Method for identifying series varieties of four famous mugwort and Nanyang mugwort and primer combination used by method

Abstract

The invention discloses a method for identifying series varieties of four famous mugwort and Nanyang mugwort and a primer combination used by the method. The primer combination is composed of a primer pair A.argyi. SSR15, a primer pair A.argyi. SSR29 and a primer pair A.argyi. SSR36, and the primer pair A.argyi. SSR36 and the primer pair A.argyi. The primer pair A.argyi. SSR15 is composed of single-stranded DNA (deoxyribonucleic acid) molecules as shown in SEQ ID NO: 1 and SEQ ID NO: 2. The primer pair A.argyi. SSR29 is composed of single-stranded DNA (deoxyribonucleic acid) molecules as shown in SEQ ID NO: 3 and SEQ ID NO: 4. The primer pair A.argyi. SSR36 is composed of single-stranded DNA (deoxyribonucleic acid) molecules as shown in SEQ ID NO: 5 and SEQ ID NO: 6. The primer combination can be used for identifying four famous mugwort, namely Nanyang mugwort No.8, Nanyang mugwort No.9, Nanyang mugwort No.10 and Nanyang mugwort No.11. The method has important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for identifying four famous mugworts and Nanyang mugwort series varieties and a primer combination used therein. Background Art

[0002] Species and variety identification is an important part of seed quality testing, and its main purpose is to determine the degree of conformity between the sample submitted for testing and the declared species or variety. With the increase in the volume of seed market trade in recent years, domestic seed operators have unilaterally pursued economic benefits and ignored quality, using other varieties to adulterate and fake, which not only caused economic losses, but also caused confusion among varieties in the market, making it difficult to distinguish between the true and the false, so that the real excellent varieties cannot be effectively protected, shortening the use period of the varieties. Therefore, accurate, reliable, fast and simple variety identification methods have attracted much attention. SSR molecular marker technology is a genetic marker technology based on nucleotide sequence variation in genetic material between individuals, and is a direct reflection of genetic polymorphism at the DNA level. Compared with other genetic marker technologies, SSR molecular marker technology has the characteristics of rich information, fast and accurate detection, and easy standardized operation, and is very suitable for plant variety identification and analysis. The Seed Law clearly stipulates that the results of variety authenticity detection using the SSR molecular marker method can be used as the basis for determining fake and inferior seeds.

[0003] Artemisia argyi H. Lév. & Vaniot is a perennial herb or slightly semi-shrubby plant of the genus Artemisia in the Asteraceae family. Artemisia is also called wormwood. The whole plant of Artemisia argyi can be used as medicine, which has the effects of warming the meridians, removing dampness, dispelling cold, antibacterial and anti-inflammatory, regulating menstruation and stopping bleeding, and enhancing immunity. It has been recorded in herbal works of various dynasties, such as "Compendium of Materia Medica" and "Shennong's Herbal Classic". Artemisia has a unique taste and is added to traditional pastries. Its young shoots and seedlings can also be eaten as vegetables. Adding wormwood leaves to ink can improve the elasticity and moisturizing properties of traditional ink, making the ink fragrant and having an antibacterial effect.

[0004] Rapid detection methods for variety identification have been established for some major crops such as corn, soybeans, wheat, and barley. In recent years, industry standards for the technical procedures for the identification of pepper, watermelon, cabbage, kale, and cucumber varieties using SSR molecular markers have emerged. So far, there have been no reports on the application of SSR markers in the variety-specific identification of mugwort. In view of the interspecificity of SSR primers, it is urgent to develop primer combinations for detecting mugwort varieties and establish corresponding detection processes. Summary of the invention

[0005] The purpose of the present invention is to identify Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11.

[0006] The present invention firstly protects a primer combination. The primer combination may include a primer pair A.argyi.SSR15, a primer pair A.argyi.SSR29 and a primer pair A.argyi.SSR36;

[0007] The primer pair A.argyi.SSR15 may be composed of a primer A.argyi.SSR15-F having a nucleotide sequence as shown in SEQ ID NO:1 and a primer A.argyi.SSR15-R having a nucleotide sequence as shown in SEQ ID NO:2;

[0008] The primer pair A.argyi.SSR29 may be composed of a primer A.argyi.SSR29-F having a nucleotide sequence as shown in SEQ ID NO:3 and a primer A.argyi.SSR29-R having a nucleotide sequence as shown in SEQ ID NO:4;

[0009] The primer pair A.argyi.SSR36 may be composed of a primer A.argyi.SSR36-F having a nucleotide sequence as shown in SEQ ID NO:5 and a primer A.argyi.SSR36-R having a nucleotide sequence as shown in SEQ ID NO:6.

[0010] In the primer combination, the 5' ends of primer A.argyi.SSR15-F, primer A.argyi.SSR29-F and primer A.argyi.SSR36-F can be modified with different fluorescent groups respectively. The primer combination composed of primers modified with different fluorescent groups is used to detect PCR amplification products by capillary electrophoresis.

[0011] In an embodiment of the present invention, primer A.argyi.SSR15-F can be modified with a HEX fluorescent group, and the fluorescent color emitted is green. Primer A.argyi.SSR29-F can be modified with a ROX fluorescent group, and the fluorescent color emitted is red. Primer A.argyi.SSR36-F can be modified with a FAM fluorescent group, and the fluorescent color emitted is blue.

[0012] Any of the above-mentioned primer combinations can specifically consist of any of the above-mentioned primer pairs A.argyi.SSR15, any of the above-mentioned primer pairs A.argyi.SSR29 and any of the above-mentioned primer pairs A.argyi.SSR36.

[0013] The use of any of the above-mentioned primer combinations in identifying Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11 also falls within the scope of protection of the present invention.

[0014] The present invention also protects a method for identifying whether the mugwort to be tested is or is a candidate for being one of Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11.

[0015] The method for identifying which of the mugwort to be tested or the candidate is Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11 protected by the present invention can be specifically method 1, which may include the following steps: using the genomic DNA of the mugwort to be tested as a template, and performing PCR amplification with any of the above-mentioned primer pairs A.argyi.SSR15, any of the above-mentioned primer pairs A.argyi.SSR29 and any of the above-mentioned primer pairs A.argyi.SSR36 to obtain a PCR amplification product; then performing the following judgment:

[0016] If the primer pair A.argyi.SSR15 is used to amplify the 111 bp amplification product, the primer pair A.argyi.SSR29 is used to amplify the 138 bp, 152 bp and 157 bp amplification products, and the primer pair A.argyi.SSR36 is used to amplify the 107 bp, 116 bp and 119 bp amplification products, then the wormwood to be tested is or is a candidate for S. argyi;

[0017] If the primer pair A.argyi.SSR15 is used to amplify an amplification product of 111 bp, the primer pair A.argyi.SSR29 is used to amplify an amplification product of 128 bp, 138 bp, 152 bp and 157 bp, and the primer pair A.argyi.SSR36 is used to amplify an amplification product of 116 bp, then the mugwort to be tested is or is a candidate for northern mugwort;

[0018] If the primer pair A.argyi.SSR15 is used to amplify the 111 bp amplification product, the primer pair A.argyi.SSR29 is used to amplify the 128 bp, 152 bp and 157 bp amplification products, and the primer pair A.argyi.SSR36 is used to amplify the 116 bp amplification product, then the mugwort to be tested is or the candidate is Qi mugwort;

[0019] If the primer pair A.argyi.SSR15 is used to amplify an amplification product of 111 bp, the primer pair A.argyi.SSR29 is used to amplify an amplification product of 138 bp and 152 bp, and the primer pair A.argyi.SSR36 is used to amplify an amplification product of 116 bp, then the mugwort to be tested is or is a candidate for Artemisia argyi;

[0020] If the primer pair A.argyi.SSR15 is used to amplify an amplification product of 111 bp, the primer pair A.argyi.SSR29 is used to amplify an amplification product of 128 bp, 138 bp and 157 bp, and the primer pair A.argyi.SSR36 is used to amplify an amplification product of 116 bp, then the mugwort to be tested is or is a candidate for Nanyang mugwort No. 8;

[0021] If the primer pair A.argyi.SSR15 is used to amplify the 111 bp amplification product, the primer pair A.argyi.SSR29 is used to amplify the 138 bp amplification product, and the primer pair A.argyi.SSR36 is used to amplify the 111 bp and 116 bp amplification products, then the mugwort to be tested is or is a candidate for Nanyang mugwort 9;

[0022] If the primer pair A.argyi.SSR15 is used to amplify the amplified products of 111bp, 128bp and 147bp, the primer pair A.argyi.SSR29 is used to amplify the amplified products of 138bp, 152bp and 157bp, and the primer pair A.argyi.SSR36 is used to amplify the amplified products of 111bp and 116bp, then the mugwort to be tested is or the candidate is Nanyang mugwort No. 10;

[0023] If the primer pair A.argyi.SSR15 can obtain amplification products of 105bp, 111bp and 123bp, the primer pair A.argyi.SSR29 can obtain amplification products of 128bp, 138bp and 152bp, and the primer pair A.argyi.SSR36 can obtain an amplification product of 116bp, then the mugwort to be tested is or the candidate is Nanyang Mugwort No. 11.

[0024] The method for identifying whether the mugwort to be tested is or is a candidate for being Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11 protected by the present invention may specifically be method 2, which may include the following steps:

[0025] (1) Using the genomic DNA of the mugwort to be tested as a template, PCR amplification is performed using any of the above primer pairs A.argyi.SSR15, any of the above primer pairs A.argyi.SSR29, and any of the above primer pairs A.argyi.SSR36 to obtain a PCR amplification product;

[0026] (2) using the genomic DNA of Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11 as templates, and using any of the above primer pairs A.argyi.SSR15, any of the above primer pairs A.argyi.SSR29 and any of the above primer pairs A.argyi.SSR36 to perform PCR amplification to obtain PCR amplification products;

[0027] (3) Make the following judgment:

[0028] If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for the mugwort to be tested are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for the wormwood to be tested, then the mugwort to be tested is or is a candidate for the wormwood to be wormwood;

[0029] If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for the tested mugwort are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for northern mugwort, then the tested mugwort is or is a candidate for northern mugwort;

[0030] If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for the tested mugwort are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for Qi mugwort, then the tested mugwort is or the candidate is Qi mugwort;

[0031] If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for the mugwort to be tested are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for Qi Ai, then the mugwort to be tested is or the candidate is Qi Ai;

[0032] If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for the tested mugwort are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for Nanyang mugwort No. 8, then the tested mugwort is or is a candidate for Nanyang mugwort No. 8;

[0033] If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for the tested mugwort are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for Nanyang mugwort No. 9, then the tested mugwort is or the candidate is Nanyang mugwort No. 9;

[0034] If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for the tested mugwort are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for Nanyang mugwort No. 10, then the tested mugwort is or is a candidate for Nanyang mugwort No. 10;

[0035] If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for the mugwort to be tested are consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for Nanyang mugwort No. 11, then the mugwort to be tested is or the candidate is Nanyang mugwort No. 11.

[0036] In any of the above methods, the PCR amplification product can be detected by electrophoresis. Preferably, the electrophoresis can be capillary electrophoresis.

[0037] The electrophoresis may also be agarose gel electrophoresis. The gel concentration of the agarose gel electrophoresis may be 1%-3% (v / v).

[0038] The present invention also protects a kit containing any of the above-mentioned primer combinations; the purpose of the kit is to identify Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11.

[0039] The present invention also protects a method for preparing the kit. The method for preparing the kit may include the step of packaging each primer in any of the above primer combinations separately.

[0040] The use of any of the above-mentioned kits in identifying Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11 also falls within the scope of protection of the present invention.

[0041] Experiments have shown that the primer combination provided by the present invention can be used to identify Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11, thereby verifying the authenticity of the eight varieties of Ai. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 1% agarose gel test results of genomic DNA of 8 Artemisia species.

[0043] Figure 2 Part of the agarose gel electrophoresis results of the PCR amplification products obtained by using primer pair 15 to amplify 8 mugwort varieties.

[0044] Figure 3 Part of the agarose gel electrophoresis results of the PCR amplification products obtained by using primer pair 29 to amplify 8 mugwort varieties.

[0045] Figure 4 Partial agarose gel electrophoresis results of PCR amplification products obtained by using primer pair 36 to amplify 8 mugwort varieties.

[0046] Figure 5 Capillary electrophoresis results of PCR amplification products obtained by amplifying A.argyi using primer pair A.argyi.SSR15.

[0047] Figure 6 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying A.argyi.SSR15 with primer pair A.argyi.

[0048] Figure 7 Capillary electrophoresis results of PCR amplification products obtained by amplifying Qi Ai using primer pair A.argyi.SSR15.

[0049] Figure 8This is the capillary electrophoresis result of the PCR amplification product obtained by using the primer pair A.argyi.SSR15 to amplify Qi Ai.

[0050] Fig. 9 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 8 using the primer pair A.argyi.SSR15.

[0051] Fig.10 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 9 using the primer pair A.argyi.SSR15.

[0052] Fig.11 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 10 using the primer pair A.argyi.SSR15.

[0053] Fig.12 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 11 using primer pair A.argyi.SSR15.

[0054] Fig.13 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying A.argyi using primer pair A.argyi.SSR29.

[0055] Fig.14 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying A.argyi.SSR29 with primer pair A.argyi.

[0056] Fig.15 Capillary electrophoresis results of PCR amplification products obtained by amplifying Qi Ai using primer pair A.argyi.SSR29.

[0057] Fig.16 This is the capillary electrophoresis result of the PCR amplification product obtained by using primer pair A.argyi.SSR29 to amplify Qi Ai.

[0058] Fig.17 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 8 using the primer pair A.argyi.SSR29.

[0059] Fig.18 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 9 using the primer pair A.argyi.SSR29.

[0060] Fig.19 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 10 using the primer pair A.argyi.SSR29.

[0061] Fig. 20 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 11 using the primer pair A.argyi.SSR29.

[0062] Fig.21 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying A.argyi using primer pair A.argyi.SSR36.

[0063] Fig. 22 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying A.argyi.SSR36 with primer pair A.argyi.

[0064] Fig.23 Capillary electrophoresis results of PCR amplification products obtained by amplifying Qi Ai using primer pair A.argyi.SSR36.

[0065] Fig.24 This is the capillary electrophoresis result of the PCR amplification product obtained by using primer pair A.argyi.SSR36 to amplify Qi Ai.

[0066] Fig.25 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 8 using the primer pair A.argyi.SSR36.

[0067] Fig.26 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 9 using primer pair A.argyi.SSR36.

[0068] Fig. 27 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 10 using primer pair A.argyi.SSR36.

[0069] Fig.28 This is the capillary electrophoresis result of the PCR amplification product obtained by amplifying Nanyang Ai No. 11 using the primer pair A.argyi.SSR36. DETAILED DESCRIPTION

[0070] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0071] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0072] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0073] QlAamp DNA Mini Kit is a product of QIAGEN, with a product number of 51306. Qubit DNA Assay Kit is a product of Invitrogen, with a product number of Q32854. 2×Precise PCR Mix is ​​a product of Sino-Science, with a product number of XS22001. Trans 2K Plus DNA Marker is a product of Quanshijin, with a product number of E065.

[0074] The names and sources of the 8 mugwort varieties in the following embodiments are shown in Table 1. Among them, Nanyang mugwort No. 8, Nanyang mugwort No. 9, Nanyang mugwort No. 10 and Nanyang mugwort No. 11 are all new varieties of Nanyang mugwort series approved by Henan Province; Hai mugwort, Bei mugwort, Qi mugwort and Qi mugwort are the four famous mugworts.

[0075] Table 1. Names and origins of eight mugwort varieties

[0076]

[0077]

[0078] Example 1: Acquisition of primer combinations and their application in identifying eight mugwort species

[0079] 1. Use QIAamp DNA Mini Kit to extract genomic DNA of the samples to be tested, and then use QubitDNA Assay Kit for DNA quantification.

[0080] The samples to be tested are 8 varieties of mugwort, namely northern mugwort leaves, sea mugwort leaves, Qi mugwort leaves, Qi mugwort leaves, Nanyang mugwort No. 8 leaves, Nanyang mugwort No. 9 leaves, Nanyang mugwort No. 10 leaves and Nanyang mugwort No. 11 leaves.

[0081] 2. After completing step 1, the genomic DNA of the sample to be tested was tested on a 1% agarose gel. The loading amount was 100 ng.

[0082] This step is performed to check DNA integrity.

[0083] Test results see Figure 1(DNAMarker is Trans 2K Plus DNAMarker). The results showed that the genomic DNA of the eight mugwort varieties was intact.

[0084] 3. After completing step 2, the genomic DNA of the sample to be tested is used as a template, and 30 primer pairs are used for PCR amplification to obtain PCR amplification products. The 30 primer pairs in this step are preliminarily screened by the inventor of this application based on the transcriptome sequencing data of existing mugwort varieties (including the above 8 mugwort varieties).

[0085] The reaction system was 25 μL, consisting of 2 μL genomic DNA of the sample to be tested, 1 μL upstream primer F aqueous solution, 1 μL downstream primer R aqueous solution, 12.5 μL 2×Precise PCR Mix and RNase Free dH2O.

[0086] The reaction program was: 94°C for 3 min; 94°C for 30 s, 50-60°C for 30 s, 72°C for 30 s, 35-40 cycles; 72°C for 5 min; 4°C∞.

[0087] 4. The PCR amplification products obtained in step 3 were respectively detected by 2% agarose gel. The loading volume was 5 μL.

[0088] The purpose of this step is to observe the polymorphism of the primer pair.

[0089] The results of partial agarose gel electrophoresis of PCR amplification products obtained by primer pair 15, primer pair 29 and primer pair 36 are shown in Table 1. Figure 2-Figure 4 (The DNAMarker is Trans 2K Plus DNAMarker.) The results showed that primer pair 15, primer pair 29 and primer pair 36 all had good polymorphism.

[0090] The upstream primers in primer pair 15, primer pair 29 and primer pair 36 are synthesized into fluorescent primers (achieved by adding fluorescent groups, such as HEX, ROX, FAM), which are sequentially composed of primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36.

[0091] The primer names, primer sequences and fluorescent colors of primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36 are shown in Table 2.

[0092] Table 2

[0093]

[0094]

[0095] 5. After completing step 4, use the genomic DNA of the sample to be tested as a template and perform PCR amplification using primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36 to obtain PCR amplification products.

[0096] The reaction system is 25 μL, consisting of 2 μL genomic DNA of the sample to be tested, 1 μL fluorescent primer F aqueous solution, 1 μL downstream primer R aqueous solution, 12.5 μL 2×Precise PCR Mix and RNase Free dH2O.

[0097] The reaction program was: 94°C for 3 min; 94°C for 30 s, 50-60°C for 30 s, 72°C for 30 s, 35-40 cycles; 72°C for 5 min; 4°C∞.

[0098] 6. Perform capillary electrophoresis on the PCR amplification products obtained in step 5 respectively.

[0099] The primer pair A.argyi.SSR15 was used to amplify Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11, respectively. The capillary electrophoresis results of the PCR amplification products were shown in the following order: Figure 5-Figure 12 .

[0100] The primer pair A.argyi.SSR29 was used to amplify Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11, respectively. The capillary electrophoresis results of the PCR amplification products were shown in the following order: Figure 13-Figure 20 .

[0101] The primer pair A.argyi.SSR36 was used to amplify Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11, respectively. The capillary electrophoresis results of the PCR amplification products were shown in the following order: Figure 21-Figure 28 .

[0102] The above experiment was repeated three times, and the banding patterns of the PCR amplification products of eight mugwort varieties were counted to obtain fingerprint maps.

[0103] The banding patterns of the PCR amplification products of the eight mugwort varieties are shown in Table 3. It can be seen that the primer combination consisting of primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36 can identify the eight mugwort varieties.

[0104] Table 3

[0105]

[0106] Example 2: Accuracy Experiment

[0107] The 8 tested mugwort leaves were taken from mugwort that had been phenotypically identified.

[0108] The name and phenotypic identification information of each tested mugwort leaf are shown in columns 1 to 2 of Table 4 .

[0109] Table 4

[0110]

[0111]

[0112] 1. Use QIAamp DNA Mini Kit to extract genomic DNA from the mugwort leaves to be tested (mugwort leaves a to be tested, mugwort leaves b to be tested, mugwort leaves c to be tested, mugwort leaves d to be tested, mugwort leaves e to be tested, mugwort leaves f to be tested, mugwort leaves g to be tested, or mugwort leaves h to be tested), and then use Qubit DNA Assay Kit for DNA quantification.

[0113] 2. After completing step 1, the genomic DNA of the mugwort leaves to be tested was tested on 1% agarose gel. The loading amount was 100 ng.

[0114] The results showed that the genomic DNA of the eight tested mugwort leaves were all intact.

[0115] 3. After completing step 2, the genomic DNA of the mugwort leaves to be tested is used as a template, and PCR amplification is performed using primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36 to obtain PCR amplification products.

[0116] The reaction system is 25 μL, consisting of 2 μL genomic DNA of the sample to be tested, 1 μL fluorescent primer F aqueous solution, 1 μL downstream primer R aqueous solution, 12.5 μL 2×Precise PCR Mix and RNase Free dH2O.

[0117] The reaction program was: 94°C for 3 min; 94°C for 30 s, 50-60°C for 30 s, 72°C for 30 s, 35-40 cycles; 72°C for 5 min; 4°C∞.

[0118] 4. Perform capillary electrophoresis on the PCR amplification products obtained in step 3. According to the capillary electrophoresis results, the band patterns of the PCR amplification products of the 8 tested mugwort leaves were obtained. The final experimental results are shown in the third column of Table 4. The details are as follows:

[0119] The tested wormwood leaf a was PCR amplified using primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36, and the PCR amplification product was subjected to capillary electrophoresis. The band pattern statistically obtained according to the capillary electrophoresis results was completely consistent with the band pattern of the sea wormwood shown in Table 3 in Example 1, indicating that the tested wormwood leaf a was indeed derived from the sea wormwood;

[0120] The tested wormwood leaf b was PCR amplified using primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36, and the PCR amplification product was subjected to capillary electrophoresis. The band pattern statistically obtained according to the capillary electrophoresis results was completely consistent with the band pattern of the northern wormwood shown in Table 3 in Example 1, indicating that the tested wormwood leaf a was indeed derived from the northern wormwood;

[0121] The tested wormwood leaf c was PCR amplified using primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36, and the PCR amplification product was subjected to capillary electrophoresis. The band pattern statistically obtained according to the capillary electrophoresis results was completely consistent with the band pattern of Qi Ai shown in Table 3 in Example 1, indicating that the tested wormwood leaf a was indeed derived from Qi Ai;

[0122] The tested wormwood leaf d was PCR amplified using primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36, and the PCR amplification product was subjected to capillary electrophoresis. The band type statistically obtained according to the capillary electrophoresis results was completely consistent with the band type of Qi Artemisia shown in Table 3 in Example 1, indicating that the tested wormwood leaf a was indeed derived from Qi Artemisia;

[0123] The tested wormwood leaf e was PCR amplified using primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36, and the PCR amplification product was subjected to capillary electrophoresis. The band pattern statistically obtained according to the capillary electrophoresis results was completely consistent with the band pattern of Nanyang Ai No. 8 shown in Table 3 in Example 1, indicating that the tested wormwood leaf a was indeed derived from Nanyang Ai No. 8;

[0124] The tested wormwood leaf f was PCR amplified using primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36, respectively, and the PCR amplification product was subjected to capillary electrophoresis. The band pattern statistically obtained according to the capillary electrophoresis results was completely consistent with the band pattern of Nanyang Ai No. 9 shown in Table 3 in Example 1, indicating that the tested wormwood leaf a was indeed derived from Nanyang Ai No. 9;

[0125] The tested wormwood leaf g was PCR amplified using primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36, and the PCR amplification product was subjected to capillary electrophoresis. The band pattern statistically obtained according to the capillary electrophoresis results was completely consistent with the band pattern of Nanyang wormwood No. 10 shown in Table 3 in Example 1, indicating that the tested wormwood leaf a was indeed derived from Nanyang wormwood No. 10;

[0126] The tested wormwood leaf h was PCR amplified using primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36, respectively. The PCR amplification products were subjected to capillary electrophoresis. The band pattern statistically obtained according to the capillary electrophoresis results was completely consistent with the band pattern of Nanyang wormwood No. 11 shown in Table 3 in Example 1, indicating that the tested wormwood leaf a was indeed derived from Nanyang wormwood No. 11.

[0127] The above results show that the method provided by the present invention can identify whether the tested mugwort leaves are Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11, thereby verifying the authenticity of the 8 mugwort varieties.

[0128] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A primer combination, comprising a primer pair A.argyi.SSR15, a primer pair A.argyi.SSR29 and a primer pair A.argyi.SSR36; The primer pair A.argyi.SSR15 consists of a primer A.argyi.SSR15-F having a nucleotide sequence as shown in SEQ ID NO:1 and a primer A.argyi.SSR15-R having a nucleotide sequence as shown in SEQ ID NO:2; The primer pair A.argyi.SSR29 consists of a primer A.argyi.SSR29-F having a nucleotide sequence as shown in SEQ ID NO:3 and a primer A.argyi.SSR29-R having a nucleotide sequence as shown in SEQ ID NO:4; The primer pair A.argyi.SSR36 consists of primer A.argyi.SSR36-F having a nucleotide sequence as shown in SEQ ID NO:5 and primer A.argyi.SSR36-R having a nucleotide sequence as shown in SEQ ID NO:

6.

2. The primer combination according to claim 1, characterized in that: The 5' ends of primer A.argyi.SSR15-F, primer A.argyi.SSR29-F and primer A.argyi.SSR36-F were modified with different fluorescent groups.

3. The primer combination according to claim 1 or 2, characterized in that: The primer combination consists of primer pair A.argyi.SSR15, primer pair A.argyi.SSR29 and primer pair A.argyi.SSR36.

4. Use of the primer combination described in any one of claims 1 to 3 in identifying Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No.

11.

5. A method for identifying whether the tested mugwort is or is a candidate for being Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11, comprising the following steps: using the genomic DNA of the tested mugwort as a template, respectively using the primer pairs A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 described in claims 1 to 3 to perform PCR amplification to obtain PCR amplification products; then performing the following judgment: If the primer pair A.argyi.SSR15 is used to amplify the 111 bp amplification product, the primer pair A.argyi.SSR29 is used to amplify the 138 bp, 152 bp and 157 bp amplification products, and the primer pair A.argyi.SSR36 is used to amplify the 107 bp, 116 bp and 119 bp amplification products, then the wormwood to be tested is or is a candidate for S. argyi; If the primer pair A.argyi.SSR15 is used to amplify an amplification product of 111 bp, the primer pair A.argyi.SSR29 is used to amplify an amplification product of 128 bp, 138 bp, 152 bp and 157 bp, and the primer pair A.argyi.SSR36 is used to amplify an amplification product of 116 bp, then the mugwort to be tested is or is a candidate for northern mugwort; If the primer pair A.argyi.SSR15 is used to amplify the 111 bp amplification product, the primer pair A.argyi.SSR29 is used to amplify the 128 bp, 152 bp and 157 bp amplification products, and the primer pair A.argyi.SSR36 is used to amplify the 116 bp amplification product, then the mugwort to be tested is or the candidate is Qi mugwort; If the primer pair A.argyi.SSR15 is used to amplify an amplification product of 111 bp, the primer pair A.argyi.SSR29 is used to amplify an amplification product of 138 bp and 152 bp, and the primer pair A.argyi.SSR36 is used to amplify an amplification product of 116 bp, then the mugwort to be tested is or is a candidate for Artemisia argyi; If the primer pair A.argyi.SSR15 is used to amplify an amplification product of 111 bp, the primer pair A.argyi.SSR29 is used to amplify an amplification product of 128 bp, 138 bp and 157 bp, and the primer pair A.argyi.SSR36 is used to amplify an amplification product of 116 bp, then the mugwort to be tested is or is a candidate for Nanyang mugwort No. 8; If the primer pair A.argyi.SSR15 is used to amplify the 111 bp amplification product, the primer pair A.argyi.SSR29 is used to amplify the 138 bp amplification product, and the primer pair A.argyi.SSR36 is used to amplify the 111 bp and 116 bp amplification products, then the mugwort to be tested is or is a candidate for Nanyang mugwort 9; If the primer pair A.argyi.SSR15 is used to amplify the amplified products of 111bp, 128bp and 147bp, the primer pair A.argyi.SSR29 is used to amplify the amplified products of 138bp, 152bp and 157bp, and the primer pair A.argyi.SSR36 is used to amplify the amplified products of 111bp and 116bp, then the mugwort to be tested is or the candidate is Nanyang mugwort No. 10; If the primer pair A.argyi.SSR15 can obtain amplification products of 105bp, 111bp and 123bp, the primer pair A.argyi.SSR29 can obtain amplification products of 128bp, 138bp and 152bp, and the primer pair A.argyi.SSR36 can obtain an amplification product of 116bp, then the mugwort to be tested is or the candidate is Nanyang Mugwort No.

11.

6. A method for identifying whether the mugwort to be tested is or is a candidate for being one of Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11, comprising the following steps: (1) Using the genomic DNA of the mugwort to be tested as a template, PCR amplification is performed using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 described in claims 1 to 3 to obtain a PCR amplification product; (2) using the genomic DNA of Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11 as templates, respectively, using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 described in claims 1 to 3 to perform PCR amplification to obtain PCR amplification products; (3) Make the following judgment: If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for the mugwort to be tested are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for the wormwood to be tested, then the mugwort to be tested is or is a candidate for the wormwood to be wormwood; If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for the tested mugwort are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for northern mugwort, then the tested mugwort is or is a candidate for northern mugwort; If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for the tested mugwort are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for Qi mugwort, then the tested mugwort is or the candidate is Qi mugwort; If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for the mugwort to be tested are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for Qi Ai, then the mugwort to be tested is or the candidate is Qi Ai; If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for the tested mugwort are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for Nanyang mugwort No. 8, then the tested mugwort is or is a candidate for Nanyang mugwort No. 8; If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for the tested mugwort are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for Nanyang mugwort No. 9, then the tested mugwort is or the candidate is Nanyang mugwort No. 9; If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for the tested mugwort are all consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 for Nanyang mugwort No. 10, then the tested mugwort is or is a candidate for Nanyang mugwort No. 10; If the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for the mugwort to be tested are consistent in size with the PCR amplification products obtained by using the primer pair A.argyi.SSR15, the primer pair A.argyi.SSR29 and the primer pair A.argyi.SSR36 respectively for Nanyang mugwort No. 11, then the mugwort to be tested is or the candidate is Nanyang mugwort No.

11.

7. The method according to claim 5 or 6, characterized in that: The PCR amplification product was detected by electrophoresis.

8. The method according to claim 7, characterized in that: The electrophoresis is capillary electrophoresis.

9. A kit comprising the primer combination according to any one of claims 1 to 3.

10. Use of the kit according to claim 9 in identifying Hai Ai, Bei Ai, Qi Ai, Qi Ai, Nanyang Ai No. 8, Nanyang Ai No. 9, Nanyang Ai No. 10 and Nanyang Ai No. 11.

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