Kit and detection method for identifying white clover and red clover seeds

Abstract

The invention discloses a kit and a detection method for identifying seeds of trifolium repens and trifolium pratense, the designed primer combination can effectively identify the seeds of trifolium repens and trifolium pratense, and the method for identifying the seeds of trifolium repens and trifolium pratense by using the primer has the characteristics of strong sensitivity, high accuracy, strong repeatability and the like; the PCR primer combination for identifying the white clover and red clover seeds also has the characteristics of short time consumption and simplicity and convenience in operation, the identification can be completed in a relatively short time under laboratory conditions, and the detection method disclosed by the invention is beneficial to rapid identification of the white clover and red clover seeds by inspectors, so that the detection efficiency is improved. Therefore, the primer combination provided by the invention has relatively high application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a kit and a detection method for identifying white clover and red clover seeds. Background Art

[0002] White clover (Trifolium repens) and red clover (T. pratense) are important legume forages, and the two have significant differences in growth characteristics: white clover is more suitable for planting in the humid climate, orchards, forests and high precipitation areas in the south, especially growing well under high humidity, rainy and mild climate conditions; red clover is more cold-resistant and suitable for planting in the northern temperate regions or mountainous areas with colder climates.

[0003] In practical applications, white clover and red clover offer both economic and ecological benefits. With the large-scale development of animal husbandry and the advancement of ecological and environmental protection, the demand for high-quality seeds and seedlings of white clover and red clover is increasing. Although white clover and red clover both belong to the genus Trifolium in the Leguminosae family, there are significant differences between the two in the aforementioned applications.

[0004] Mature white clover plants have the characteristics of low creeping growth, soft and juicy leaves with good palatability, suitable as high-quality grazing grass; white clover has a strong nitrogen fixation ability, and can improve soil fertility and promote ecological restoration through symbiosis with rhizobia; it has a wide adaptability, is shade-tolerant and trampling-resistant, and prefers clay and acid-resistant soils. It has a shallow root system and can be used to improve topsoil. It is resistant to waterlogging but not drought-resistant; in terms of the ecological environment, white clover is often used in urban greening.

[0005] Mature red clover plants grow upright, are tall, and have a large biomass, making them suitable for mowing and making hay or silage. Compared with white clover, they contain higher crude protein. Red clover adapts to neutral to slightly acidic soils, has deep roots, and can be used to improve deep soils. It is drought-resistant but not resistant to long-term waterlogging. In terms of the ecological environment, red clover is often used for ecological restoration. In addition, red clover grows fast and can form high-yield forage communities. In addition to being used as feed, it also has certain medicinal value, such as anti-osteoporosis effects.

[0006] However, due to the similar morphology of white clover and red clover seeds, they are difficult to distinguish with the naked eye and are easily mixed during production. They may be mistakenly planted in unsuitable habitats due to different ecological adaptability, resulting in low survival rates and reduced yields, affecting planting benefits; they may also affect the quality and selling price of forage due to differences in nutritional quality, causing economic losses and damaging market reputation. At present, the identification of white clover and red clover seeds mainly relies on traditional morphological observation, but this method is time-consuming and labor-intensive, easily affected by human factors, and has low accuracy. With the rapid development of molecular technology, the use of molecular means to develop more efficient and accurate identification technologies can achieve rapid, accurate and convenient identification of white clover and red clover seeds, which is of great significance for ensuring the production quality and promotion and application of forage seeds.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The invention aims to provide a kit and a detection method for identifying white clover and red clover seeds.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides a PCR primer pair for distinguishing white clover and red clover seeds, wherein the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO.2.

[0011] In a second aspect, the present invention provides the use of the above-mentioned PCR primer pair in preparing a kit for identifying white clover and red clover seeds.

[0012] In a third aspect, the present invention provides the use of the above-mentioned PCR primer pair in identifying white clover and red clover seeds.

[0013] In a fourth aspect, the present invention provides a kit for identifying white clover and red clover seeds, comprising the above-mentioned PCR primer pair.

[0014] In a fifth aspect, the present invention provides the use of the above-mentioned kit in identifying white clover and red clover seeds.

[0015] In a sixth aspect, the present invention provides a method for identifying white clover and red clover seeds, comprising:

[0016] Extract genomic DNA from the seeds to be tested, then use the extracted DNA as a template to perform PCR amplification using the above kit, and then detect the PCR product by agarose gel electrophoresis;

[0017] If the band size amplified by electrophoresis is 162bp, the sample to be tested is white clover seeds; if the band size amplified by electrophoresis is 178bp, the sample to be tested is red clover seeds; if the band size amplified by electrophoresis is both 162bp and 178bp, the sample to be tested is a mixture of white clover and red clover seeds.

[0018] The present invention has the following beneficial effects:

[0019] The primer combination designed by the present invention can effectively identify white clover and red clover seeds, and the method for identifying white clover and red clover seeds using the primers has the characteristics of high sensitivity, high accuracy, strong repeatability, etc.; the identification of white clover and red clover seeds using the PCR primer combination of the present invention is also characterized by short time consumption and simple operation, and the identification can be completed in a short time under laboratory conditions. The detection method of the present invention helps inspectors to quickly identify white clover and red clover seeds. Therefore, the primer combination provided by the present invention has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 These are morphological comparisons of white clover and red clover at different growth stages, where (a) to (f) show white clover on the left and red clover on the right; (a) is a comparison of white clover and red clover seeds; (b) is a comparison of single white clover and red clover seeds; (c) is a comparison of 15-day-old seedlings of white clover and red clover; (d) is a phenotypic comparison of 65-day-old white clover and red clover leaves; (e) is a 100-day-old white clover and red clover leaf leaves; (f) is a phenotypic comparison of 100-day-old white clover and red clover; except for the scale bar in Figure (b), which represents 0.5 mm, the scale bars in the rest are 2 cm;

[0022] Figure 2 The PCR identification results of white clover and red clover seeds in Example 4 are shown. Numbers 1-20 in the figure represent 20 individual plants of white clover and red clover, "M" represents DNA marker, and "H2O" represents the negative control group using ddH2O as the template;

[0023] Figure 3The PCR identification results between white clover and red clover seeds of different germplasms in Example 5 are shown. Numbers 1-10 for white clover represent 10 different germplasms of white clover, and numbers 1-8 for red clover represent 8 different germplasms of red clover. "M" represents DNA Marker, and "H2O" represents the negative control group using ddH2O as the template.

[0024] Figure 4 The PCR identification results of the mixed seeds of white clover and red clover in Example 6; in the figure: 100:0, 90:10, 75:25, 50:50, 25:75, 10:90 and 0:100 represent the ratios of the mixed genomic DNA templates of white clover and red clover, "M" represents DNA Marker, and "H2O" represents the negative control group using ddH2O as the template;

[0025] Figure 5 The following are the sequence alignments and partial sequencing peaks of white clover and red clover in Example 7; (a) shows the sequence alignment of white clover and red clover using DNAman, where the shaded area indicates sequence identity, and the unshaded area indicates sequence differences caused by single nucleotide polymorphisms and insertions and deletions; (b) shows the partial sequencing peaks of white clover and red clover;

[0026] Figure 6 This is a comparison of the melting curves in Example 8. In the figure: the blue line represents the melting curves of four white clover germplasms, the red line represents the melting curves of four red clover germplasms, and the black line represents the melting curve of the negative control group using ddH2O as the template. DETAILED DESCRIPTION

[0027] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0028] The morphology of white clover and red clover at different stages Figure 1 As shown, in order to quickly, accurately and efficiently distinguish white clover and red clover from the seed stage, the present invention screened a target region in the genome of each plant based on the characteristics of the gene sequences of the two plants, and designed a pair of primers for distinguishing white clover and red clover seeds. The sequence of the target region of white clover is shown in SEQ ID NO.3; the sequence of the target region of red clover is shown in SEQ ID NO.4.

[0029] The primers designed for these two sequences are:

[0030] Trifolium-F(5′-3′): TATGACGGAAAACGATATATGAG-SEQ ID NO.1;

[0031] Trifolium-R(5'-3'): TAAAAATTTGAAAAGAAAAAAGTCC-SEQ ID NO. 2.

[0032] Based on the above primer set, the present invention can provide a kit for identifying white clover and red clover seeds, which contains the above primer pair and other conventional reagents for PCR amplification, including PCR amplification reagents, DNA template and ddH2O.

[0033] Among them, PCR amplification reagents include Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, and other commonly used auxiliary reagents.

[0034] In addition to the PCR amplification reagents provided herein, other commercially available products of the same type can also be used in the PCR reaction system. Simply add the specified amount of DNA template, ddH2O, and primers before amplification to perform PCR amplification. This requires that the positive control sample amplify the correct band or curve, while the negative control sample exhibits no amplified band or curve.

[0035] By amplifying and detecting the DNA of the test sample, it was found that when the test sample was white clover seeds, the size of the electrophoresis amplified band was 162bp; when the test sample was red clover seeds, the size of the electrophoresis amplified band was 178bp; when the test sample was a mixture of white clover and red clover seeds, amplified bands of both 162bp and 178bp were present.

[0036] Since the above detection primers or kit can be used to identify whether the seed sample to be tested is white clover, red clover or a mixture of the two, the present invention also provides a method for identifying white clover and red clover seeds, which comprises the following steps:

[0037] (1) Extracting genomic DNA from seeds to be tested;

[0038] (2) using the DNA obtained in step (1) as a template and the above primer pair to perform a PCR amplification reaction;

[0039] (3) taking the PCR amplification product and detecting it by agarose gel electrophoresis;

[0040] (4) Determine the variety of the seeds to be tested based on the bands that appear in the electrophoresis strips.

[0041] In the above detection method, the reaction system of PCR amplification can be adjusted according to the actual situation of the experiment. The reaction system can be a 10 μL system, a 20 μL system, a 50 μL system, a 100 μL system, or other systems.

[0042] Among them, when the reaction system is 10 μL, its specific components are: 5 μL of PCR amplification reagent, 0.5 μL of forward primer, 0.5 μL of reverse primer, 0.5 μL of template DNA, and 3.5 μL of ddH2O; among them, the concentration of template DNA is 100 ng / μL; the concentrations of forward primer and reverse primer are 10 μM.

[0043] The corresponding PCR amplification reaction program was as follows: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 10 s, annealing at 52.7°C for 30 s, extension at 68°C for 30 s, 35 cycles, and extension at 68°C for 5 min.

[0044] When the reaction system is 50 μL, its specific components are: 25 μL of PCR amplification reagent, 2.5 μL of forward primer, 2.5 μL of reverse primer, 5 μL of template DNA, and 15 μL of ddH2O; among them, the concentration of template DNA is 100 ng / μL; the concentrations of forward primer and reverse primer are 10 μM.

[0045] The corresponding PCR amplification reaction program was as follows: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 10 s, annealing at 54°C for 30 s, extension at 68°C for 30 s, 35 cycles, and extension at 68°C for 5 min.

[0046] The primers provided by the present invention can also be used to detect the sample to be tested by other methods, such as qRT-PCR. Those skilled in the art can design a suitable amplification system and reaction conditions according to the actual situation of the experiment, which will not be described in detail here.

[0047] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0048] Example 1

[0049] This embodiment is a primer combination for identifying white clover and red clover seeds, wherein the sequence of the forward primer of the primer combination is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO.2.

[0050] Trifolium-F(5′-3′): TATGACGGAAAACGATATATGAG-SEQ ID NO.1;

[0051] Trifolium-R(5'-3'): TAAAAATTTGAAAAGAAAAAAGTCC-SEQ ID NO. 2.

[0052] Example 2

[0053] This embodiment is a kit for identifying white clover and red clover seeds, the components of which include the primer combination of Example 1, PCR amplification reagents and ddH2O.

[0054] Example 3

[0055] This example is a method for identifying white clover and red clover seeds, which uses the primers of Example 1 for detection, and the specific steps are as follows:

[0056] S1. Collect seeds and germinate them using the single-layer filter paper method at 25°C for 6-8 days until the seedlings are relatively strong. Extract genomic DNA from the germinated seeds using the SDS lysis method.

[0057] S2. PCR detection of genomic DNA from white clover seeds using the primer pair shown in SEQ ID NO.1-2:

[0058] The total volume of the PCR detection system was 10 μL, including 5 μL of 2× SsoRobust Green Taq PCR ProMix (Guangzhou Yingzan Biotechnology Co., Ltd., product number: T111A), 0.5 μL of primer Trifolium-F (concentration: 10 μM), 0.5 μL of primer Trifolium-R (concentration: 10 μM), 3.5 μL of ddH2O, and 0.5 μL of white clover DNA template (100 ng / μL).

[0059] Amplification conditions were as follows: (1) pre-denaturation at 98°C for 2 min; (2) denaturation at 98°C for 10 s; (3) annealing at 52.7°C for 30 s; (4) extension at 68°C for 30 s; (5) 35 cycles of steps 2-4; (6) extension at 68°C for 5 min.

[0060] S3. Using the primer pair shown in SEQ ID NO.1-2, genomic DNA of red clover seeds was detected by PCR:

[0061] The total volume of the PCR detection system was 10 μL, which included 5 μL of 2× SsoRobust Green Taq PCR ProMix (Guangzhou Yingzan Biotechnology Co., Ltd., product number: T111A), 0.5 μL of primer Trifolium-F (concentration of 10 μM), 0.5 μL of primer Trifolium-R (concentration of 10 μM), 3.5 μL of ddH O, and 0.5 μL of red clover DNA template (100 ng / μL);

[0062] Amplification conditions were as follows: (1) pre-denaturation at 98°C for 2 min; (2) denaturation at 98°C for 10 s; (3) annealing at 52.7°C for 30 s; (4) extension at 68°C for 30 s; (5) 35 cycles of steps 2-4; (6) extension at 68°C for 5 min.

[0063] S4. Electrophorese the PCR products of white clover and red clover on the same 3% agarose gel using 2 μL / 50 mL of nucleic acid dye at 120 V for 50 min. Photograph under UV light.

[0064] S5. The sample with an electrophoresis amplified band size of 162 bp is white clover seeds; the sample with an amplified band size of 178 bp is red clover seeds.

[0065] Example 4

[0066] To verify whether the primer pair in Example 1 can identify any variety of white clover and red clover seeds, one germplasm sample from each of 10 white clover and 8 red clover germplasms was used as the test material (as shown in Table 1). Using the single-layer filter paper germination method at 25°C, germination was carried out for 6-8 days until the seedlings were relatively strong. Genomic DNA from 20 individual seedlings of each of the two germplasms was extracted using the SDS lysis method. This DNA was diluted to a concentration of 100 ng / μL and used as a template for PCR amplification using the primer pair in Example 1.

[0067] Table 1 Information of the two test materials

[0068]

[0069]

[0070] The PCR amplification method was the same as in Example 3. Twenty PCR products of white clover and red clover were electrophoresed on the same 3% agarose gel, with 2 μL / 50 mL of nucleic acid dye and an electrophoresis voltage of 120 V. The gel was run for 50 min and photographed under UV light.

[0071] Figure 2It shows that using the genomic DNA of 20 single seeds each of white clover (Rivanda) and red clover (Xuanli) as templates, there are two types of electrophoresis amplification bands, both of which are between 150bp and 200bp in size, and the electrophoresis amplification fragment of white clover (Rivanda) is smaller than that of red clover (Xuanli). Therefore, it can be judged that the band size of 162bp corresponds to white clover seeds, and the band size of 178bp corresponds to red clover seeds.

[0072] Example 5

[0073] To further verify whether the primer pairs in Example 1 can identify any variety of white clover and red clover seeds, this example selected 10 white clover seed germplasms and 8 red clover seed germplasms as test materials (as shown in Tables 2 and 3). Using the single-layer filter paper germination method at 25°C, the seeds were germinated for 6-8 days until the seedlings were relatively strong. Twenty individual seedlings from each germplasm were mixed for genomic DNA extraction. The DNA was diluted to a concentration of 100 ng / μL and used as a template for PCR amplification using the primers in Example 1.

[0074] Table 2. Description of the 10 white clover seed germplasms tested

[0075]

[0076] Table 3. Description of the 8 red clover seed germplasms tested

[0077]

[0078]

[0079] The PCR amplification method was the same as in Example 3. 18 PCR products of white clover and red clover were electrophoresed on the same 3% agarose gel, with 2 μL / 50 mL of nucleic acid dye and an electrophoresis voltage of 120 V. The gel was run for 50 min and photographed under UV light.

[0080] The results are as follows Figure 3 As shown, using genomic DNA from 10 accessions of white clover and 8 accessions of red clover as templates, there are two types of electrophoresis amplification bands, both of which are between 150bp and 200bp in size, and the electrophoresis amplification fragments of the 10 accessions of white clover are all smaller than the electrophoresis amplification fragments of the 8 accessions of red clover. Therefore, it can be judged that the band size of 162bp corresponds to white clover seeds, and the band size of 178bp corresponds to red clover seeds.

[0081] Example 6

[0082] In order to verify whether the primer pair in Example 1 can identify mixed seeds, in this example, DNA of white clover and red clover were mixed in ratios of 100:0, 90:10, 75:25, 50:50, 25:75, 10:90 and 0:100, respectively, and ddH2O was used as a template as a control, and PCR amplification was performed using the primer pair in Example 1.

[0083] PCR detection was performed on genomic DNA mixed in different ratios using the primer pairs in Example 1; the total volume of the PCR detection system was 10 μL, including 5 μL of 2×SsoRobust Green Taq PCR ProMix (Guangzhou Yingzan Biotechnology Co., Ltd., product number: T111A), 0.5 μL of primer Trifolium-F (concentration: 10 μM), 0.5 μL of primer Trifolium-R (concentration: 10 μM), 3.5 μL of ddH2O, and 0.5 μL of white clover DNA template (100 ng / μL);

[0084] The touchdown PCR amplification conditions were as follows: (1) pre-denaturation at 98°C for 2 min; (2) denaturation at 98°C for 10 s; (3) initial annealing at 57°C for 30 s; (4) extension at 68°C for 30 s; (5) in steps 2-4, cycles 1-7 were performed, with step (3) decreasing 1°C after each cycle, and finally completing 8-35 cycles at 50°C; (6) extension at 68°C for 5 min;

[0085] The PCR products were electrophoresed on the same 3% agarose gel, with 2 μL / 50 mL of nucleic acid dye and an electrophoresis voltage of 100 V for 60 min, and the images were taken under UV light.

[0086] The sizes of the electrophoresis amplified bands were all between 150bp and 200bp. The amplified band of 162bp was from white clover; the amplified band of 178bp was from red clover.

[0087] The PCR product was detected by 3% agarose gel electrophoresis and the results were as follows:

[0088] Figure 4 The mixed template shows that the concentration of genomic DNA from white clover seeds decreases from left to right, and the electrophoretic band gradually darkens. The concentration of genomic DNA from red clover seeds increases from left to right, and the electrophoretic band gradually brightens. The electrophoretic amplified fragments from white clover are smaller than those from red clover. The negative control group using ddH2O as the template has no electrophoretic amplified bands.

[0089] Example 7

[0090] To further verify the effectiveness of the designed primers, the amplification results of white clover and red clover were sequenced in this example. It was predicted that the red clover would have 16 more base pairs than the white clover.

[0091] PCR amplification was performed using 100 ng / μL white clover genomic DNA as a template using Trifolium-F and Trifolium-R primers. PCR system and method:

[0092] The total volume of the PCR detection system was 50 μL, including 2× SsoRobust Green Taq PCR ProMix (Guangzhou Yingzan Biotechnology Co., Ltd., product number: T111A) 25 μL, primer Trifolium-F 2.5 μL (concentration of 10 μM), primer Trifolium-R 2.5 μL (concentration of 10 μM), ddH2O 15 μL and DNA template (100 ng / μL) 5 μL.

[0093] Amplification conditions were as follows: (1) pre-denaturation at 98°C for 2 min; (2) denaturation at 98°C for 10 s; (3) annealing at 54°C for 30 s; (4) extension at 68°C for 30 s; (5) 35 cycles of steps 2-4; (6) extension at 68°C for 5 min. The above system was repeated three times, and the three replicates were mixed and then analyzed by electrophoresis.

[0094] The PCR amplification products were detected by 3% agarose gel electrophoresis and sent to Shanghai Shenggong Bioengineering Co., Ltd. for sequencing.

[0095] PCR amplification was performed using 100 ng / μL Trifolium genomic DNA as a template using Trifolium-F and Trifolium-R primers. PCR system and method:

[0096] The total volume of the PCR detection system was 50 μL, including 2× SsoRobust Green Taq PCR ProMix (Guangzhou Yingzan Biotechnology Co., Ltd., product number: T111A) 25 μL, primer Trifolium-F 2.5 μL (concentration of 10 μM), primer Trifolium-R 2.5 μL (concentration of 10 μM), ddH2O 15 μL and DNA template (100 ng / μL) 5 μL.

[0097] Amplification conditions were as follows: (1) pre-denaturation at 98°C for 2 min; (2) denaturation at 98°C for 10 s; (3) annealing at 54°C for 30 s; (4) extension at 68°C for 30 s; (5) 35 cycles of steps 2-4; (6) extension at 68°C for 5 min. The above system was repeated three times, and the three replicates were mixed and then analyzed by electrophoresis.

[0098] The PCR amplification products were detected by 3% agarose gel electrophoresis and sent to Shanghai Shenggong Bioengineering Co., Ltd. for sequencing.

[0099] from Figure 5 It can be seen that the sequencing results are consistent with the expected results.

[0100] Example 8

[0101] In order to verify the specificity and reliability of the primer pair of Example 1, it was used for melting curve (qRT-PCR amplicon melting) analysis:

[0102] Four samples each of the white clover and red clover template DNAs in Example 5 were randomly selected as qRT-PCR template DNAs. The qRT-PCR system and method were as follows:

[0103] The total volume of the qRT-PCR detection system was 10 μL, including 5 μL of 2× Realab Green PCR Fastmixture (Lanbolide Biotechnology, product number: R0202-02), 0.2 μL of primer Trifolium-F (concentration of 10 μM), 0.2 μL of primer Trifolium-R (concentration of 10 μM), 3.6 μL of ddH2O, and 1 μL of DNA template (100 ng / μL).

[0104] The amplification conditions were as follows: (1) pre-denaturation at 95°C for 30 s; (2) denaturation at 95°C for 10 s; (3) annealing and extension at 65°C for 30 s; (4) 40 cycles of the 2-3 step; (5) melting curve analysis, 95°C for 30 s, heating rate 4.4°C / s, then 40°C for 30 s, heating rate 1.5°C / s, heating rate from 40°C to 95°C at 0.06°C / s, fluorescence was collected 10 times per degree, and finally at 37°C for 30 s, cooling rate 1.5°C / s.

[0105] The results are as follows Figure 6 As shown, it can be seen from the melting curves that the four samples of white clover and red clover can be amplified, and no other curves are amplified, which proves that the amplification products obtained by amplification using the primers of the present invention are specific and uniform.

[0106] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A PCR primer pair for distinguishing white clover and red clover seeds, characterized in that: The sequence of the forward primer in the PCR primer pair is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO.

2.

2. Use of the PCR primer pair as claimed in claim 1 in preparing a kit for identifying white clover and red clover seeds.

3. Use of the PCR primer pair as claimed in claim 1 in distinguishing white clover and red clover seeds.

4. A kit for identifying white clover and red clover seeds, characterized in that: Comprising the PCR primer pair according to claim 1.

5. The kit according to claim 4, characterized in that The kit also includes PCR amplification reagents, DNA template and ddH2O.

6. The kit according to claim 5, characterized in that The PCR amplification reagents include Taq DNA polymerase, dNTPs, MgCl2 and reaction buffer.

7. Use of the kit according to any one of claims 4 to 6 in identifying white clover and red clover seeds.

8. A method for identifying white clover and red clover seeds, characterized in that: include: Extracting genomic DNA from the seeds to be tested, then using the extracted DNA as a template to perform PCR amplification using the kit according to any one of claims 4 to 6, and then detecting the PCR product by agarose gel electrophoresis; If the band size amplified by electrophoresis is 162bp, the sample to be tested is white clover seeds; if the band size amplified by electrophoresis is 178bp, the sample to be tested is red clover seeds; if the band sizes amplified by electrophoresis are both 162bp and 178bp, the sample to be tested is a mixture of white clover and red clover seeds.

9. The method according to claim 8, characterized in that The reaction program of PCR amplification was as follows: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 10 s, annealing at 52.7°C for 30 s, extension at 68°C for 30 s, 35 cycles, and extension at 68°C for 5 min.

10. The method according to claim 8, characterized in that The PCR amplification reaction program was as follows: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 10 s, annealing at 54°C for 30 s, extension at 68°C for 30 s, 35 cycles, and extension at 68°C for 5 min.

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