A primer for identifying camphor tree varieties and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a primer for identifying camphor tree varieties and its application. The primer includes CC12-F and CC12-R; the nucleotide sequences of CC12-F and CC12-R are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The primers designed by the present invention can detect and identify different camphor tree varieties, and the detection results are accurate. The results can be judged after the primers of the present invention are simply processed, PCR amplified and STR detected on the samples. Generally, the whole detection process can be completed within 5 hours, which provides a high degree of reliability for the detection results.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a primer for identifying camphor tree varieties and application thereof. Background Art

[0002] Camphor tree is an evergreen broad-leaved tree of the Lauraceae family, mainly distributed in southern and southwestern provinces of my country. Camphor tree has strong ability to absorb smoke and dust, conserve water resources, consolidate soil and prevent sand, and beautify the environment. The camphor tree has a large crown and dense shade, and its majestic appearance makes it an excellent tree species for urban greening. The branches and leaves of camphor tree can be used to extract essential oils for use in medicine and spice industry. With the emphasis on variety protection system, the number of applications for camphor tree variety rights has increased rapidly, which has brought challenges to the protection and identification of new varieties.

[0003] At present, the specificity test of camphor tree varieties mainly adopts morphological identification method, which has the disadvantage of long identification cycle; secondly, the traits investigated by morphological method are mostly quantitative traits, which are controlled by micro-effect polygenes and are easily affected by the environment; in addition, the homogenization of new variety breeding technology and the frequent exchange of basic breeding materials have caused the narrow genetic basis, and the morphological differences between varieties are getting smaller and smaller, resulting in insufficient morphological traits for variety identification, which increases the difficulty of variety identification. Therefore, there is an urgent need for a fast and effective variety identification method to clarify the "identity" of the variety and provide technical support for the identification of new plant varieties and the crackdown on variety infringement. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a primer for identifying camphor tree varieties and its application, which specifically adopts the following technical scheme:

[0005] In a first aspect of the present invention, a primer for identifying camphor tree varieties is provided, the primers comprising CC12-F and CC12-R; the nucleotide sequences of CC12-F and CC12-R are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0006] SEQ ID NO.1: 5'-AGTTGGGGTCTTGTGTGCCT-3';

[0007] SEQ ID NO. 2: 5'-TGCGAAACTGCTTGGGCT-3'.

[0008] The second aspect of the present invention provides a kit for detecting camphor tree varieties, the kit comprising the above-mentioned primers for identifying camphor tree varieties.

[0009] As a further preferred embodiment, the above kit further comprises Taq enzyme, 10×Taq buffer containing MgCl 2 , dNTP mixed solution and water.

[0010] A third aspect of the present invention provides a method for identifying camphor tree varieties, comprising the following steps:

[0011] S1: Extraction of DNA from camphor tree samples to be tested;

[0012] S2: using the camphor tree sample DNA to be tested as a template, using the above primers to perform PCR amplification to obtain a PCR product of the camphor tree to be tested;

[0013] S3: Use capillary electrophoresis to detect and analyze the STR site peak of the PCR product, and determine the camphor tree variety based on the STR site peak.

[0014] As a further preferred embodiment, the reaction system for the above-mentioned PCR amplification is: 2.5 μL of 10×Taq buffer containing MgCl2, 0.5 μL of 5 μM dNTP mixture, 0.5 μL of 10 μM primer CC12-F, 0.5 μL of 10 μM primer CC12-R, 0.2 μL of 5 U / μL Taq enzyme, 1 μL of 20 ng / μL-50 ng / μL DNA, and water is added to a total volume of 25 μL.

[0015] As a further preferred embodiment, the reaction procedure of the above-mentioned PCR amplification is: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 10 cycles, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, and repair and extension at 72°C for 10 min.

[0016] As a further preferred embodiment, in the above S1, the Ezup column-type plant tissue genomic DNA extraction kit is used to extract the DNA of the camphor tree sample to be tested.

[0017] The fourth aspect of the present invention is the application of the above primers in the construction of SSR fingerprint of Cinnamomum camphora.

[0018] A fifth aspect of the present invention provides the use of the above-mentioned primers for identifying camphor tree varieties or the above-mentioned kit in identifying camphor tree varieties.

[0019] As a further preferred embodiment, the above-mentioned camphor tree varieties include Guizhang, Huai'naoxiang, Jilong No. 1, borneol No. 1, lemonxiang, Qianyexiang, Ruyu, Shenggan, Xianggui, Yuhuang, and Yongjin.

[0020] The beneficial effects of the present invention are:

[0021] (1) The primers designed by the present invention can be used to detect and identify different camphor tree varieties, and the detection results are accurate, providing a high degree of reliability for the detection results.

[0022] (2) The method of the present invention can determine the result after simple sample processing, PCR amplification and STR detection. Generally, the entire detection process can be completed within 5 hours.

[0023] (3) The method of the present invention is convenient for sampling. Leaves, buds, flowers and other tissues or organs can all be used as experimental materials. Seedlings, large seedlings and perennial plants can all be sampled without being restricted by season or location.

[0024] (4) Molecular identification of camphor tree seedlings can avoid the phenomenon of mixing good and bad seedlings during seedling production and sales, and provide technical support for the screening of similar varieties of new camphor tree varieties and the crackdown on variety infringement. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown is the capillary electrophoresis peak diagram of Cinnamomum camphora;

[0026] Figure 2 Shown is the capillary electrophoresis peak diagram of Huai Nao Xiang;

[0027] Figure 3 Shown is the capillary electrophoresis peak diagram of Jilong No. 1;

[0028] Figure 4 Shown is the capillary electrophoresis peak diagram of borneol No. 1;

[0029] Figure 5 Shown is the capillary electrophoresis peak diagram of lemon fragrance;

[0030] Figure 6 Shown is the capillary electrophoresis peak diagram of Chibaxiang;

[0031] Figure 7 Shown is the capillary electrophoresis peak diagram of Yuyu;

[0032] Figure 8 Shown is the capillary electrophoresis peak diagram of Sheng Gan;

[0033] Fig. 9 Shown is the capillary electrophoresis peak diagram of cinnamon;

[0034] Fig.10 Shown is the capillary electrophoresis peak diagram of Yuhuang;

[0035] Fig.11 Shown is the capillary electrophoresis peak diagram of Yongjin. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments to clearly and completely describe the concept, specific structure and technical effects of the present invention, so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other without conflict.

[0037] Example 1

[0038] Acquisition of a primer for identifying camphor tree varieties

[0039] According to the existing camphor genome data, MISA software was used to search for short tandem repeat sequence sites, and Primer 5.0 software was used for primer design. Finally, 99,599 pairs of primers were successfully designed, 40 pairs of primers were randomly synthesized, 14 pairs of which were polymorphic. 11 camphor varieties were amplified by PCR using 14 pairs of primers. Finally, a pair of core primers with good polymorphism and high repeatability was selected as a special primer for camphor variety identification, named CC12, with an optimal annealing temperature of 58°C. The primer sequence is as follows:

[0040] Primer CC12-F: 5′-AGTTGGGTCTTGTGTGCCT-3′;

[0041] Primer CC12-R: 5′-TGCGAAACTGCTTGGGCT-3′.

[0042] Example 2

[0043] A method for identifying camphor tree varieties comprises the following steps:

[0044] (1) Extraction of template DNA: DNA of the target 11 camphor tree samples was extracted separately using the Ezup column-type plant tissue genomic DNA extraction kit (batch number: B518261) from Sangon Biotechnology.

[0045] (2) Using CC12 primer pair, extract DNA as template and perform PCR amplification (the PCR amplification procedure and amplification reaction system are shown in Tables 1 and 2):

[0046] (3) The PCR product is subjected to capillary electrophoresis to determine the fragment size (STR detection). The specific process is as follows:

[0047] Use a pipette to draw a mixture of 990 μL of highly deionized formamide (HIDI) and 10 μL of molecular weight internal standard (LIZ500), add it to a 96-well reaction plate, 10 μL per well, place the 96-well plate in a plate centrifuge, centrifuge at 1200 rpm for 15 seconds, add 1 μL of camphor tree sample to the corresponding well of the 96-well plate; place the 96-well plate in a plate centrifuge, centrifuge at 1200 rpm for 15 seconds; seal the 96-well plate with a sealing film, shake, place the 96-well plate in a plate centrifuge, centrifuge at 1200 rpm for 30 seconds; place it in a PCR instrument, the denaturation program is 98°C, 5 minutes, and immediately place the 96-well plate on an ice-water mixture to cool rapidly after the program ends; place the 96-well plate in a plate centrifuge, centrifuge at 1200 rpm for 15 seconds, and use a genetic analyzer model 3730xl to detect STR samples;

[0048] (4) Genemapper software was used to analyze the capillary electrophoresis results. The abscissa of the amplified fragment peak graph was the fragment size, and the ordinate was the fluorescence intensity value. The allelic variation size data of the homozygous site was recorded as X, and the allelic variation data of the heterozygous site was recorded as X / Y. The primer pair CC12 can amplify 11 alleles, which can completely distinguish 11 camphor tree varieties. The specific amplification results are shown in Tables 3 and Figure 1-Figure 11 .

[0049] Table 1 Polymerase chain reaction (PCR) amplification procedure

[0050]

[0051] Table 2 Amplification reaction system

[0052]

[0053] Table 3 STR locus peak statistics of 11 camphor tree varieties

[0054]

[0055] As can be seen from the above table, different STR locus peaks were obtained in the varieties after CC12 amplification, which can accurately distinguish 11 camphor tree varieties.

[0056] Example 3

[0057] A method for rapid identification of camphor tree varieties using CC12 primers

[0058] (1) Extraction of template DNA: DNA of the camphor tree sample to be tested was extracted using the Ezup column-type plant tissue genomic DNA extraction kit (Batch No.: B518261) from Sangon Biotechnology;

[0059] (2) Using CC12 primer pair, extract DNA as template and perform PCR amplification (the PCR amplification procedure and amplification reaction system are shown in Tables 1 and 2):

[0060] (3) The PCR product is subjected to capillary electrophoresis to determine the fragment size (STR detection). The specific process is as follows:

[0061] Use a pipette to draw a mixture of 990 μL of highly deionized formamide (HIDI) and 10 μL of molecular weight internal standard (LIZ500), add it to a 96-well reaction plate, 10 μL per well, place the 96-well plate in a plate centrifuge, centrifuge at 1200 rpm for 15 seconds, add 1 μL of camphor tree sample to the corresponding well of the 96-well plate; place the 96-well plate in a plate centrifuge, centrifuge at 1200 rpm for 15 seconds; seal the 96-well plate with a sealing film, shake, place the 96-well plate in a plate centrifuge, centrifuge at 1200 rpm for 30 seconds; place it in a PCR instrument, the denaturation program is 98°C, 5 minutes, and immediately place the 96-well plate on an ice-water mixture to cool rapidly after the program ends; place the 96-well plate in a plate centrifuge, centrifuge at 1200 rpm for 15 seconds, and use a genetic analyzer model 3730xl to detect STR samples;

[0062] (4) Genemapper software was used to analyze SSR amplification data, and capillary electrophoresis was used to detect amplified fragment peaks. The horizontal axis was the fragment size, and the vertical axis was the fluorescence intensity value. The allelic variation size data of the homozygous site was recorded as X, and the allelic variation data of the heterozygous site was recorded as X / Y. The primer pairs were used to detect 20 unknown samples to be tested. The test results are shown in Table 4, and the identification accuracy rate was 100%.

[0063] Table 4 Identification results of 20 camphor tree samples

[0064]

[0065] According to the above table, when facing unknown camphor tree variety samples, the specific camphor tree variety can be accurately determined by the loci using the primers and methods of the present invention.

[0066] Although the description of the present invention has been quite detailed and specifically describes several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad possible interpretation of these claims in view of the prior art by reference to the appended claims, thereby effectively covering the intended scope of the present invention. In addition, the above description of the present invention is based on the embodiments foreseeable by the inventor, and its purpose is to provide a useful description, and those non-substantial changes to the present invention that have not yet been foreseen may still represent equivalent changes to the present invention.

Claims

1. A method for identifying camphor tree varieties, characterized in that: The following steps are involved: S1: Extraction of DNA from camphor tree samples to be tested; S2: using the DNA of the camphor tree sample to be tested as a template, using primers to perform PCR amplification to obtain the PCR product of the camphor tree to be tested; S3: using capillary electrophoresis to detect and analyze the STR site peak of the PCR product, and determining the camphor tree variety according to the STR site peak; The primers include CC12-F and CC12-R; the nucleotide sequences of CC12-F and CC12-R are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

2. The method according to claim 1, characterized in that The reaction system for the PCR amplification is: 2.5 μL of 10×Taq buffer containing MgCl2, 0.5 μL of 5 μM dNTP mixture, 0.5 μL of 10 μM primer CC12-F, 0.5 μL of 10 μM primer CC12-R, 0.2 μL of 5 U / μL Taq enzyme, 1 μL of 20 ng / μL-50 ng / μL DNA, and water is added to a total volume of 25 μL.

3. The method according to claim 1, characterized in that: The reaction procedure of the PCR amplification was as follows: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, for 10 cycles, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for 30 cycles, and repair and extension at 72°C for 10 min.

4. The method according to claim 1, characterized in that: In S1, the Ezup column-type plant tissue genomic DNA extraction kit was used to extract the DNA of the camphor tree samples to be tested.

5. An application of primers for identifying camphor tree varieties in the construction of camphor tree SSR fingerprints, characterized in that: The primers include CC12-F and CC12-R; the nucleotide sequences of CC12-F and CC12-R are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

6. Application of primers for detecting SSR molecular markers of camphor trees in identification of camphor tree varieties, characterized in that: The primers include CC12-F and CC12-R; the nucleotide sequences of CC12-F and CC12-R are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

7. The use according to claim 6, characterized in that: The camphor tree varieties include Guizhang, Huai'naoxiang, Jilong No. 1, borneol No. 1, lemonxiang, Qianyexiang, Ruyu, Shenggan, Xianggui, Yuhuang and Yongjin.

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