Primer and method for rapidly identifying variety of camellia oleifera
By using RAPD molecular marker technology and primer design, a rapid identification method for Camellia oleifera varieties was constructed, which solved the problem of distinguishing Camellia oleifera varieties and achieved low-cost and efficient variety identification. It is applicable to the differentiation of a variety of Camellia oleifera varieties and the addition of new varieties.
Patent Information
- Application Number
- CN202511214901.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-28
- Publication Date
- 2025-11-07
AI Technical Summary
Existing technologies are insufficient to effectively distinguish Camellia oleifera varieties, especially under the influence of asexual reproduction and environmental factors, which leads to serious problems of synonymy and homonymy. Furthermore, SNP marker methods are costly and complex to operate, making it difficult to quickly and accurately identify new varieties.
Using RAPD molecular marker technology, 10 primers were designed for PCR amplification to construct an artificial variety identification map. Specific bands were used to distinguish Camellia oleifera varieties, and rapid identification was achieved through primer and band information.
It enables rapid and accurate identification of camellia oleifera varieties, reduces costs, improves identification efficiency, overcomes environmental impacts, is applicable to the differentiation of a large number of varieties and the addition of new varieties, and protects the interests of breeders and forest farmers.
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Figure CN120905430A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of plant molecular markers, more particularly, it relates to a primer and method for rapidly identifying Camellia oleifera varieties. BACKGROUND
[0002] Camellia oleifera is a unique high-quality woody oil crop in China, and has become an important force for ensuring national edible oil safety, promoting farmers' income and ecological protection and coordinated development. The healthy and sustainable development of Camellia oleifera industry, and the innovation and application of good seed are of great importance. In recent years, with the systematic and in-depth breeding work, high-yield and high-quality national and provincial Camellia oleifera varieties have emerged.
[0003] However, due to the strong ability of asexual reproduction of Camellia oleifera and frequent exchange between varieties, combined with the fact that the management of Camellia oleifera seedling market is not perfect, the phenomenon of "same thing different name" caused by the same variety being approved in different provinces and "same name different thing" caused by using inferior products as good ones has occurred, which seriously damages the interests of breeders, seed companies and forest farmers. Although the promulgated forestry industry standard "Plant Specificity, Uniformity and Stability (DUS) Test Camellia oleifera" (2016) provides certain technical indicators and methods for the identification of Camellia oleifera varieties, these indicators and methods are based on phenotypic traits. Phenotypic traits are greatly affected by the environment, making it difficult to effectively distinguish or identify numerous varieties. Moreover, many Camellia oleifera breeding parents are increasingly concentrated in a few excellent varieties or lines, making the bred varieties more similar in more traits, and thus difficult to distinguish.
[0004] DNA molecular markers overcome the shortcomings of traditional morphological markers, cytological markers and biological markers which are easily affected by external factors. Although there is a study on using SNP markers to construct a DNA fingerprint of Camellia oleifera, the development cost of the markers is high, and KASP typing or DNA sequence sequencing is required for later detection, which is complicated and costly. Moreover, if new varieties or lines are to be identified, new SNP markers need to be mined again through genome sequencing, and all varieties need to be re-encoded, so the operation amount is very large and the cost is high.
[0005] RAPD (Random Amplified Polymorphic DNA) molecular markers have a long history of development, and are one of the earliest DNA marker technologies. They have the characteristics and advantages of being fast, simple, not easily affected by external environment and not requiring prior knowledge of genome sequence, and have been widely used in variety classification research, genetic basis research of germplasm resources, genetic linkage markers, genetic map construction and other aspects. SUMMARY
[0006] The technical problem to be solved by the present application is to provide a Camellia oleifera variety identification method which is simple to operate, low in cost and accurate in identification result, and a primer for the method.
[0007] The application discloses a method for rapidly identifying camellia oleifera varieties.
[0008] Step 1: extracting genomic DNA of camellia oleifera varieties;
[0009] Step 2: performing PCR amplification by using RAPD primers in the following table:
[0010] SEQ TD NO Primer Sequence (5'-3') Annealing temperature (°C) 1 Y-35 ATGACTTCTCG 46.6 2 Y-40 AGCGCTATCGT 45.5 3 Y-41 TGCCTCCTCAG 47.3 4 Y-42 CGCGCTATGCC 45.2 5 Y-47 ACGAGCGATAC 44.5 6 Y-48 ACCTGGCACAC 47.1 7 Y-52 CGATGGTGGTC 45.4 8 Y-53 GCGTGATGTCT 46.6 9 Y-54 CGCTGATGTTC 44.5 10 Y-57 TCGCTCAACTG 42.3
[0011] Step 3: constructing an artificial variety identification drawing according to specific bands generated by each RAPD primer amplification;
[0012] Step 4: identifying camellia oleifera varieties according to the MCID.
[0013] Preferably, in step 1, the extracted DNA is detected by 1% agarose gel electrophoresis, and the concentration and purity of the DNA are measured by using a spectrophotometer; and all the DNA is diluted to 20 ng / μL.
[0014] Preferably, in step 2, the reaction system of PCR amplification is 20 μL in total volume, including 2 μL of 10×Buffer, 1.5 mmol of dNTP, 40 ng of template DNA, 3.75 mmol of MgCl2, 1 U of Taq enzyme and 5 μmol of RAPD primer.
[0015] Preferably, in step 2, the PCR reaction program is as follows: 95 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 30 s, annealing for 60 s according to the specific annealing temperature of the primer, 72 ℃ extension for 2 min, 40 cycles, and finally 72 ℃ extension for 10 min.
[0016] Preferably, in step 2, the PCR amplification product is electrophoresed in 1.5% agarose gel for 40 min, EB-stained, observed under an ultraviolet lamp and photographed.
[0017] Preferably, in step 3, the construction of the artificial variety identification drawing includes:
[0018] Taking the primer as a node, the node is followed by the differentiation results of the primer on different camellia oleifera varieties, and the information of the amplification spectrum bands used for differentiation is marked on the branch of the node;
[0019] According to the presence or absence of specific bands amplified by the primer, the camellia oleifera varieties are grouped, and the presence or absence of specific bands is respectively indicated by "+" and "-".
[0020] Further PCR amplification is performed on each group of camellia oleifera varieties by using other primers, and the groups are continuously grouped according to polymorphic bands, until all camellia oleifera varieties are identified and differentiated.
[0021] The application has the beneficial effects that:
[0022] 1.The application uses RAPD molecular marker technology to artificially draw a variety identification schematic diagram, and realizes the identification and differentiation between varieties according to the polymorphic bands after PCR amplification. Unlike the method of using computer software to draw digital fingerprint maps and using statistical software for cluster analysis, the application more directly shows the primer and its polymorphic bands that distinguish the varieties, and has small workload and strong practicability, especially when a large number of test varieties are used, the method is simpler and more practical, and can be used for rapid identification of a large number of camellia oleifera varieties.
[0023] 2.The application greatly improves the utilization efficiency of primers, and 10 primers can distinguish 61 camellia oleifera varieties. In the case of known varieties, the corresponding primers can be found on the MCID, and according to the presence or absence and size information of the characteristic bands, it can be clearly and completely embodied how the varieties are distinguished.
[0024] 3.When a new camellia oleifera variety or strain is added, the existing 10 primers can be directly used for PCR amplification, if the result is different from the existing varieties, it can be directly supplemented to the MCID; if it is found that the new variety is difficult to be distinguished from the existing varieties by 10 primers, new primers need to be added for identification. The method has small workload, high efficiency and long-term effectiveness.
[0025] 4.The application overcomes the disadvantage that phenotypic identification is greatly affected by the environment, compared with the SNP marker method, the cost is low, the operation is simple, and it is not limited by time, season, seedling age, cultivation environment and management measures, which can effectively improve the identification efficiency of camellia oleifera varieties and protect the legal rights and interests of breeders and farmers. BRIEF DESCRIPTION OF DRAWINGS
[0026] Figure 1 PCR amplification results of primer Y35 in 61 camellia oleifera varieties.
[0027] Figure 2 PCR amplification results of some varieties in the first group using primer Y40 (A), and PCR amplification results of two subgroups using Y41 (B) and Y47 (C).
[0028] Figure 3 MCID of 61 camellia oleifera varieties constructed by using 10 primers.
[0029] Figure 4 Identification results of randomly selected 8 camellia oleifera varieties for verification; A: “Changlin No. 21”, “Changlin No. 44”; B: “Changlin No. 55”, “Changlin No. 4”; C: “Huangshan No. 7”, “Miyouxuza No. 22”; D: “Changlin No. 39”, “Zhelin No. 1”. DETAILED DESCRIPTION
[0030] The subject matter described herein will now be discussed with reference to example implementations. It should be understood that the discussion of these implementations is merely meant to provide a better understanding of the subject matter described herein and can include changes, modifications, additions or omissions of the functions and arrangements of the elements discussed without departing from the scope of the present description. Various examples can omit, substitute, or add various procedures or components as appropriate. Also, it should be understood that instead of a single arrangement of elements, a plurality of arrangements can be performed, and each arrangement can involve a different grouping of the elements discussed. Also, the methods described can be implemented in hardware, software, or a combination of both.
[0031] The present application designs RAPD primers for the gene sequence of hexaploid Camellia oleifera, and determines 10 RAPD primers after screening. After extracting the DNA of Camellia oleifera varieties, the RAPD primers are used for PCR amplification one by one, and the manual cultivar identification diagram (MCID) corresponding to the distinguished varieties is constructed according to the specific band. The primer and specific band for distinguishing between different varieties can be clearly indicated from the MCID, and the method shows strong practicability. The method is similar to a library retrieval system, and is convenient for searching for the related information required for identification, finding the corresponding primer sequence and the size and presence or absence of the amplified band, and then performing targeted variety identification. The present application greatly improves the utilization efficiency of the primers, and 10 primers can distinguish 61 Camellia oleifera varieties. In the case of known varieties, the corresponding primers can be searched on the MCID, and according to the presence or absence and size information of the characteristic bands, it can be clearly and completely shown how the varieties are distinguished. When a new Camellia oleifera variety or strain is added, the existing 10 primers can be directly used for PCR amplification, and if the result has different bands from the existing 61 Camellia oleifera varieties, the new variety can be directly supplemented to the MCID. If it is found that the new variety is difficult to be distinguished from the existing 61 varieties by using the 10 primers, new primers need to be added for distinguishing and identifying. The method has small workload, high efficiency and long-term effectiveness.
[0032] Example 1
[0033] A method for rapidly identifying Camellia oleifera varieties, comprising the following steps:
[0034] Step 1: Extracting the genomic DNA of Camellia oleifera varieties; the extracted DNA is detected by 1% agarose gel electrophoresis, and the concentration and purity of the DNA are measured by a spectrophotometer, and all the DNA concentrations are diluted to 20 ng / μL.
[0035] Step 2: Using the RAPD primers in the following table for PCR amplification:
[0036]
[0037] The reaction system of PCR amplification is 20 μL in total volume, including 2 μL of 10×Buffer, 1.5 mmol of dNTP, 40 ng of template DNA, 3.75 mmol of MgCl2, 1 U of Taq enzyme and 5 μmol of RAPD primer;
[0038] The PCR reaction program is as follows: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30 s, annealing for 60 s according to the specific annealing temperature of the primer, extension at 72 ℃ for 2 min, 40 cycles, and finally extension at 72 ℃ for 10 min;
[0039] The PCR amplification product is electrophoresed in a 1.5% agarose gel for 40 min, stained with EB, and observed and photographed under an ultraviolet lamp.
[0040] Step 3: Constructing an artificial variety identification map according to the specific bands generated by amplification of each RAPD primer;
[0041] The construction of the artificial variety identification map includes:
[0042] Taking the primer as a node, the node is followed by the differentiation results of the primer for different camellia varieties, and the information of the amplification spectrum band used for differentiation is labeled on the node branch;
[0043] According to the presence or absence of specific bands amplified by the primer, the camellia varieties are grouped, and the presence or absence of specific bands is represented by "+" and "-", respectively;
[0044] Further PCR amplification of each group of camellia varieties is carried out by using other primers, and the groups are continued to be grouped according to the polymorphic bands, until all camellia varieties are identified and differentiated.
[0045] Step 4: Identifying the camellia varieties according to the MCID.
[0046] Example 2
[0047] In this example, 61 camellia varieties (Table 1) are collected, and genomic DNA is extracted from all experimental varieties. Referring to the genome of hexaploid Camellia oleifera, 10 RAPD primers (Table 2) are designed. The selected primers are used for PCR amplification, and the specificity of the amplified bands is counted. Finally, an artificial variety identification map (MCID) of 61 camellia varieties is constructed, so as to realize the rapid differentiation of 61 camellia varieties at the DNA level.
[0048] Table 1 Name of the tested camellia varieties
[0049]
[0050] Table 2 Information of 10 RAPD primers for identifying 61 camellia varieties
[0051]
[0052] 1. Extraction of DNA
[0053] Genomic DNA of 61 Camellia oleifera varieties was extracted by using DNA extraction kit, and the extracted DNA was free of gel-like material, protein and phenolic substance, and was suitable for PCR amplification. The DNA was detected by 1% agarose gel electrophoresis, and the concentration and purity of the DNA were determined by using NanoDrop, and all the DNA concentrations were diluted to 20 ng / μL.
[0054] 2. PCR amplification
[0055] The reaction system of PCR amplification was 20 μL, including 10×Buffer 2 μL, dNTP 1.5 mmol, template DNA 40 ng, MgCl2 3.75 mmol, Taq enzyme 1 U, RAPD primer 5 μmol, and ddH2O was added to 20 μL. The PCR reaction program was as follows: 95 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 30 s, the annealing temperature was determined according to the specific primer (see Table 2), the annealing time was 60 s, 72 ℃ extension for 2 min, 40 cycles, and finally 72 ℃ extension for 10 min. The amplification product was electrophoresed in 1.5% agarose gel for 40 min, EB stained, and observed and photographed under ultraviolet lamp.
[0056] 3. Construction of MCID
[0057] The MCID of 61 Camellia oleifera varieties was constructed according to the specific bands amplified by 10 primers. The MCID is a node with primers, and the node is followed by the differentiation results of different Camellia oleifera varieties by the primer. The information of the amplification spectrum band used for differentiation is labeled on the node branch. The specific steps are as follows:
[0058] Firstly, 61 Camellia oleifera varieties were divided into two groups according to the presence or absence of the band amplified by primer Y35 at 900 bp, and the presence or absence of specific band was represented by "+" and "-", respectively. The first group was + 900 bp, and the second group was - 900 bp. Figure 1 Then, all the varieties in the above two groups were identified by using other primers. The first group and the second group could be divided into three groups by using the polymorphic bands with sizes of 1900 bp and 600 bp amplified by primer Y40, respectively.
[0059] Taking the 29 Camellia oleifera varieties in the first group as an example, they were divided into three subgroups by using Y40 primer for amplification (part of samples). Figure 2 A) The "Dabianshan No. 5", "Huangshan No. 1" and "Huangshan No. 3" in the second subgroup were differentiated by + 1900 bp and - 600 bp, and then further distinguished by the 1100 bp polymorphic band of primer Y41. Figure 2B). Finally, "Huangshan 1" and "Dabianshan 5" were distinguished by 200bp polymorphic bands of primer Y47 Figure 2 C). The first and third subgroups can be distinguished by primers Y41, Y42, Y47, Y48, etc.
[0060] The results show that there is a close relationship between the specific bands of 29 camellia varieties in the first group distinguished by primer Y35, the primers used and the varieties identified, which can be used to identify the 29 varieties. The 32 varieties in another group distinguished by primer Y35 are identified in turn according to this method, and all 61 camellia varieties can be distinguished by using 10 primers. Finally, the identification map of 61 camellia varieties, i.e. camellia variety MCID (Camellia Identification Map) is drawn according to the primers and the corresponding bands. Figure 3
[0061] 4. Verification of variety identification results
[0062] To further verify the reliability and practicability of the MCID for identifying camellia varieties, four groups of a total of 8 varieties ("Changlin 21", "Changlin 44", "Changlin 55", "Changlin 4", "Huangshan 7", "Minyouza 22", "Changlin 39", "Zhelin 1") were randomly selected for verification. According to the positions of these varieties in the MCID, it can be found that the four groups of varieties can be distinguished by using primers Y57, Y48 and Y47.
[0063] The PCR amplification results show that "Changlin 21" and "Changlin 44"; "Changlin 55" and "Changlin 4" can be distinguished by two bands of 550bp and 400bp amplified by primer Y57 Figure 4 A, B), "Huangshan 7" and "Minyouza 22" can be distinguished by a band of 900bp amplified by primer Y48 Figure 4 C), and "Changlin 39" and "Zhelin 1" can be distinguished by a band of 400bp amplified by primer Y47 Figure 4 Figure 4 D). The results of using the three specific primers to distinguish the four groups of varieties are consistent with the expected results of the MCID, which shows the reliability and practicability of the MCID.
[0064] As can be seen from the above examples, the primers and method for rapidly identifying camellia varieties provided by the present application are simple to operate, low in cost and accurate in identification results, which can effectively solve the problem of difficult identification of camellia varieties in the prior art, and have important application value for camellia germplasm resource management, new variety protection, seedling market supervision, etc.
[0065] The above describes the embodiments of the present application, but the embodiments are not limited to the above specific embodiments, and the above specific embodiments are only illustrative but not restrictive, and the ordinary skilled in the art can make more equivalent embodiments under the inspiration of the embodiments, which are all within the protection scope of the embodiments.
Claims
1. A primer for rapidly identifying Camellia oleifera varieties, characterized in that, The primers are as shown in SEQ TD NO. 1, SEQ TD NO. 2, SEQ TD NO. 3, SEQ TD NO. 4, SEQ TD NO. 5, SEQ TD NO. 6, SEQ TD NO. 7, SEQ TD NO. 8, SEQ TD NO. 9, and SEQ TD NO.
10.
2. A method for rapidly identifying Camellia oleifera varieties, characterized in that, The method comprises the following steps: Step 1: Extracting the genomic DNA of Camellia oleifera varieties; Step 2: Performing PCR amplification using the RAPD primers listed below: SEQ TD NO. 1, SEQ TD NO. 2, SEQ TD NO. 3, SEQ TD NO. 4, SEQ TD NO. 5, SEQ TD NO. 6, SEQ TD NO. 7, SEQ TD NO. 8, SEQ TD NO. 9, and SEQ TD NO. 10; Step 3: Constructing an artificial variety identification map according to the specific bands generated by each RAPD primer amplification; Step 4: Identifying the Camellia oleifera varieties according to the MCID.
3. The method for rapidly identifying Camellia oleifera varieties according to claim 2, characterized in that, In step 1, the extracted DNA is detected by 1% agarose gel electrophoresis, and the concentration and purity of the DNA are measured by a spectrophotometer. All the DNA concentrations are diluted to 20 ng / μL.
4. The method for rapidly identifying Camellia oleifera varieties according to claim 1, characterized in that, In step 2, the reaction system for PCR amplification is 20 μL in total volume, including 10×Buffer 2 μL, dNTP 1.5 mmol, template DNA 40 ng, MgCl2 3.75 mmol, Taq enzyme 1 U, and RAPD primer 5 μmol.
5. The method for rapidly identifying Camellia oleifera varieties according to claim 1, characterized in that, In step 2, the PCR reaction program is as follows: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, annealing for 60 s according to the specific annealing temperature of the primer, 72℃ extension for 2 min, 40 cycles, and finally 72℃ extension for 10 min.
6. The method for rapidly identifying Camellia oleifera cultivars according to claim 1, characterized in that, In step 2, the PCR amplification products are electrophoresed in 1.5% agarose gel for 40 min, stained with EB, and observed and photographed under a UV lamp.
7. The method for rapidly identifying Camellia oleifera cultivars according to claim 1, characterized in that, In step 3, the construction of the artificial variety identification map includes: Taking the primer as a node, the node is followed by the differentiation results of the primer for different Camellia oleifera varieties, and the information of the amplification bands used for differentiation is labeled on the branch of the node; According to the presence or absence of specific bands amplified by the primer, the Camellia oleifera varieties are grouped, and the presence or absence of specific bands is represented by "+" and "-", respectively; Using other primers, the Camellia oleifera varieties in each group are further subjected to PCR amplification, and the grouping is continued according to the polymorphic bands until all the Camellia oleifera varieties are identified and differentiated.