the PCR amplification results of the Artemisia argyi sample to be tested were compared with the amplification results of Zheng Ai No.

2. If the band results were inconsistent, the Artemisia argyi sample to be tested was determined to be a "false hybrid". If the band results are consistent, the tested Artemisia argyi is determined to be Zheng Ai No.

2.

2. The method for identifying Zheng Ai No. 2 pseudohybrids based on SCoT molecular markers as described in claim 1, characterized in that, In step (2), the PCR amplification process uses a 15 μL reaction system designed as follows: Primers, 1.0 μL; 2x Tag Master Mix, 9μL; DNA template, 3 μL, 50 ng / µl; The remaining portion was supplemented with ddH2O to a final volume of 15 μL; PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 90 s, 50℃ annealing for 60 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 8 min.

3. The method for identifying Zheng Ai No. 2 pseudohybrids based on SCoT molecular markers as described in claim 2, characterized in that, After amplification, the reaction products were detected by 1.2% agarose gel electrophoresis.

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