PCR (Polymerase Chain Reaction) detection reagent for distinguishing different peanut varieties and application of PCR detection reagent

Abstract

The invention belongs to the technical field of biology, and discloses a PCR (Polymerase Chain Reaction) detection reagent for distinguishing different peanut varieties and application of the PCR detection reagent, a large number of specific primers are developed on the basis of genome data of Arachis duranensis and Arachis ipaensis, 5 pairs of primers and 7 pairs of primers are respectively screened from the Arachis duranensis and the Arachis ipaensis, and DNA (Deoxyribose Nucleic Acid) of the Arachis duranensis and the Arachis ipaensis is extracted for PCR verification. The primer disclosed by the invention is high in specificity, and the primer can provide a new tool for distinguishing the Arachis duranensis from the Arachis ipaensis, species evolution and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a PCR detection reagent for distinguishing different peanut varieties and an application thereof. Background Art

[0002] Peanut ( Peanut ) originated in South America and is an important protein and vegetable oil plant; at the same time, peanut vines and other by-products also have feeding value. Cultivated peanuts are allotetraploid plants, derived from wild diploid peanuts. Arachis duranensis and Arachis ipaensis Natural hybridization is formed by chromosome doubling.

[0003] Compared with wild peanuts, cultivated peanuts have low genetic diversity, which leads to weaker biological and abiotic resistance. Therefore, introducing wild peanut gene resources into cultivated peanuts is an important means to improve the biological and abiotic resistance of cultivated peanuts. Traditionally, the use of distant hybridization technology, that is, hybridization of wild diploid peanuts with cultivated peanuts, can solve the problem of low genetic diversity of peanuts, thereby improving the resistance of peanuts, such as disease resistance and drought resistance. Among them, the use of colchicine treatment Arachis duranensis and Arachis ipaensis , doubling the chromosomes to tetraploid; then, hybridizing with cultivated peanuts can also improve the resistance of peanuts. For example, by constructing artificial tetraploid peanuts[ A. duranensis × A. ipaensis ] 4× When hybridized with peanuts, the offspring can significantly improve drought tolerance. Arachis duranensis and Arachis ipaensis have similar phenotypes and genomic sequences. Arachis duranensis and Arachis ipaensis There are challenges. Although genomic methods can be used to distinguish Arachis duranensis and Arachis ipaensis and identification of artificial tetraploid peanut[ A. duranensis × A. ipaensis ] 4× However, the genome sequencing cycle is about 3 months, and the cost is still more than 50,000 yuan, which is time-consuming and expensive. Therefore, it is urgent to develop a PCR detection reagent to distinguish Arachis duranensis and Arachis ipaensis . Summary of the invention

[0004] In order to solve the above problems, the present invention provides a PCR detection reagent for distinguishing different peanut varieties and its application.

[0005] The present invention is achieved through the following technical solutions: A PCR detection reagent for distinguishing different peanut varieties, comprising one or more of the specific primers AdJT9P7, AdDT22E, AdYJ0J8, AdFIS15, AdJ4978, AiW94MM, AiZ32X2, Ai6Y57C, AiRV4HN, Ai930A9, AiH8Y97 and AiQKV15: The forward primer of AdJT9P7 is shown as SEQ ID NO.1, and the reverse primer is shown as SEQ ID NO.2.

[0006] The forward primer of AdDT22E is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ID NO.4.

[0007] The forward primer of AdYJ0J8 is shown as SEQ ID NO.5, and the reverse primer is shown as SEQ ID NO.6.

[0008] The forward primer of AdFIS15 is shown in SEQ ID NO.7, and the reverse primer is shown in SEQ ID NO.8.

[0009] The forward primer of AdJ4978 is shown in SEQ ID NO.9, and the reverse primer is shown in SEQ ID NO.10.

[0010] The forward primer of AiW94MM is shown as SEQ ID NO.11, and the reverse primer is shown as SEQ ID NO.12.

[0011] The forward primer of AiZ32X2 is shown in SEQ ID NO.13, and the reverse primer is shown in SEQ ID NO.14.

[0012] The forward primer of Ai6Y57C is shown in SEQ ID NO.15, and the reverse primer is shown in SEQ ID NO.16.

[0013] The forward primer of AiRV4HN is shown in SEQ ID NO.17, and the reverse primer is shown in SEQ ID NO.18.

[0014] The forward primer of Ai930A9 is shown in SEQ ID NO.19, and the reverse primer is shown in SEQ ID NO.20.

[0015] The forward primer of AiH8Y97 is shown as SEQ ID NO.21, and the reverse primer is shown as SEQ ID NO.22.

[0016] The forward primer of AiQKV15 is shown in SEQ ID NO.23, and the reverse primer is shown in SEQ ID NO.24.

[0017] Preferably, the peanut varieties to be distinguished are Arachis duranensis and Arachis ipaensis .

[0018] The PCR detection reagent for distinguishing different peanut varieties is effective in distinguishing wild peanuts Arachis duranensis and Arachis ipaensis Application in.

[0019] The PCR detection reagent for distinguishing different peanut varieties is Arachis duranensis and Arachis ipaensis Application in kinship identification.

[0020] Preferably, the specific method is as follows: (1) Extract DNA from leaves of unknown samples.

[0021] (2) Using the DNA extracted in step (1) as a template, PCR amplification is performed using the PCR detection reagent.

[0022] (3) Determine the results based on the amplified bands, as follows: The specific primer AdJT9P7 amplified a band with a size of 471 bp, which was determined to be Arachis duranensis , no band is judged as Arachis ipaensis .

[0023] The specific primer AdDT22E amplified a band with a size of 324 bp, which was determined to be Arachis duranensis , no band is judged as Arachis ipaensis .

[0024] The specific primer AdYJ0J8 amplified a band with a size of 413 bp, which was determined to be Arachis duranensis , no band is judged as Arachis ipaensis .

[0025] The specific primer AdFIS15 amplified a band with a size of 302 bp, which was determined to be Arachis duranensis , no band is judged as Arachis ipaensis .

[0026] The specific primer AdJ4978 amplified a band with a size of 210 bp, which was determined to be Arachis duranensis , no band is judged as Arachis ipaensis .

[0027] The specific primer AiW94MM amplified a band with a size of 712 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis .

[0028] The specific primer AiZ32X2 amplified a band with a size of 824 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis .

[0029] The specific primer Ai6Y57C amplified a band with a size of 792 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis .

[0030] The specific primer AiRV4HN amplified a band with a size of 981 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis .

[0031] The specific primer Ai930A9 amplified a band with a size of 709 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis .

[0032] The specific primer AiH8Y97 amplified a band with a size of 572 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis .

[0033] The specific primer AiQKV15 amplified a band with a size of 344 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis .

[0034] Preferably, the PCR system is: DNA template 1 μL~3 μL, 2×Master PCR Mix 9 μL~11 μL, Forward primer 0.4 μL~0.6 μL, Reverse primer 0.4 μL~0.6 μL, and water is added to 19 μL~21 μL.

[0035] Preferably, the PCR system is: 1 μL DNA template, 10 μL 2×Master PCR Mix, 0.5 μL Forward primer, 0.5 μL Reverse primer, and water is added to 20 μL.

[0036] Preferably, the PCR program is: 93°C-95°C, 5 min, 93°C-95°C, 29s-31s, 58°C-62°C, 29s-31s, 71°C-73°C, 1 min-2 min, 35 cycles, 71°C-73°C, 4 min-6 min. The annealing temperature is 60°C.

[0037] Preferably, the PCR program is: 94°C, 5 min, 94°C, 30 s, 60°C, 30 s, 72°C, 1 min, 35 cycles, 72°C, 5 min. The annealing temperature is 60°C.

[0038] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a PCR detection reagent for distinguishing different peanut varieties, wherein the PCR detection reagent is composed of one or more of the specific primers AdJT9P7, AdDT22E, AdYJ0J8, AdFIS15, AdJ4978, AiW94MM, AiZ32X2, Ai6Y57C, AiRV4HN, Ai930A9, AiH8Y97 and AiQKV15. The present invention utilizes the completed comparative genomics method to compare Arachis duranensis and Arachis ipaensis The genome sequences of Arachis duranensis and Arachis ipaensis The specific DNA sequences were identified and specific amplification primers were designed for these specific sequences. The specificity and amplification efficiency of the primers were verified one by one using the PCR method. The above 12 specific primers were screened out and can be used as PCR detection reagents to distinguish different peanut varieties. Arachis duranensis and Arachis ipaensis Identification of two wild peanuts. Using this PCR detection reagent to detect two wild peanuts, you only need to extract DNA for PCR amplification, which can be completed in 1 day and costs less than 1,000 yuan. The primers are highly specific and can accurately identify and distinguish Arachis duranensis and Arachis ipaensis Two wild peanut species provide new tools for species evolution and more. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 The present invention is 12 pairs of specific primers in Arachis duranensis and Arachis ipaensis Amplification results at the genomic level: M represents DNA marker DL2000; AdJT9P7-AiQKV15 represents 12 pairs of primers in Arachis duranensis and Arachis ipaensis The amplification results. Among them, Ad represents Arachis duranensis , Ai said Arachis ipaensis . DETAILED DESCRIPTION

[0041] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0043] The inventive concept of the present invention is as follows: Arachis duranensis and Arachis ipaensis have similar phenotypes and genomic sequences. Arachis duranensis and Arachis ipaensis There are challenges. Although genomic methods can be used to distinguish Arachis duranensis and Arachis ipaensis and identification of artificial tetraploid peanut[ A. duranensis × A. ipaensis ] 4× However, the genome sequencing cycle is about 3 months and the cost is still more than 50,000 yuan, which is time-consuming and expensive.

[0044] Based on this, the present invention provides a PCR detection reagent for distinguishing different peanut varieties, and the PCR detection reagent is composed of one or more of the specific primers AdJT9P7, AdDT22E, AdYJ0J8, AdFIS15, AdJ4978, AiW94MM, AiZ32X2, Ai6Y57C, AiRV4HN, Ai930A9, AiH8Y97 and AiQKV15. The present invention uses the completed comparative genomics method to compare Arachis duranensis and Arachis ipaensis The genome sequences of Arachis duranensis and Arachis ipaensis The specific DNA sequences were identified and specific amplification primers were designed for these specific sequences. The specificity and amplification efficiency of the primers were verified one by one using the PCR method. The above 12 specific primers were screened out and can be used as PCR detection reagents to distinguish different peanut varieties. Arachis duranensis and Arachis ipaensis Identification of two wild peanuts. Using this PCR detection reagent to detect two wild peanuts, you only need to extract DNA for PCR amplification, which can be completed in 1 day and costs less than 1,000 yuan. The primers are highly specific and can accurately identify and distinguish Arachis duranensis and Arachis ipaensis Two wild peanut species provide new tools for species evolution and more.

[0045] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0046] The Chinese name of 2×Master PCR Mix is ​​2×PCR premix.

[0047] The Chinese name of Forward primer is forward primer.

[0048] The Chinese name of Reverse primer is reverse primer.

[0049] Embodiment 1, Arachis duranensis and Arachis ipaensis Identification of specific sequences and design of amplification primers Arachisduranensis , gene version number is V14167.gnm2.J7QH and Arachis ipaensis The genome sequence and GFF3 file of gene version number K30076.gnm2.1GWY were downloaded from the PeanutBase public database https: / / dev.peanutbase.org / download. The local BLAST program was used to Arachis duranensis The amino acid sequence of Arachis ipaensisThe amino acid sequence of was used as the database, and BLASTp was run with the E-value set to 10 -4 ,Record Arachis duranensis There is no sequence matching result in the database. Arachis ipaensis The amino acid sequence of Arachis duranensis The amino acid sequence of was used as the database, and BLASTp was run with the E-value set to 10 -4 ,Record Arachis ipaensis There are no sequences with matching results in the database. These amino acid sequences are named genome-specific amino acids.

[0050] According to the GFF3 file Arachis duranensis and Arachis ipaensis The DNA sequence corresponding to the specific amino acid was extracted from the genome. Then, the local BLAST program was used to Arachis duranensis The DNA sequence of the specific amino acid of Arachis ipaensis The genome sequence of was used as the database, and BLASTn was run with an E-value of 10 -9 ,Record Arachis duranensis There is no sequence alignment in the database. Similarly, use the local BLAST program to Arachis ipaensis The DNA sequence of the specific amino acid of Arachis duranensis The genome sequence of was used as the database, and BLASTn was run with an E-value of 10 -9 ,Record Arachis ipaensis There is no sequence matching result in the database. The obtained sequence is a genome-specific DNA gene. A total of 32 specific genes were obtained, and the names of the specific genes are shown in Table 1.

[0051] Primer priemier 5 software was used to design specific amplification primers for 32 specific gene sequences, and all specific primers were synthesized by Qingke Biotechnology Co., Ltd.

[0052] Table 1 Arachis duranensis and Arachis ipaensis Genome-specific genes

[0053] Example 2: Differentiation Arachis duranensis and Arachis ipaensis Screening of species-specific primers Arachis duranensis V14167 strain and Arachis ipaensisPlants of K30076 strain were planted in a greenhouse. The leaves of healthy plants were selected and DNA was extracted using the Tiangen Plant DNA Extraction Kit from Beijing, China. Three biological replicates were set up. The specific operation method was referred to the instructions of the kit. The DNA concentration was detected by UV spectrophotometer, and the DNA integrity was detected by 1% agarose electrophoresis. DNA that met the requirements of PCR amplification was used for subsequent experiments. Other raw materials, reagents, and methods used were obtained through conventional means in the field unless otherwise specified.

[0054] The PCR system was: 1 μL DNA template, 10 μL 2×Master PCR Mix, 0.5 μL Forward primer, 0.5 μL Reverse primer, and water was added to 20 μL.

[0055] The PCR program was: 94°C, 5 min, 94°C, 30 s, 60°C, 30 s, 72°C, 1 min, 35 cycles, 72°C, 5 min. The annealing temperature was 60°C. Arachis duranensis and Arachis ipaensis The specific primer information and amplified fragment information are shown in Table 2.

[0056] The PCR amplification products were detected by 1% agarose gel electrophoresis. If the amplified band was consistent with the predicted band size, it was a specific amplification product. After verification, a total of 5 pairs of amplification products were obtained. Arachis duranensis Specific primers for amplification Arachis ipaensis Specific primers such as Figure 1 As shown, Figure 1 Some primers have primer dimers, which are all below 100 bp and do not belong to the target band; primers without primer dimers are considered to have higher accuracy. These primers can be used to distinguish Arachis duranensis and Arachis ipaensis .

[0057] Table 2 Classification Arachis duranensis and Arachis ipaensis Specific primers

[0058] Example 3: Differentiation Arachis duranensis and Arachis ipaensis Application of species-specific primers 1. Sample source Collect 12 existing healthy Arachis duranensis Plant leaves, and 12 existing healthy Arachis ipaensis The leaves of plants, mixed and disordered, making it impossible to distinguish Arachis duranensis and Arachis ipaensis Species are numbered 1 to 24.

[0059] 2. Nucleic acid extraction DNA was extracted using the Tiangen Plant DNA Extraction Kit from Beijing, China. The specific operation method is as follows.

[0060] (1) Take about 100 mg of fresh plant tissue and grind it thoroughly with liquid nitrogen.

[0061] (2) Quickly transfer the ground powder to a centrifuge tube pre-filled with 700 μL of 65°C preheated buffer GP1. Quickly invert to mix, then place the centrifuge tube in a 65°C water bath for 20 min. Invert the tube several times to mix the sample.

[0062] (3) Add 700 μL of chloroform, mix thoroughly, and centrifuge at 12,000 rpm for 5 min.

[0063] (4) Carefully transfer the upper aqueous phase obtained in the previous step into a new centrifuge tube, add 700 μL of buffer GP2, and mix thoroughly.

[0064] (5) Transfer the mixed liquid into adsorption column CB3, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0065] (6) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0066] (7) Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0067] (8) Repeat step 7.

[0068] (9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, and discard the waste liquid. Leave the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0069] (10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50 μL of elution buffer TE to the middle of the adsorption membrane, leave it at room temperature for 2 min, centrifuge it at 12000 rpm for 2 min, and collect the solution in a centrifuge tube.

[0070] DNA concentration was detected by ultraviolet spectrophotometer, and DNA integrity was detected by 1% agarose electrophoresis. DNA that met the requirements of PCR amplification was used for subsequent experiments. Other raw materials, reagents, and methods used were obtained by conventional means in the art unless otherwise specified. PCR amplification was performed on sample DNA using the 12 pairs of specific primers verified in Example 2.

[0071] The PCR system was: 1 μL DNA template, 10 μL 2×Master PCR Mix, 0.5 μL Forward primer, 0.5 μL Reverse primer, and water was added to 20 μL.

[0072] The PCR program was as follows: 94°C, 5 min, 94°C, 30 s, 60°C, 30 s, 72°C, 1 min, 35 cycles, 72°C, 5 min. The annealing temperature was 60°C.

[0073] Molecular sequencing was used as a control, and the results are shown in Table 3.

[0074] Table 3 Molecular sequencing detection control results

[0075] It should be noted that the PCR system of the present invention is: DNA template 1μL~3μL, 2×Master PCR Mix 9μL~11μL, Forward primer 0.4μL~0.6μL, Reverse primer 0.4μL~0.6μL, water is added to 19μL~21μL; the PCR program is: 93℃~95℃, 5min, 93℃~95℃, 29s~31s, 58℃~62℃, 29s~31s, 71℃~73℃, 1min~2min, 35 cycles, 71℃~73℃, 4min~6min, annealing temperature is 60℃, all of which can distinguish wild peanuts. Arachis duranensis and Arachis ipaensis .

[0076] In summary, the present invention has developed a Arachis duranensis and Arachis ipaensis Specific primers for distinguishing the two species; the efficiency and reliability of the specific primers were verified by PCR amplification. Arachis duranensis and Arachis ipaensis The genome sequencing of two wild peanut species can be completed in one day at a cost of less than 1,000 yuan. The genome sequencing cycle is about 3 months and the cost is more than 50,000 yuan. Arachis duranensis and Arachis ipaensis, Arachis duranensis and of Arachis ipaensis It provides new tools for studies such as kinship identification and species evolution.

[0077] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0078] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A PCR detection reagent for distinguishing different peanut varieties, characterized in that: The PCR detection reagent is composed of one or more of the specific primers AdJT9P7, AdDT22E, AdYJ0J8, AdFIS15, AdJ4978, AiW94MM, AiZ32X2, Ai6Y57C, AiRV4HN, Ai930A9, AiH8Y97 and AiQKV15; The forward primer of AdJT9P7 is shown in SEQ ID NO.1, and the reverse primer is shown in SEQ ID NO.2; The forward primer of AdDT22E is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ID NO.4; The forward primer of AdYJ0J8 is shown in SEQ ID NO.5, and the reverse primer is shown in SEQ ID NO.6; The forward primer of AdFIS15 is shown in SEQ ID NO.7, and the reverse primer is shown in SEQ ID NO.8; The forward primer of AdJ4978 is shown in SEQ ID NO.9, and the reverse primer is shown in SEQ ID NO.10; The forward primer of AiW94MM is shown in SEQ ID NO.11, and the reverse primer is shown in SEQ ID NO.12; The forward primer of AiZ32X2 is shown in SEQ ID NO.13, and the reverse primer is shown in SEQ ID NO.14; The forward primer of Ai6Y57C is shown in SEQ ID NO.15, and the reverse primer is shown in SEQ ID NO.16; The forward primer of AiRV4HN is shown in SEQ ID NO.17, and the reverse primer is shown in SEQ ID NO.18; The forward primer of Ai930A9 is shown in SEQ ID NO.19, and the reverse primer is shown in SEQ ID NO.20; The forward primer of AiH8Y97 is shown in SEQ ID NO.21, and the reverse primer is shown in SEQ ID NO.22; The forward primer of AiQKV15 is shown in SEQ ID NO.23, and the reverse primer is shown in SEQ ID NO.

24.

2. The PCR detection reagent for distinguishing different peanut varieties according to claim 1, characterized in that: The peanut varieties distinguished are Arachis duranensis and Arachis ipaensis .

3. The PCR detection reagent for distinguishing different peanut varieties as claimed in claim 1 is effective in distinguishing wild peanut Arachis duranensis and Arachis ipaensis Application in.

4. The use according to claim 3, characterized in that The specific method is as follows: (2) Extract DNA from unknown leaf samples; (3) using the DNA extracted in step (1) as a template and performing PCR amplification using the PCR detection reagent described in claim 1; (4) Determine the results based on the amplified bands, as follows: The specific primer AdJT9P7 amplified a band with a size of 471 bp, which was determined to be Arachis duranensis , no band is judged as Arachis ipaensis ; The specific primer AdDT22E amplified a band with a size of 324 bp, which was determined to be Arachis duranensis , no band is judged as Arachis ipaensis ; The specific primer AdYJ0J8 amplified a band with a size of 413 bp, which was determined to be Arachis duranensis , no band is judged as Arachis ipaensis ; The specific primer AdFIS15 amplified a band with a size of 302 bp, which was determined to be Arachis duranensis , no band is judged as Arachis ipaensis ; The specific primer AdJ4978 amplified a band with a size of 210 bp, which was determined to be Arachis duranensis , no band is judged as Arachis ipaensis ; The specific primer AiW94MM amplified a band with a size of 712 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis ; The specific primer AiZ32X2 amplified a band with a size of 824 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis ; The specific primer Ai6Y57C amplified a band with a size of 792 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis ; The specific primer AiRV4HN amplified a band with a size of 981 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis ; The specific primer Ai930A9 amplified a band with a size of 709 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis ; The specific primer AiH8Y97 amplified a band with a size of 572 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis ; The specific primer AiQKV15 amplified a band with a size of 344 bp, which was determined to be Arachis ipaensis , no band is judged as Arachis duranensis .

5. The use according to claim 4, characterized in that The PCR system is: DNA template 1 μL~3 μL, 2×Master PCR Mix 9 μL~11 μL, Forward primer 0.4 μL~0.6 μL, Reverse primer 0.4 μL~0.6 μL, and water is added to 19 μL~21 μL.

6. The use according to claim 5, characterized in that The PCR system is: 1 μL DNA template, 10 μL 2×MasterPCR Mix, 0.5 μL Forward primer, 0.5 μL Reverse primer, and water is added to 20 μL.

7. The use according to claim 4, characterized in that The PCR program is: 93°C~95°C, 5 min, 93°C~95°C, 29s~31s, 58°C~62°C, 29s~31s, 71°C~73°C, 1min~2min, 35 cycles, 71°C~73°C, 4min~6min, and the annealing temperature is 60°C.

8. The use according to claim 7, characterized in that The PCR program was: 94°C, 5 min, 94°C, 30 s, 60°C, 30 s, 72°C, 1 min, 35 cycles, 72°C, 5 min, and the annealing temperature was 60°C.

Images