Primer group for identifying Houttuynia cordata variety Changpo No.1 based on SSR (Simple Sequence Repeat) marker, application of primer group and variety and purity identification method
By using SSR labeled primer sets with strong specificity and high polymorphism, the Houttuynia cordata species Changbanpo No. 1 and its purity were quickly identified, which solved the problems of low identification accuracy and complex operation in the prior art, and achieved efficient and accurate variety and purity identification.
Patent Information
- Application Number
- CN202510515142.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-23
- Publication Date
- 2025-07-29
AI Technical Summary
In the prior art, the variety and purity identification of the Houttuynia cordata No. 1, the Houttuynia cordata species mainly relies on morphological markers, cytologic markers and biochemical markers, and has defects such as low accuracy, complex operation and poor repeatability.
The SSR-labeled primer set (cbp-4, cbp-16, cbp-38) with strong specificity and high polymorphism were used to construct the fingerprint of Houttuynia cordata and transform it into binary data through PCR amplification and electrophoresis analysis, so as to quickly identify the Houttuynia cordata species Changbanpo No. 1 and its purity.
It has achieved efficient, fast and low-cost identification and purity confirmation of Houttuynia cordata varieties, with high accuracy and no interference from external factors, and provides reliable identification methods to support the protection and development and utilization of germplasm resources.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biometric identification, and particularly relates to a primer set for identifying the Houttuynia cordata variety Changbanpo No. 1 based on SSR markers, its application, variety and purity identification method. Background Art
[0002] Houttuynia cordata is a perennial herb of the genus Houttuynia in the Saururaceae family. As a traditional Chinese herbal medicine and Chinese herbal tea, Houttuynia cordata has a long history, and its medicinal history dates back more than 2,000 years. The planting area of Houttuynia cordata in Dangyang City, Hubei Province is 32,000 mu. In 2022, the annual output value exceeded 600 million yuan. Among them, the sandy soil in Lianghe Town is very suitable for the growth of Houttuynia cordata, with a planting area of 25,000 mu and an annual output of about 1.3 million tons, and it has been awarded the title of "Hometown of Houttuynia cordata in China". The Houttuynia cordata variety "Changbanpo No. 1" was systematically selected from the local varieties of farmers in Lianghe Town, Dangyang City. It has the characteristics of high yield, good resistance and excellent quality. The mu yield is as high as 12,000 catties, which is more than 3 times that of the cultivated Houttuynia cordata varieties in other provinces, and the quality of the underground rhizomes is high, with great market competitiveness and promotion value. Therefore, how to protect the variety rights of Changbanpo No. 1 is particularly important. At present, the identification of the variety and purity of Changbanpo No. 1 mainly relies on methods such as morphological markers, cytological markers and biochemical markers. However, these markers have various defects such as low accuracy, complex operation and poor repeatability. Summary of the Invention
[0003] The present invention aims to provide a primer set for identifying the Houttuynia cordata variety Changbanpo No. 1 based on SSR markers, its application, variety and purity identification method. The SSR marker primer set has strong specificity, high polymorphism and good resolution, can quickly identify the Houttuynia cordata variety Changbanpo No. 1, and can also quickly confirm the variety purity of the sample to be tested.
[0004] To solve the above problems, the present invention adopts the following technical solutions:
[0005] The present invention provides a primer set for identifying the Houttuynia cordata variety Changbanpo No. 1 based on SSR markers, and the primer set is composed of cbp-4, cbp-16, and cbp-38: where:
[0006] cbp-4F: 5’-ATGTTTCCCTGACGACGACG-3’, as shown in SEQ ID NO: 1;
[0007] cbp-4R: 5’-AAGCAAAGGAGAGGTCGTGG-3’, as shown in SEQ ID NO: 2;
[0008] cbp-16F: 5’-GGTGGGTTCTTTCTTTCAATGC-3’, as shown in SEQ ID NO: 3;
[0009] cbp-16R: 5’-CATGGGCCTTTACAAGACGC-3’, as shown in SEQ ID NO: 4;
[0010] cbp-38F: 5’-TTGTTCTCGCAATCAAACCC-3’, as shown in SEQ ID NO: 5;
[0011] cbp-38R: 5’-ACGAAAACAACAAGGAGGAGG-3’, as shown in SEQ ID NO: 6.
[0012] Provide an application of the above primer set in the variety identification or purity identification of Houttuynia cordata Thunb. cv. Changbanpo No. 1.
[0013] Provide a method for identifying the variety of Houttuynia cordata Thunb. cv. Changbanpo No. 1, specifically including the following steps:
[0014] 1) Take Houttuynia cordata Thunb. cv. Changbanpo No. 1 and the Houttuynia cordata Thunb. to be tested, and extract the standard genome and sample genome respectively;
[0015] 2) Use the above primer set to perform PCR amplification on the standard genome and sample genome respectively to obtain the standard PCR product and sample PCR product, then perform electrophoresis analysis, and count the electrophoresis bands to obtain the fingerprint maps of Houttuynia cordata Thunb. cv. Changbanpo No. 1 and the Houttuynia cordata Thunb. to be tested;
[0016] 3) Take the fingerprint map of Houttuynia cordata Thunb. cv. Changbanpo No. 1 as the standard map, compare the fingerprint map of the Houttuynia cordata Thunb. to be tested with the standard map. When the number of different alleles between samples ≥ 1, it is determined as different varieties; when the number of different alleles between samples = 0, it is determined as the same variety.
[0017] According to the above scheme, in step 2), the reaction system for PCR amplification is: template DNA 1.5 μL, forward primer 0.5 μL, reverse primer 0.5 μL, 2×TSINGKE Master Mix 5 μL, ddH2O 2.5 μL.
[0018] According to the above scheme, in step 2), the reaction program for PCR amplification is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s; annealing for 50 s; extension at 72°C for 1 min; denaturation, annealing, and extension are repeated in cycles for 35 times; extension at 72°C for 10 min; preservation at 4°C.
[0019] According to the above scheme, in step 2), perform electrophoresis of the standard PCR product in a 6% polyacrylamide gel, take pictures after silver staining, and count the electrophoresis bands to obtain the fingerprint maps of Houttuynia cordata Thunb. cv. Changbanpo No. 1 and the Houttuynia cordata Thunb. to be tested.
[0020] According to the above scheme, in step 3), the fingerprint spectrum of the houttuynia cordata to be tested and the standard spectrum are respectively converted into binary data to obtain the molecular identity card of the houttuynia cordata to be tested and the molecular identity card of houttuynia cordata No. 1 of Changbanpo, and then compare and identify whether the houttuynia cordata to be tested is houttuynia cordata No. 1 of Changbanpo; wherein: the method for obtaining the molecular identity card is as follows: if there is a band in the fingerprint spectrum, it is recorded as 1, if there is no band, it is recorded as 0, and the unclear band is recorded after being judged according to the actual situation. The fingerprint spectrum is converted into binary data to obtain the molecular identity cards of houttuynia cordata No. 1 of Changbanpo and the houttuynia cordata to be tested.
[0021] Preferably, the molecular identity card of houttuynia cordata No. 1 of Changbanpo is 0001111000110110101011.
[0022] Provide a method for identifying the purity of houttuynia cordata variety Changbanpo No. 1, which specifically includes the following steps:
[0023] 1) Take multiple samples of houttuynia cordata No. 1 of Changbanpo with the purity to be tested, and extract the sample genomes respectively;
[0024] 2) Use the above primer set to perform PCR amplification on multiple sample genomes respectively to obtain sample PCR products, and then perform electrophoresis analysis, and count the electrophoresis bands to obtain the sample spectrum;
[0025] 3) Take the bands commonly possessed by most samples of houttuynia cordata No. 1 of Changbanpo as the characteristic bands of houttuynia cordata No. 1 of Changbanpo, and compare the sample spectrum with the characteristic bands. When the number of different alleles between samples ≥ 1, it is determined as different varieties, indicating that houttuynia cordata No. 1 of Changbanpo has mutated; when the number of different alleles between samples = 0, it is determined as the same variety, that is, houttuynia cordata No. 1 of Changbanpo; wherein the purity = the number of individuals of the same variety / the total number of individuals identified × 100%.
[0026] According to the above scheme, in step 2), the reaction system for PCR amplification is: 1.5 μL of template DNA, 0.5 μL of forward primer, 0.5 μL of reverse primer, 5 μL of 2×TSINGKE Master Mix, and 2.5 μL of ddH2O.
[0027] According to the above scheme, in step 2), the reaction program for PCR amplification is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s; annealing for 50 s; extension at 72°C for 1 min; the cycle of denaturation, annealing, and extension is repeated 35 times; extension at 72°C for 10 min; preservation at 4°C.
[0028] According to the above scheme, in step 2), the standard PCR product is electrophoresed in a 6% polyacrylamide gel, stained with silver and photographed for recording, and the electrophoresis bands are counted to obtain the sample spectrum.
[0029] The present invention has the following beneficial effects:
[0030] The present invention provides a primer set for identifying the Houttuynia cordata variety Changbanpo No. 1 based on SSR markers, which consists of cbp-4, cbp-16, and cbp-38. This SSR marker primer set has strong specificity, high polymorphism, and good resolution. It can quickly identify the Houttuynia cordata variety Changbanpo No. 1 and can also rapidly confirm the variety purity of the test sample. The whole process is efficient, fast, and low-cost, and is not interfered by external factors. It is a reliable identification method, providing strong technical support for the protection, development, and utilization of the germplasm resources of Houttuynia cordata Changbanpo No. 1. Description of the Drawings
[0031] Figure 1 This is the binary molecular identity card of Houttuynia cordata Changbanpo No. 1 obtained in Example 1 of the present invention.
[0032] Figure 2 This is the binary molecular identity card of Houttuynia cordata from different original places in Example 1 of the present invention.
[0033] Figure 3 This is the amplified electrophoresis bands of three pairs of primers for 22 samples of Houttuynia cordata Changbanpo No. 1 to be identified for purity in Example 2 of the present invention; where M is DNA Marker, S1 - S22 are 22 samples of Houttuynia cordata Changbanpo No. 1, and △ is a different species. Detailed Embodiments
[0034] The following further describes the present application in detail with reference to the drawings. It should be noted that the following specific embodiments are only used to illustrate the present application and do not constitute a limitation on its protection scope. The present invention is elaborated with the help of examples to facilitate understanding of its characteristics and advantages. The provided examples are only used to explain the inventive method and do not limit the rest of the content. Among them, the experimental methods in the examples are all conventional methods unless otherwise specified, and the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified. Those skilled in the art can make non-essential improvements and adjustments to the present application according to the above application content.
[0035] Example 1
[0036] A total of 8 samples of Houttuynia cordata leaf samples from different original places were collected from the variety resource field of Hubei Houttuynia cordata Agricultural Industry Development Co., Ltd. The information table of the Houttuynia cordata leaf samples is shown in Table 1, and they were stored in self-sealing bags filled with anhydrous silica gel. The total DNA was extracted using the plant genomic DNA kit from Tiangen Biochemical Technology (Beijing) Co., Ltd.
[0037] Table 1. Information Table of Houttuynia cordata Leaf Samples
[0038]
[0039]
[0040] PCR amplification: The sequences of 3 pairs of SSR primers are as follows:
[0041] cbp-4F: 5’-ATGTTTCCCTGACGACGACG-3’
[0042] cbp-4R: 5’-AAGCAAAGGAGAGGTCGTGG-3’
[0043] cbp-16F: 5’-GGTGGGTTCTTTCTTTCAATGC-3’
[0044] cbp-16R: 5’-CATGGGCCTTTACAAGACGC-3’
[0045] cbp-38F: 5’-TTGTTCTCGCAATCAAACCC-3’
[0046] cbp-38R: 5’-ACGAAAACAACAAGGAGGAGG-3’
[0047] The 10 μL reaction system is as follows: 1.5 μL of template DNA, 0.5 μL of forward primer, 0.5 μL of reverse primer, 5 μL of 2×TSINGKE Master Mix, and 2.5 μL of ddH2O.
[0048] PCR reaction procedure: The amplification reaction is carried out on a PCR instrument according to the following procedure: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s; annealing for 50 s; extension at 72°C for 1 min; the cycle of denaturation, annealing, and extension is repeated 35 times; extension at 72°C for 10 min; preservation at 4°C.
[0049] The amplified products are detected by 6% polyacrylamide gel electrophoresis. According to the results of the silver-stained polyacrylamide gel electrophoresis diagram, the acrylamide gel and the glass plate are placed under the viewing lamp for statistics. The loci are counted, and the fingerprint maps of various varieties of Houttuynia cordata are drawn. Then, the fingerprint maps are converted into binary data. Bands are recorded as "1", and bands without are recorded as "0". Blurred bands are recorded after discrimination according to the actual situation, and the molecular identity cards of various varieties of Houttuynia cordata are constructed. The results are as Figure 1 and 2As shown below. Among them: The molecular ID of Houttuynia cordata 'Changbanpo No. 1' is 0001111000110110101011; the molecular ID of Houttuynia cordata from Jiangyou City, Sichuan Province is 01101010000101011111011; the molecular ID of Houttuynia cordata from Gong'an County, Hubei Province is 00111110000001101000011; the molecular ID of Houttuynia cordata from Qiubei County, Yunnan Province is 10011110000011111101011; the molecular ID of Houttuynia cordata from Chongqing City is 00000110000000011010010; the molecular ID of Houttuynia cordata from Jixi City, Anhui Province is 00000110000000011100010; the molecular ID of Houttuynia cordata from Jinhua City, Zhejiang Province is 01111101101001001001100; the molecular ID of Houttuynia cordata from Lishui City, Zhejiang Province is 00011100010001001001100. Through the above 3 pairs of SSR primer sets, these 8 varieties of Houttuynia cordata can be distinguished, and the discrimination rate is 100%.
[0050] Example 2
[0051] A total of 22 leaf samples of Houttuynia cordata 'Changbanpo No. 1' were collected from Zhaizha Village, Lianghe Town, Dangyang City. The information table of the Houttuynia cordata leaf samples is shown in Table 2, and they were stored in a self-sealing bag filled with anhydrous silica gel. The total DNA was extracted using the Plant Genomic DNA Kit from Tiangen Biochemical Technology (Beijing) Co., Ltd.
[0052] Table 2. Information Table of Houttuynia cordata Leaf Samples
[0053]
[0054] PCR Amplification: The sequences of 3 pairs of SSR primers are as follows:
[0055] cbp-4F: 5’-ATGTTTCCCTGACGACGACG-3’
[0056] cbp-4R: 5’-AAGCAAAGGAGAGGTCGTGG-3’
[0057] cbp-16F: 5’-GGTGGGTTCTTTCTTTCAATGC-3’
[0058] cbp-16R: 5’-CATGGGCCTTTACAAGACGC-3’
[0059] cbp-38F: 5’-TTGTTCTCGCAATCAAACCC-3’
[0060] cbp-38R: 5’-ACGAAAACAACAAGGAGGAGG-3’
[0061] The 10 μL reaction system is as follows: 1.5 μL of template DNA, 0.5 μL of forward primer, 0.5 μL of reverse primer, 5 μL of 2×TSINGKE Master Mix, and 2.5 μL of ddH2O.
[0062] PCR reaction procedure: The amplification reaction was carried out on a PCR instrument according to the following procedure: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s; annealing for 50 s; extension at 72°C for 1 min; the cycle of denaturation, annealing, and extension was repeated 35 times; extension at 72°C for 10 min; stored at 4°C.
[0063] The amplified products were detected by 6% polyacrylamide gel electrophoresis. According to the results of the silver-stained polyacrylamide gel electrophoresis pattern, the acrylamide gel and the glass plate were placed under an illuminator for viewing and counting. The bands commonly possessed by most of the Houttuynia cordata samples of Changbanpo No. 1 were used as the characteristic bands of Houttuynia cordata of Changbanpo No. 1. The bands of each sample were compared with the characteristic bands. The amplified bands of 22 samples are as Figure 3 shown. If the number of different alleles between samples ≥ 1, it was determined as different varieties, indicating that Houttuynia cordata of Changbanpo No. 1 had mutated; if the number of different alleles between samples = 0, it was determined as the same variety, that is, Houttuynia cordata of Changbanpo No. 1; among them, purity = the number of individuals of the same variety / the total number of individuals identified × 100%. It can be comprehensively calculated from the identification situations of each pair of primers that the variety purity of the 22 Houttuynia cordata samples of Changbanpo No. 1 collected in this experiment = 19 / 22 × 100% = 86.36%. Figure 3 It should be understood that for those of ordinary skill in the art, improvements or modifications can be made according to the above description, and all such improvements and modifications should fall within the protection scope of the appended claims of the present invention.
[0064]
Claims
1. A primer set for identifying the variety Changbanpo No. 1 of Houttuynia cordata Thunb. based on SSR markers, characterized in that, The primer set consists of cbp-4, cbp-16, and cbp-38 Composition: Among them: cbp-4 includes a forward primer and a reverse primer whose nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; cbp-16 includes a forward primer and a reverse primer whose nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively; cbp-38 includes a forward primer and a reverse primer whose nucleotide sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively.
2. The application of the primer set according to claim 1 in the variety identification or purity identification of Houttuynia cordata Thunb. cv. Changbanpo No.
1.
3. A variety identification method for the Houttuynia cordata variety Changbanpo No. 1, characterized in that, Specifically, it includes the following steps: 1) Take Houttuynia cordata Thunb. cv. Changbanpo No. 1 and the Houttuynia cordata Thunb. to be tested, and extract the standard genome and sample genome respectively; 2) Use the primer set according to claim 1 to perform PCR amplification on the standard genome and sample genome obtained in step 1) respectively to obtain a standard PCR product and a sample PCR product, then perform electrophoresis analysis, and count the electrophoresis bands to obtain the fingerprint maps of Houttuynia cordata Thunb. cv. Changbanpo No. 1 and the Houttuynia cordata Thunb. to be tested; 3) Take the fingerprint map of Houttuynia cordata Thunb. cv. Changbanpo No. 1 as the standard map, compare the fingerprint map of the Houttuynia cordata Thunb. to be tested with the standard map. When the number of differential alleles between samples ≥ 1, it is determined as different varieties; when the number of differential alleles between samples = 0, it is determined as the same variety.
4. The variety identification method according to claim 3, characterized in that In the said step 2), the reaction system for PCR amplification is: template DNA 1.5 μL, forward primer 0.5 μL, reverse primer 0.5 μL, 2×TSINGKE Master Mix 5 μL, ddH2O 2.5 μL; the reaction program for PCR amplification is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s; annealing for 50 s; extension at 72°C for 1 min; the cycle of denaturation, annealing, and extension is repeated 35 times; extension at 72°C for 10 min; preservation at 4°C.
5. The variety identification method according to claim 3, characterized in that Perform electrophoresis of the standard PCR product in a 6% polyacrylamide gel, take pictures after silver staining and record, and count the electrophoresis bands to obtain the fingerprint maps of Houttuynia cordata Thunb. cv. Changbanpo No. 1 and the Houttuynia cordata Thunb. to be tested.
6. The variety identification method according to claim 3, characterized in that, In the said step 3), convert the fingerprint map of the Houttuynia cordata Thunb. to be tested and the standard map into binary data respectively to obtain the molecular ID card of the Houttuynia cordata Thunb. to be tested and the molecular ID card of Houttuynia cordata Thunb. cv. Changbanpo No. 1, and compare and identify whether the Houttuynia cordata Thunb. to be tested is Houttuynia cordata Thunb. cv. Changbanpo No. 1; among them: the acquisition method of the molecular ID card is: if there is a band in the fingerprint map, it is recorded as 1, if there is no band, it is recorded as 0, and if it is missing, it is recorded as 2. Convert the fingerprint map into binary data to obtain the molecular ID cards of Houttuynia cordata Thunb. cv. Changbanpo No. 1 and the Houttuynia cordata Thunb. to be tested.
7. The variety identification method according to claim 6, characterized in that, The molecular ID card of Houttuynia cordata Thunb. cv. Changbanpo No. 1 is 0001111000110110101011.
8. A method for purity identification of the Houttuynia cordata variety Changbanpo No. 1, characterized in that, Specifically, it includes the following steps: 1) Take multiple Houttuynia cordata Thunb. cv. Changbanpo No. 1 with the purity of the variety to be tested, and extract the sample genome respectively; 2) Use the primer set according to claim 1 to perform PCR amplification on the multiple sample genomes obtained in step 1) respectively to obtain sample PCR products, then perform electrophoresis analysis, and count the electrophoresis bands to obtain the sample maps; 3) The spectral band shared by most samples of Houttuynia cordata No. 1 from Changbanpo was used as the characteristic spectral band of Houttuynia cordata No. 1 from Changbanpo. The sample profile was compared with the characteristic spectral band. When the number of differential alleles between samples was ≥1, the samples were judged to be different varieties, indicating that Houttuynia cordata No. 1 from Changbanpo had mutated; when the number of differential alleles between samples was = 0, the samples were judged to be the same variety, i.e., Houttuynia cordata No. 1 from Changbanpo. Purity = number of individuals of the same variety / total number of individuals identified × 100%.
9. The purity identification method according to claim 8, characterized in that, In the step 2), the reaction system for PCR amplification is: 1.5 μL of template DNA, 0.5 μL of forward primer, 0.5 μL of reverse primer, 5 μL of 2×TSINGKE Master Mix, and 2.5 μL of ddH2O; the reaction procedure for PCR amplification is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s; annealing for 50 s; extension at 72°C for 1 min; repeated cycles of denaturation, annealing, and extension for 35 times; extension at 72°C for 10 min; and storage at 4°C.
10. The purity identification method according to claim 8, characterized in that, In the step 2), the standard PCR product is electrophoresed in a 6% polyacrylamide gel, silver-stained and photographed, and the electrophoresis bands are counted to obtain a sample profile.