Primer combination for obtaining standard DNA fingerprint spectrum database of cinnamomum variety and application of primer combination

Abstract

The invention discloses a primer combination for obtaining a standard DNA fingerprint database of cinnamomum species and application of the primer combination, and relates to the technical field of biology. The primer combination comprises 14 pairs of primers, and the nucleotide sequences of the 14 pairs of primers are respectively shown as SEQ ID NO.1-SEQ ID NO.28; the invention also provides a standard DNA fingerprint database of the cinnamomum species and a construction method thereof, and provides application of the primer combination or the standard DNA fingerprint database of the cinnamomum species in identification of the cinnamomum species, and a kit for identification of the cinnamomum species. The method has the beneficial effects that 14 pairs of primers with relatively stable difference and good polymorphism are screened, a cinnamomum variety standard DNA fingerprint database can be directly applied to authenticity identification of cinnamomum varieties, particularly screening of similar varieties in a cinnamomum new variety test guide, and the long-term problems of long time consumption, tedious steps and the like in cinnamomum variety identification are solved; and the cinnamomum camphora variety identification is not limited by space-time conditions any more.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer combination for obtaining a standard DNA fingerprint library of Cinnamomum varieties and an application thereof. Background Art

[0002] The review of new plant varieties needs to pass the Distinctness, Uniformity and Stability test, namely the DUS test, to determine whether it meets the basic requirements for variety protection. The DUS test requires observation and evaluation over multiple growing seasons, and the process is time-consuming. Secondly, the test mainly relies on the comparison of phenotypic characteristics, which may ignore some differences at the gene level and thus fail to fully reflect the genetic diversity of the variety. In addition, the DUS test is sensitive to environmental conditions, and the test results in different regions may vary, affecting its consistency and reliability. Finally, for some specific plants or complex traits, the existing DUS test standards may not be sufficient to cover all the characteristics, which may cause some new varieties to fail the test. Therefore, in practical applications, the DUS test needs to be combined with other technical means, such as molecular marker technology, to improve its accuracy and efficiency. Compared with morphological characteristics, molecular markers have the advantages of good stability, no environmental influence, and rapid detection. Therefore, DNA molecular marker technology has broad application prospects in the testing, identification and protection of new plant varieties.

[0003] Cinnamomum plants are important members of the Lauraceae family, widely distributed in tropical and subtropical regions, and have important ecological and economic value. In recent years, a variety of new varieties of Cinnamomum have been bred through biological breeding technology. At present, the review of Cinnamomum varieties mainly adopts the traditional DUS test method, which has good applicability for ornamental varieties, but has poor applicability for some traits that cannot be directly observed. DNA molecular markers have been used to carry out some genetic diversity evaluation studies in camphor trees, but there have been no reports on variety review and identification protection. The construction of fingerprints of standard varieties of Cinnamomum through DNA molecular marker technology can not only provide guidance for the scientificity and accuracy of the review of new plant varieties, but also provide technical support for variety infringement cases. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and to provide a primer combination for obtaining a standard DNA fingerprint library of cinnamon varieties and its application, specifically including providing a set of primer pairs for obtaining a standard DNA fingerprint library of cinnamon varieties, and a standard DNA fingerprint library of cinnamon varieties constructed using the set of primer pairs and a construction method. The 14 pairs of complete primer pairs of the present invention and the cinnamon standard DNA fingerprint library constructed by the cinnamon standard DNA fingerprint library have the advantages of good repeatability, stability and reliability, and rapid detection, and provide a basis for identifying cinnamon varieties using SSR molecular marker technology. The standard DNA fingerprint library of cinnamon varieties of the present invention can be directly applied to the authenticity identification of cinnamon varieties, solving the long-term problems of long-term identification of cinnamon varieties and cumbersome steps, so that the identification of cinnamon varieties is no longer restricted by time and space conditions. The standard DNA fingerprint library of cinnamon varieties of the present invention can be used for the screening of standard varieties and similar varieties in the test standard of the industry standard LY / T 3121-2019 "Guide to the Specificity, Consistency and Stability Test of New Plant Varieties Cinnamon", providing a scientific basis for the scientificity and accuracy of the review of new plant varieties.

[0005] The technical solution of the present invention is as follows: The first aspect of the present invention provides a primer combination for obtaining a standard DNA fingerprint library of Cinnamomum varieties, the primer combination comprising 14 pairs of primers, the nucleotide sequences of the upstream primers and the downstream primers of the 14 pairs of primers are as follows: Primer 1: SEQ ID NO. 1, SEQ ID NO. 2; Primer 2: SEQ ID NO. 3, SEQ ID NO. 4; Primer 3: SEQ ID NO. 5, SEQ ID NO. 6; Primer 4: SEQ ID NO. 7, SEQ ID NO. 8; Primer 5: SEQ ID NO. 9, SEQ ID NO. 10; Primer 6: SEQ ID NO. 11, SEQ ID NO. 12; Primer 7: SEQ ID NO. 13, SEQ ID NO. 14; Primer 8: SEQ ID NO. 15, SEQ ID NO. 16; Primer 9: SEQ ID NO. 17, SEQ ID NO. 18; Primer 10: SEQ ID NO. 19, SEQ ID NO. 20; Primer 11: SEQ ID NO. 21, SEQ ID NO. 22; Primer 12: SEQ ID NO. 23, SEQ ID NO. 24; Primer 13: SEQ ID NO. 25, SEQ ID NO. 26; Primer 14: SEQ ID NO. 27, SEQ ID NO. 28.

[0006] The second aspect of the present invention provides the use of the primer combination in constructing a standard DNA fingerprint library of Cinnamomum varieties.

[0007] The third aspect of the present invention provides a method for constructing a standard DNA fingerprint library of Cinnamomum varieties, comprising the following steps: S1. Extract DNA from Cinnamomum plant samples; S2, using the extracted DNA as a template and the primer combination to perform fluorescent PCR amplification to obtain an amplified product; S3, performing capillary electrophoresis on the amplified product, and analyzing the capillary electrophoresis result to obtain fluorescent SSR data; S4. Convert the fluorescent SSR data into digital and / or letter codes to represent the molecular identity card of each Cinnamomum variety, and construct a standard DNA fingerprint library of Cinnamomum varieties.

[0008] Preferably, the specific steps of S2 include: Using the extracted DNA as a template, adding a fluorescent marker gene to the 5' end of the forward primers of primers 1 to 14, performing fluorescent PCR amplification, and obtaining an amplified product; The PCR amplification reaction system included: 2×TSINGKE Master Mix 10 μL, forward primer, reverse primer 0.15 μL each, DNA 1 μL, ddH2O to 20 μL; PCR amplification program: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, 35 cycles; final extension at 72 °C for 5 min.

[0009] Preferably, the specific steps of S3 include: Take 10 μL of the mixed solution and 0.5 μL of the amplified product, add them to the PCR plate, heat at 95°C for 5 min, cool at -20°C, and centrifuge at 4000 rpm to mix; After capillary electrophoresis detection using ABI 3730 xl genetic analyzer, the locus data were read using Gene mapper software to obtain fluorescent SSR data.

[0010] Preferably, the specific steps of S4 include: The fluorescent SSR data of primers 1 to 14 are assigned numbers 0-9 and X according to the size of the genomic fragment from small to large; The assignments are arranged in the order of primer 1 to primer 14 to form a molecular identity card with 14 character strings, and a standard DNA fingerprint library of Cinnamomum varieties is constructed.

[0011] Preferably, the assignment method in S4 includes: The fluorescent SSR data of primers 1 to 14 include a total of 124 allele numbers; The fluorescent SSR data of primer 1 includes 11 allele numbers, and the 11 allele numbers are assigned values ​​of 0-9 and X in order from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 2 includes 8 allele numbers, and the 8 allele numbers are assigned values ​​0-7 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 3 includes 7 allele numbers, and the 7 allele numbers are assigned values ​​0-6 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 4 includes 10 allele numbers, and the 10 allele numbers are assigned values ​​0-9 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 5 includes 9 allele numbers, and the 9 allele numbers are assigned values ​​0-8 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 6 includes 9 allele numbers, and the 9 allele numbers are assigned values ​​0-8 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 7 includes 10 allele numbers, and the 10 allele numbers are assigned values ​​0-9 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 8 includes 5 allele numbers, and the 5 allele numbers are assigned values ​​0-4 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 9 includes 7 allele numbers, and the 7 allele numbers are assigned values ​​0-6 from small to large according to the size of the genome fragment; The fluorescent SSR data of the primer 10 includes 11 allele numbers, and the 11 allele numbers are assigned values ​​0-9 and X in order from small to large according to the size of the genome fragment; The fluorescent SSR data of the primer 11 includes 10 allele numbers, and the 10 allele numbers are assigned values ​​0-9 from small to large according to the size of the genome fragment; The fluorescent SSR data of the primer 12 includes 10 allele numbers, and the 10 allele numbers are assigned values ​​0-9 from small to large according to the size of the genome fragment; The fluorescent SSR data of the primer 13 includes 9 allele numbers, and the 9 allele numbers are assigned values ​​of 0-8 from small to large according to the size of the genome fragment; The fluorescent SSR data of the primer 14 includes 8 allele numbers, and the 8 allele numbers are assigned values ​​0-7 from small to large according to the size of the genome fragment.

[0012] The fourth aspect of the present invention provides a standard DNA fingerprint library of Cinnamomum varieties, which is obtained by the construction method.

[0013] The fifth aspect of the present invention provides the use of the primer combination or the standard DNA fingerprint library of Cinnamomum varieties in identifying Cinnamomum varieties or screening similar varieties in the test guide for new Cinnamomum varieties.

[0014] The similar variety is the variety with the smallest difference in properties and morphology from the target variety among all known varieties. In a broad sense, similar varieties can be understood as varieties of the same plant species that are close to and similar to the target variety in properties and morphology. In the field of new plant varieties, similar varieties have different meanings in different scenarios. For example, in the national standard "General Guidelines for Specificity, Consistency and Stability Testing of New Plant Varieties" GB / T 19557.1-2004, the specified comparison objects are the application variety and similar varieties. The similar varieties here refer to field observations. It is impossible to plant and compare all known varieties with the application variety. Therefore, only the variety with the smallest expected property difference among the known varieties can be selected for planting comparison.

[0015] Specifically, the methods for screening similar varieties in the industry standard LY / T 3121-2019 "Specificity, consistency and stability testing of new plant varieties for Cinnamomum" include: S1. Extract DNA from samples of new Cinnamomum species; S2, using the extracted DNA as a template, using 14 pairs of primers to perform fluorescent PCR amplification to obtain the amplified product; S3, performing capillary electrophoresis on the amplified product and analyzing the capillary electrophoresis result to obtain fluorescent SSR data; S4, converting the fluorescent SSR data into the same digital and / or alphabetical codes as those used in the standard DNA fingerprint library of the Cinnamomum species for encoding and sorting, to obtain a molecular ID card of the sample; If the molecular ID number of the sample is more than 70% identical to the molecular ID number of the Cinnamomum variety in the standard DNA fingerprint library of the Cinnamomum variety, then the sample is a candidate similar variety.

[0016] More specifically, according to the standard DNA fingerprint library of camphor varieties in Example 2 of the present invention, there are currently 13 camphor tree varieties in the fingerprint library. If the molecular ID number of the sample is consistent with the molecular ID number of any of the 13 camphor tree varieties by 10 to 13 digits and their positions, the sample is a similar variety.

[0017] The sixth aspect of the present invention provides a kit for identifying Cinnamomum varieties or screening similar varieties in the testing guide for new Cinnamomum varieties, comprising the primer combination.

[0018] The present invention has at least one of the following beneficial effects: The present invention screened 14 pairs of primers with good polymorphism from the designed 100 pairs of SSR primers, and used these 14 pairs of primers to construct a standard DNA fingerprint library of Cinnamomum, which has the advantages of good repeatability, stability and reliability, and rapid detection, and provides a basis for using SSR molecular marker technology to identify Cinnamomum varieties.

[0019] The standard DNA fingerprint library of Cinnamomum varieties constructed by the present invention can be directly applied to the authenticity identification of Cinnamomum varieties, solving the long-standing problems of long-term time-consuming and cumbersome steps in the identification of Cinnamomum varieties, so that the identification of Cinnamomum varieties is no longer restricted by time and space conditions. The standard DNA fingerprint library of Cinnamomum varieties of the present invention can be used for the screening of similar varieties in the industry standard LY / T 3121-2019 "Guide to the Specificity, Consistency and Stability Testing of New Plant Varieties Cinnamomum", providing a scientific basis for the scientificity and accuracy of the review of new plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the amplified fragment of primer CC6 in Huai Nao Xiang.

[0021] Figure 2 This is the amplified fragment of primer CC7 in Huai Nao Xiang.

[0022] Figure 3 This is the amplified fragment of primer CC8 in Huai Nao Xiang.

[0023] Figure 4 This is the amplified fragment of primer CC12 in Jilong 1.

[0024] Figure 5 This is the amplified fragment of primer CC16 in Huai Nao Xiang Hao.

[0025] Figure 6 This is the amplified fragment of primer CC21 in Jilong 1.

[0026] Figure 7 This is the amplified fragment of primer CC30 in Jilong 1.

[0027] Figure 8 This is the amplified fragment of primer CC31 in Ruyu.

[0028] Fig. 9 This is the amplified fragment of primer CC32 in Shenggan.

[0029] Fig.10 This is the amplified fragment of primer CC33 in Yongjin.

[0030] Fig.11 This is the amplified fragment of primer CC34 in Chibaxiang.

[0031] Fig.12 This is the amplified fragment of primer CC35 in Cinnamomum cassia.

[0032] Fig.13 This is the amplified fragment of primer CC37 in Yuhuang.

[0033] Fig.14 This is the amplified fragment of primer CC39 in Cinnamomum cassia. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Example 1 This embodiment provides a method for obtaining and screening a primer combination for obtaining a standard DNA fingerprint library of Cinnamomum species, comprising the following steps: 1.1 Test materials As shown in Table 1, the 13 Cinnamomum varieties tested were collected from the Nanchang Station of the National New Variety Testing Station of Forestry and Grassland Plants of Jiangxi Academy of Forestry Sciences in April 2024. Two single plants of each variety were randomly selected, and mature leaves without diseases and insect pests were put into self-sealing bags and stored in a -80℃ refrigerator for later use.

[0036] Table 1 Test materials 1.2 DNA extraction The genomic DNA of the test materials was extracted using a plant genomic DNA rapid extraction kit (B518231, Sangon Biotech (Shanghai) Co., Ltd.), the DNA quality was tested by agarose gel electrophoresis, and the DNA concentration was tested using a nucleic acid protein analyzer (Eppendorf, Germany) and stored at -80°C for future use.

[0037] 1.3 SSR Marker Development Taking the Cinnamomum camphora genome as a reference, the SSR information loci of Cinnamomum camphora were analyzed by Tbtools SSRminer plug-in, and then 100 pairs of SSR primers were designed by Tbtools Batch Target Region Primer Design for polymorphism screening.

[0038] Different fluorescent marker genes were added to the 5' end of the forward primer of 100 pairs of SSR primers, and Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize them. Fluorescent primer PCR reaction system: 10 μL of ready-to-use PCR premix (2×TSINGKE Master Mix), 0.15 μL of each forward and reverse primer, 1 μL of DNA, and ddH2O to 20 μL. PCR amplification program: 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; finally 72 ℃ extension for 5 min.

[0039] 1.4 Capillary electrophoresis detection The PCR products were mixed according to different fluorescent markers, and the PCR products were detected by capillary fluorescence electrophoresis using a 3730XL sequencer at Sangon Biotech (Shanghai) Co., Ltd.

[0040] 10 μl of Mix (HIDI Formamide buffer and GS500 LIZ internal standard mixed at a volume ratio of 130:1) and 0.5 μl of the above SSR amplification product were mixed and added to the PCR plate, heated at 95°C for pre-denaturation for 5 min, cooled at -20°C, and centrifuged at 4000 rpm to mix. After capillary electrophoresis detection using an ABI 3730 xl genetic analyzer (Thermo, USA), the site data was read using Genemapper software.

[0041] Through capillary fluorescence electrophoresis detection, SSR primers with good polymorphism and high amplification success rate were finally selected for subsequent data analysis.

[0042] 1.5 Data Analysis The GenAlEx software (version 6.501) was used to calculate genetic diversity parameters such as observed allele (Na), effective allele (Ne), Shannon information index (I), observed heterozygosity (Ho), and expected heterozygosity (He).

[0043] The results are as follows: The amplification information of the 14 primer pairs with high amplification success rates is shown in Table 2. After fluorescence capillary electrophoresis, each of the 14 primer pairs could obtain accurate DNA fragment sizes in Cinnamomum species.

[0044] Table 2 SSR primer sequence information The genetic diversity parameter statistics of the 14 SSR loci are shown in Table 3. Among them, the genomic DNA of the Cinnamomum varieties was amplified using the 14 pairs of SSR primers screened, and a total of 124 alleles were detected. The number of observed alleles at each locus was 5-11, and the average number of alleles per primer pair was 8.86. Among them, primers CC6 and CC33 had the largest number of alleles, and primer CC31 had the smallest number of alleles. The Shannon information index I value ranged from 0.97 to 2.23, with an average of 1.84. The observed heterozygosity Ho value of the primers ranged from 0.31 to 0.92, with an average of 0.62. The expected heterozygosity He value ranged from 0.52 to 0.88, with an average of 0.79.

[0045] Table 3 Statistics of genetic diversity parameters of 14 SSR loci Example 2 This embodiment provides a method for constructing a standard DNA fingerprint library of Cinnamomum varieties, comprising the following steps: S1, extracting DNA from the 13 Cinnamomum plant samples in Example 1, using the same extraction method as in Example 1; S2, using the extracted DNA as a template, and using the 14 pairs of primers in Example 1 to perform fluorescent PCR amplification to obtain an amplified product; Figure 1 to Figure 14 These are the amplified fragments of some primers in Cinnamomum species.

[0046] The specific steps include: Using the extracted DNA as a template, different fluorescent marker genes were added to the 5' end of the forward primers of primers 1 to 14 selected in Example 1, respectively, and fluorescent PCR amplification was performed to obtain an amplified product; Fluorescent primer PCR reaction system: 10 μL of ready-to-use PCR premix (2×TSINGKE Master Mix), 0.15 μL of each forward and reverse primer, 1 μL of DNA, and ddH2O to 20 μL. PCR amplification program: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; and finally 72°C extension for 5 min.

[0047] S3, performing capillary electrophoresis on the amplified product and analyzing the capillary electrophoresis result to obtain fluorescent SSR data; The specific steps include: 10 μL of mixed solution (HIDI Formamide buffer and GS500 LIZ internal standard mixed at a volume ratio of 130:1) and 0.5 μL of the above SSR amplification product were mixed and added to the PCR plate, heated at 95°C for pre-denaturation for 5 min, cooled at -20°C, and centrifuged at 4000 rpm to mix. After capillary electrophoresis detection by ABI 3730 xl Genetic Analyzer (Thermo, USA), the site data was read using Gene Mapper software.

[0048] S4. Convert the fluorescent SSR data into digital and / or letter codes to represent the molecular identity card of each Cinnamomum variety, and construct a standard DNA fingerprint library of Cinnamomum varieties.

[0049] The specific steps include: (1) Assignment: The genotypes amplified by the 14 pairs of primers were assigned values. According to Table 3, a total of 124 alleles were detected, and the number of observed alleles at each site was 5 to 11. That is, since the number of observed alleles at each site was at most 11, only 11 values ​​were required. In this embodiment, the numbers 0-9 and X were used for assignment.

[0050] Therefore, for sites with 11 observed alleles, values ​​of 0-9 and X are assigned according to the size of the genomic fragment from small to large. For sites with less than 11 observed alleles, values ​​of 0-9 are assigned according to the size of the genomic fragment from small to large.

[0051] The specific assignment method is as follows: The fluorescent SSR data of primer 1 (CC6) included 11 allele numbers, which were assigned values ​​of 0-9 and X from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 2 (CC7) included 8 allele numbers, which were assigned values ​​of 0 to 7 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 3 (CC8) included 7 allele numbers, which were assigned values ​​of 0 to 6 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 4 (CC12) included 10 allele numbers, which were assigned values ​​of 0 to 9 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 5 (CC16) included 9 allele numbers, which were assigned values ​​of 0 to 8 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 6 (CC21) included 9 allele numbers, which were assigned values ​​of 0 to 8 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 7 (CC30) included 10 allele numbers, which were assigned values ​​of 0 to 9 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 8 (CC31) included 5 allele numbers, which were assigned values ​​of 0 to 4 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 9 (CC32) included 7 allele numbers, which were assigned values ​​of 0 to 6 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 10 (CC33) included 11 allele numbers, which were assigned values ​​0-9 and X from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 11 (CC34) included 10 allele numbers, which were assigned values ​​of 0 to 9 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 12 (CC35) included 10 allele numbers, which were assigned values ​​of 0 to 9 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 13 (CC37) included 9 allele numbers, which were assigned values ​​of 0 to 8 from small to large according to the size of the genomic fragment; The fluorescent SSR data of primer 14 (CC39) included 8 allele numbers, which were assigned values ​​of 0-7 from small to large according to the size of the genomic fragment.

[0052] (2) Sorting: In this embodiment, the assignments are sorted in order from primer 1 to primer 14, that is, the assignments (0-9 and X) of the CC6 site are sorted in order as the first position, the assignments (0-7) of the CC7 site are sorted in order as the second position, the assignments (0-6) of the CC8 site are sorted in order as the third position, and so on. The assignments (0-7) of the CC39 site are sorted in order as the 14th position.

[0053] According to the above-mentioned assignment and sorting method, a string of 14 numbers or numbers and letters is formed as the molecular identity card of the sample, and a standard DNA fingerprint library of Cinnamomum varieties is constructed.

[0054] The results are as follows: Table 4 shows the genotype assignment and sorting criteria amplified by 14 pairs of primers.

[0055] Table 4 Genotype assignment and ranking criteria for the loci of the 14 primer pairs According to the assignment and sorting criteria in Table 4 and combined with the genotypes of camphor tree varieties, the identity cards of 13 camphor tree varieties were obtained, as shown in Table 5.

[0056] Table 5 Identity cards of 13 camphor tree species In summary, the present invention screened 14 pairs of primers, and used these 14 pairs of primers to construct a standard DNA fingerprint library of Cinnamomum varieties. The 14 pairs of primers of the present invention and the standard DNA fingerprint library of Cinnamomum constructed therefrom were used to identify Cinnamomum varieties, which has the advantages of good repeatability, stability and reliability, and rapid detection, and provides a basis for identifying Cinnamomum varieties using SSR molecular marker technology.

[0057] The standard DNA fingerprint library of Cinnamomum varieties of the present invention can be directly applied to the authenticity identification of Cinnamomum varieties, solving the long-standing problems of long-term time-consuming and complicated steps in the identification of Cinnamomum varieties, and making the identification of Cinnamomum varieties no longer restricted by time and space conditions.

[0058] The standard DNA fingerprint library of Cinnamomum varieties of the present invention can be used for screening similar varieties in the industry standard LY / T 3121-2019 "Guide to Specificity, Consistency and Stability Testing of New Plant Varieties - Cinnamomum", providing a scientific basis for the scientificity and accuracy of the review of new plant varieties.

[0059] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A primer combination for obtaining a standard DNA fingerprint library of Cinnamomum species, characterized in that: The primer combination includes 14 pairs of primers, and the nucleotide sequences of the upstream primers and downstream primers of the 14 pairs of primers are as follows: Primer 1: SEQ ID NO. 1, SEQ ID NO. 2; Primer 2: SEQ ID NO. 3, SEQ ID NO. 4; Primer 3: SEQ ID NO. 5, SEQ ID NO. 6; Primer 4: SEQ ID NO. 7, SEQ ID NO. 8; Primer 5: SEQ ID NO. 9, SEQ ID NO. 10; Primer 6: SEQ ID NO. 11, SEQ ID NO. 12; Primer 7: SEQ ID NO. 13, SEQ ID NO. 14; Primer 8: SEQ ID NO. 15, SEQ ID NO. 16; Primer 9: SEQ ID NO. 17, SEQ ID NO. 18; Primer 10: SEQ ID NO. 19, SEQ ID NO. 20; Primer 11: SEQ ID NO. 21, SEQ ID NO. 22; Primer 12: SEQ ID NO. 23, SEQ ID NO. 24; Primer 13: SEQ ID NO. 25, SEQ ID NO. 26; Primer 14: SEQ ID NO. 27, SEQ ID NO.

28.

2. Use of the primer combination described in claim 1 in constructing a standard DNA fingerprint library for Cinnamomum varieties.

3. A method for constructing a standard DNA fingerprint library of Cinnamomum species, characterized in that: The following steps are involved: S1. Extract DNA from Cinnamomum plant samples; S2. Using the extracted DNA as a template, and using the primer combination of claim 1 to perform fluorescent PCR amplification to obtain an amplified product; S3, performing capillary electrophoresis on the amplified product, and analyzing the capillary electrophoresis result to obtain fluorescent SSR data; S4. Convert the fluorescent SSR data into digital and / or letter codes to represent the molecular identity card of each Cinnamomum variety, and construct a standard DNA fingerprint library of Cinnamomum varieties.

4. The construction method according to claim 3, characterized in that: The specific steps of S2 include: Using the extracted DNA as a template, adding a fluorescent marker gene to the 5' end of the forward primers of primers 1 to 14 of claim 1, performing fluorescent PCR amplification, and obtaining an amplified product; The PCR amplification reaction system included: 2×TSINGKE Master Mix 10 μL, forward primer, reverse primer 0.15 μL each, DNA 1 μL, ddH2O to 20 μL; PCR amplification program: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, 35 cycles; final extension at 72 °C for 5 min.

5. The construction method according to claim 3, characterized in that: The specific steps of S3 include: Take 10 μL of the mixed solution and 0.5 μL of the amplified product, add them to the PCR plate, heat at 95°C for pre-denaturation for 5 min, cool at -20°C, and centrifuge at 4000 rpm to mix; After capillary electrophoresis detection using ABI 3730 xl genetic analyzer, the locus data were read using Gene mapper software to obtain fluorescent SSR data.

6. The construction method according to claim 3, characterized in that: The specific steps of S4 include: The fluorescent SSR data of primers 1 to 14 are assigned numbers 0-9 and X according to the size of the genomic fragment from small to large; The assignments are arranged in the order of primer 1 to primer 14 to form a molecular identity card with 14 character strings, and a standard DNA fingerprint library of Cinnamomum varieties is constructed.

7. The construction method according to claim 6, characterized in that: The assignment methods in S4 include: The fluorescent SSR data of primers 1 to 14 include a total of 124 allele numbers; The fluorescent SSR data of primer 1 includes 11 allele numbers, and the 11 allele numbers are assigned values ​​of 0-9 and X in order from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 2 includes 8 allele numbers, and the 8 allele numbers are assigned values ​​0-7 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 3 includes 7 allele numbers, and the 7 allele numbers are assigned values ​​0-6 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 4 includes 10 allele numbers, and the 10 allele numbers are assigned values ​​0-9 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 5 includes 9 allele numbers, and the 9 allele numbers are assigned values ​​0-8 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 6 includes 9 allele numbers, and the 9 allele numbers are assigned values ​​0-8 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 7 includes 10 allele numbers, and the 10 allele numbers are assigned values ​​0-9 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 8 includes 5 allele numbers, and the 5 allele numbers are assigned values ​​0-4 from small to large according to the size of the genome fragment; The fluorescent SSR data of primer 9 includes 7 allele numbers, and the 7 allele numbers are assigned values ​​0-6 from small to large according to the size of the genome fragment; The fluorescent SSR data of the primer 10 includes 11 allele numbers, and the 11 allele numbers are assigned values ​​0-9 and X in order from small to large according to the size of the genome fragment; The fluorescent SSR data of the primer 11 includes 10 allele numbers, and the 10 allele numbers are assigned values ​​0-9 from small to large according to the size of the genome fragment; The fluorescent SSR data of the primer 12 includes 10 allele numbers, and the 10 allele numbers are assigned values ​​0-9 from small to large according to the size of the genome fragment; The fluorescent SSR data of the primer 13 includes 9 allele numbers, and the 9 allele numbers are assigned values ​​of 0-8 from small to large according to the size of the genome fragment; The fluorescent SSR data of the primer 14 includes 8 allele numbers, and the 8 allele numbers are assigned values ​​0-7 from small to large according to the size of the genome fragment.

8. A standard DNA fingerprint library of Cinnamomum species, characterized in that: Obtained by the construction method described in any one of claims 4 to 7.

9. Use of the primer combination of claim 1 or the standard DNA fingerprint library of Cinnamomum varieties of claim 8 in identifying Cinnamomum varieties or screening similar varieties in the test guide for new Cinnamomum varieties, characterized in that: The similar variety is the variety that has the smallest difference in properties and morphology from the target variety among all known varieties.

10. A kit for identifying Cinnamomum species or screening similar species in the test guide for new Cinnamomum species, characterized in that: Containing the primer combination as described in claim 1, the similar variety is the variety with the smallest difference in properties and morphology from the target variety among all known varieties.

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