SSR (Simple Sequence Repeat) core primer combination for identifying tea tree variety Danxia 8, kit and application

Abstract

The invention discloses an SSR (Simple Sequence Repeat) core primer combination for identifying a tea tree variety 'Danxia 8', a kit and application. According to the invention, three pairs of SSR core primers are screened by utilizing an SSR fluorescence labeling detection technology, and the three pairs of SSR core primers can be used for rapidly identifying and detecting 'Danxia No.8' from other tea tree varieties. The SSR core primer combination disclosed by the invention can be used for identifying the variety of Danxia No.8, the uncertainty of identification based on external morphological characteristics is overcome, the result is reliable and visual, the detection efficiency is high, the operation is simple and convenient, and meanwhile, the popularization, utilization and protection of the variety of Danxia No.8 are also facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of variety resource identification and germplasm innovation, and in particular to an SSR core primer combination, a kit and an application thereof for identifying the tea variety "Danxia No. 8". Background Art

[0002] "Danxia No. 8" is an excellent tea tree variety selected from the natural variant of the wild population of Renhua white tea. It is characterized by: the tree is half-open, the leaves are obliquely arranged, lanceolate, green, flat, inward-folded, wavy, gradually pointed, sharp teeth, and hard. The flowers are solitary, with single petals, 5 petals of milky white, compound pistil, located in the center of the stamen group, and 3 shallow lobes of the style. It is suitable for making famous red and white tea. The black tea is tight, fine, uniform and has a sharp seedling, full of golden hair, with a strong and lasting compound rose fragrance, a strong and refreshing taste, and a bright dark red soup; the white tea has a slender and straight bud tip, white hair is full of white hair, and the soup is apricot yellow and bright, with a sweet, mellow and fresh taste, and a rich fragrance with magnolia fragrance.

[0003] The traditional method of identifying "Danxia No. 8" is that the identifier makes a judgment based on its external morphology and other characteristics based on years of professional experience. This is easily affected by factors such as the growth years, cultivation measures, development stages, cultivation measures and environmental conditions of "Danxia No. 8", and the identification results are extremely inaccurate. Therefore, in order to better identify the variety of "Danxia No. 8", developing an accurate and rapid method suitable for identifying the variety of "Danxia No. 8" has become a technical problem that technicians in this field urgently need to solve.

[0004] Molecular marker technology can identify varieties quickly, accurately and efficiently, and the use of molecular marker methods has good application prospects in variety-specific evaluation and protection. Summary of the invention

[0005] In order to overcome the above-mentioned defects and shortcomings in the prior art, the present invention provides an SSR core primer combination, a kit and an application for identifying the tea variety "Danxia No. 8".

[0006] The first object of the present invention is to provide an SSR core primer combination for identifying the tea variety "Danxia No. 8".

[0007] The second object of the present invention is to provide the use of the above-mentioned SSR core primer combination in the preparation of an identification product of the tea variety "Danxia No. 8".

[0008] The third object of the present invention is to provide a product comprising the above-mentioned SSR core primer combination for use in identifying the tea variety "Danxia No. 8".

[0009] The fourth object of the present invention is to provide a kit for identifying the tea variety "Danxia No. 8".

[0010] A fifth object of the present invention is to provide a method for identifying the tea variety "Danxia No. 8".

[0011] To achieve the above-mentioned purpose, the present invention provides an SSR core primer combination for identifying the tea variety "Danxia No. 8", wherein the SSR core primer combination comprises SSR core primers with nucleotide sequences shown as DX8.SSR-1-F, DX8.SSR-1-R, DX8.SSR-2-F, DX8.SSR-2-R, DX8.SSR-3-F and DX8.SSR-3-R.

[0012] The 5' end of the SSR core primer shown in the nucleotide sequence DX8.SSR-1-F, the 5' end of the SSR core primer shown in the nucleotide sequence DX8.SSR-2-F and / or the 5' end of the SSR core primer shown in the nucleotide sequence DX8.SSR-3-F is labeled with a fluorescent reporter group.

[0013] The fluorescent reporter group is FAM, HEX, TAMRA or ROX.

[0014] Application of any of the above-mentioned SSR core primer combinations in the preparation of an identification product for the tea variety "Danxia No. 8".

[0015] Application of a product comprising any of the above-mentioned SSR core primer combinations in identifying the tea variety "Danxia No. 8".

[0016] A kit for identifying the tea variety "Danxia No. 8", comprising any of the above-mentioned SSR core primer combinations.

[0017] The kit also includes HSTaq DNA polymerase, dNTPs, 10×Buffer, a positive control reference substance, a negative control reference substance, and ultrapure water.

[0018] A method for identifying the tea variety "Danxia No. 8" is provided, using any of the above-mentioned SSR core primer combinations or kits for identification.

[0019] The method comprises the following steps: S1. Extracting genomic DNA from the tea tree sample to be tested; S2. Using the genomic DNA extracted in step S1 as a template, PCR amplification is performed using any of the above-mentioned SSR core primer combinations; S3. Typing the PCR amplification product obtained in step S2, and performing band discrimination on the typing result: The SSR core primers shown by the nucleotide sequences of DX8.SSR-1-F and DX8.SSR-1-R amplify two characteristic bands of 260bp and 264bp, the SSR core primers shown by the nucleotide sequences of DX8.SSR-2-F and DX8.SSR-2-R amplify two characteristic bands of 420bp and 423bp, and the SSR core primers shown by the nucleotide sequences of DX8.SSR-3-F and DX8.SSR-3-R amplify two characteristic bands of 272bp and 287bp, indicating that the tea tree sample to be tested is "Danxia No. 8"; the appearance of other bands indicates that the tea tree sample to be tested is not "Danxia No. 8".

[0020] The typing method described in step S3 is to perform capillary electrophoresis on the PCR amplification product; wherein the PCR amplification, the total reaction system is 10μL, including: 10×Buffer I 1μL, 2.5 mMdNTPs 0.8μL, 2.5μM upstream primer 0.6μL, 2.5μM downstream primer 0.6μL, TAKARA HSTaq 0.1μL, DNA 1μL, ddH2O filler 10μL; PCR amplification reaction conditions are: 95℃ pre-denaturation for 5min; 94℃ denaturation for 30s; 60℃ annealing for 30s for 35 cycles; 72℃ extension for 30s; and finally 60℃ extension for 30min.

[0021] Advantages and effects of the present invention: The present invention discloses an SSR core primer combination, a kit and an application for identifying a tea variety "Danxia No. 8". The present invention utilizes SSR fluorescent marker detection technology to screen and obtain three pairs of SSR core primers, which can quickly identify and detect "Danxia No. 8" from other tea varieties. The SSR core primer combination disclosed in the present invention can perform variety identification on "Danxia No. 8", overcomes the uncertainty of identification based on external morphological characteristics, has reliable and intuitive results, high detection efficiency, and is easy to operate. It is also beneficial to the promotion, utilization and protection of the variety "Danxia No. 8". BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Capillary electrophoresis bands of 21 tea varieties / lines amplified by the specific SSR core primers DX8.SSR-1-F and DX8.SSR-1-R in Example 1.

[0023] Figure 2 Capillary electrophoresis bands of 21 tea varieties / lines amplified by the specific SSR core primers DX8.SSR-2-F and DX8.SSR-2-R in Example 1.

[0024] Figure 3Capillary electrophoresis bands of 21 tea varieties / lines amplified by the specific SSR core primers DX8.SSR-3-F and DX8.SSR-3-R in Example 1.

[0025] Figure 4 Capillary electrophoresis bands of 31 tea varieties / lines amplified by the specific SSR core primers DX8.SSR-1-F and DX8.SSR-1-R in Example 2.

[0026] Figure 5 Capillary electrophoresis bands of 31 tea varieties / lines amplified by the specific SSR core primers DX8.SSR-2-F and DX8.SSR-2-R in Example 2.

[0027] Figure 6 Capillary electrophoresis bands of 31 tea varieties / lines amplified by the specific SSR core primers DX8.SSR-3-F and DX8.SSR-3-R in Example 2. DETAILED DESCRIPTION

[0028] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0029] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] Example 1 Screening and obtaining of specific primer sequences of “Danxia No. 8” 1. Experimental Methods 1. SSR primer design The genome sequence of the tea plant genome database (http: / / tpia.teaplants.cn / ) was searched using MISA software to retrieve the SSR sites in the genome sequence. Primer 5.0 was used to batch design 200 pairs of SSR primers, which were synthesized by Beijing Reader Gene Technology Co., Ltd.

[0031] 2. Extraction of DNA from tea varieties A preliminary screening was conducted using 21 tea varieties / lines with similar genetic and phenotypical characteristics, including Danxia No. 8, Huangkeng Baimao No. 5, Luokeng-2, Danxia No. 9, and the hybrid offspring of Danxia No. 8 and Qingshan No. 1 (numbered A16~A21). The tea varieties / lines used in the preliminary screening are shown in Table 1 (taken from the Guangdong Tea Germplasm Resource Bank).

[0032] Table 1 21 tea varieties / lines used in the preliminary screening

[0033] DNA was extracted from the 21 tea varieties / lines (Table 1) used in the preliminary screening. The specific steps of DNA extraction were as follows: a) Take tea leaves and add liquid nitrogen to grind them thoroughly, and weigh about 100 mg of the ground powder.

[0034] b) Quickly add 400 μL of GPS buffer and 10 μL of RNase A to the ground powder, vortex quickly to mix, and place the centrifuge tube in a 65°C water bath for 15 min. Invert the centrifuge tube several times to mix the sample during the water bath.

[0035] c) Add 100 μL of buffer GPA, vortex for 1 min, centrifuge at 12,000 rpm for 5 min, transfer the supernatant to the filter column CS, then centrifuge at 12,000 rpm for 1 min, and transfer the filtrate to a new centrifuge tube.

[0036] d) Add an equal volume of anhydrous ethanol and mix thoroughly. Flocculent precipitation may appear at this time.

[0037] e) Transfer the solution and flocculent precipitate obtained in the previous step to RNase-Free adsorption column CR2, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the RNase-Free adsorption column CR2 in a collection tube.

[0038] f) Add 550 μL of deproteinizing solution RD to the RNase-Free adsorption column CR2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the RNase-Free adsorption column CR2 in a collection tube.

[0039] g) Add 700 μL of rinse solution PW to the RNase-Free adsorption column CR2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the RNase-Free adsorption column CR2 in a collection tube.

[0040] h) Repeat step g.

[0041] i) Place the RNase-Free adsorption column CR2 back into the collection tube, centrifuge at 12,000 rpm for 2 min, discard the collection tube, and then transfer the RNase-Free adsorption column CR2 to a new centrifuge tube and dry it at room temperature for 5-10 min.

[0042] j) Add 50-100 μL of elution buffer TB to the RNase-Free adsorption column CR2, place at room temperature for 3-5 minutes, centrifuge at 12000 rpm for 2 minutes, collect the solution into a centrifuge tube, and obtain the tea tree genomic DNA.

[0043] 3. PCR Amplification The tea tree genomic DNA extracted in step 2 is used as a template, and PCR amplification is performed using the SSR fluorescent marker detection technology, wherein the primers include an upstream primer with a fluorescent reporter group (the fluorescent reporter group is FAM, HEX, TAMRA, or ROX, and the FAM fluorescent marker is used in the present invention) labeled at the 5' end, and a downstream primer. The PCR amplification guided by the upstream primer with a fluorescent reporter group labeled at the 5' end produces a PCR product with fluorescence.

[0044] For PCR amplification, the total reaction system was 10 μL, including: 10×Buffer I 1 μL, 2.5 mM dNTPs 0.8 μL, 2.5 μM upstream primer 0.6 μL, 2.5 μM downstream primer 0.6 μL, TAKARA HSTaq 0.1 μL, DNA 1 μL, and ddH2O to make up 10 μL.

[0045] The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s; annealing at 60°C for 30 s for 35 cycles; extension at 72°C for 30 s; and finally extension at 60°C for 30 min.

[0046] 4. Typing test Capillary electrophoresis detection: 9 μL of molecular weight internal standard and formamide mixture (volume ratio of 0.5:8.5) and 1.0 μL of PCR product were added to each well of a 96-well plate; denaturation was performed at 95°C for 3 min, and typing was performed using a 3730XL DNA analyzer.

[0047] 5. Data Analysis The raw data files obtained by 3730XL DNA analyzer were imported into the analysis software genemapper ID 3.2 for band discrimination analysis.

[0048] 2. Experimental Results Three pairs of specific SSR core primers were screened out from 200 pairs of SSR primers, namely DX8.SSR-1-F, DX8.SSR-1-R, DX8.SSR-2-F, DX8.SSR-2-R, DX8.SSR-3-F and DX8.SSR-3-R. The primer sequences are shown in Table 2.

[0049] Table 2 Specific SSR core primer sequences

[0050] The statistical results of capillary electrophoresis bands of 21 tea varieties / lines (Table 1) amplified by these three pairs of specific SSR core primers are shown in Tables 3 and Figures 1 to 3 As shown in the figure, the results show that "Danxia No. 8" has characteristic bands, specifically: (1) The specific SSR core primers DX8.SSR-1-F and DX8.SSR-1-R amplified two characteristic bands of 260 bp and 264 bp respectively; (2) Specific SSR core primers DX8.SSR-2-F and DX8.SSR-2-R amplified two characteristic bands of 420 bp and 423 bp; (3) The specific SSR core primers DX8.SSR-3-F and DX8.SSR-3-R amplified two characteristic bands of 272 bp and 287 bp.

[0051] The above results showed that the three pairs of specific SSR core primers could amplify characteristic bands for the tea variety "Danxia No. 8", so "Danxia No. 8" could be distinguished from the other 20 related and phenotypically similar tea varieties / lines.

[0052] Table 3 Statistics of capillary electrophoresis bands of 21 tea varieties / lines by three pairs of specific SSR core primers

[0053] Example 2 Further validation of 3 pairs of specific SSR core primers 1. Experimental Methods The three pairs of specific SSR core primers DX8.SSR-1-F and DX8.SSR-1-R, DX8.SSR-2-F and DX8.SSR-2-R, and DX8.SSR-3-F and DX8.SSR-3-R for identifying "Danxia No. 8" were further verified, and 30 tea varieties currently promoted and applied in production were selected for amplification detection simultaneously with "Danxia No. 8". The details of the 31 tea varieties are shown in Table 4.

[0054] Table 4 31 tea varieties currently being promoted and applied in production

[0055] The extraction method, PCR amplification, typing detection and data analysis method of tea variety DNA are carried out according to Example 1.

[0056] 2. Experimental Results The statistical results of capillary electrophoresis bands of 31 tea varieties (Table 4) amplified by three pairs of specific SSR core primers are shown in Tables 5 and Figures 4 to 6 As shown, the results show that only Danxia No. 8 has characteristic bands, specifically: (1) The specific SSR core primers DX8.SSR-1-F and DX8.SSR-1-R amplified two characteristic bands of 260 bp and 264 bp respectively; (2) Specific SSR core primers DX8.SSR-2-F and DX8.SSR-2-R amplified two characteristic bands of 420 bp and 423 bp; (3) The specific SSR core primers DX8.SSR-3-F and DX8.SSR-3-R amplified two characteristic bands of 272 bp and 287 bp.

[0057] The above results indicate that the three pairs of specific SSR core primers can amplify specific alleles for the tea variety "Danxia No. 8", and therefore can distinguish "Danxia No. 8" from the other 30 tea varieties currently promoted and applied in production.

[0058] Table 5 Statistics of capillary electrophoresis bands of 31 tea varieties promoted and applied

[0059] Example 3 A method for identifying "Danxia No. 8" using specific SSR core primer combination 1. Extract DNA of tea variety "Danxia No. 8" according to the method of Example 1; 2. Using the DNA extracted in step 1 as a template, PCR amplification was performed using three pairs of specific SSR core primers DX8.SSR-1-F and DX8.SSR-1-R, DX8.SSR-2-F and DX8.SSR-2-R, DX8.SSR-3-F and DX8.SSR-3-R. The sequences of the three pairs of specific SSR core primers are as follows, wherein the 5' end of the upstream primer is labeled with a fluorescent reporter group (the fluorescent reporter group can be FAM, HEX, TAMRA or ROX, and the HEX fluorescent label is used in the present invention): DX8.SSR-1-F:GTAGACTACGCTTGGTTGCCAT; DX8.SSR-1-R:CTTGTGCGAATCTTGAGACTCTAG; DX8.SSR-2-F:TCCACAACGACAACACCAACA; DX8.SSR-2-R:GCAGATACAGAAACGACCAGGTC; DX8.SSR-3-F: TCATGAATATCACAAACCTGGTAACT; DX8.SSR-3-R:CCTCAGATTGACCTGCATGAGTT; 3. PCR amplification method was carried out according to Example 1: The PCR product obtained in step 2 is subjected to typing detection and data analysis, and the typing detection and data analysis methods are both performed in accordance with Example 1.

[0060] 4. Interpretation of results: If the amplification products of the three pairs of specific SSR core primers show the following characteristic bands, it indicates that the tea variety to be tested is "Danxia No. 8"; if the following characteristic bands do not appear, it indicates that the tea variety to be tested is not "Danxia No. 8": (1) The specific SSR core primers DX8.SSR-1-F and DX8.SSR-1-R amplified two characteristic bands of 260 bp and 264 bp respectively; (2) Specific SSR core primers DX8.SSR-2-F and DX8.SSR-2-R amplified two characteristic bands of 420 bp and 423 bp; (3) The specific SSR core primers DX8.SSR-3-F and DX8.SSR-3-R amplified two characteristic bands of 272 bp and 287 bp.

[0061] Example 4

[0062] A kit for identifying the tea variety "Danxia No. 8" 1. Composition (1) Three pairs of specific SSR core primers, wherein the 5' end of the upstream primer is labeled with a fluorescent reporter group (the fluorescent reporter group can be FAM, HEX, TAMRA or ROX, and the HEX fluorescent label is used in the present invention): DX8.SSR-1-F:GTAGACTACGCTTGGTTGCCAT; DX8.SSR-1-R:CTTGTGCGAATCTTGAGACTCTAG; DX8.SSR-2-F:TCCACAACGACAACACCAACA; DX8.SSR-2-R:GCAGATACAGAAACGACCAGGTC; DX8.SSR-3-F: TCATGAATATCACAAACCTGGTAACT; DX8.SSR-3-R:CCTCAGATTGACCTGCATGAGTT; (2) HSTaq DNA polymerase, dNTPs, 10×Buffer, positive control reference, negative control reference, and ultrapure water.

[0063] 2. Usage The detection and result interpretation were performed according to the method of Example 3.

[0064] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An SSR core primer combination for identifying the tea variety "Danxia No. 8", characterized in that: The SSR core primer combination comprises SSR core primers represented by nucleotide sequences of DX8.SSR-1-F, DX8.SSR-1-R, DX8.SSR-2-F, DX8.SSR-2-R, DX8.SSR-3-F and DX8.SSR-3-R; wherein the primer sequence of DX8.SSR-1-F is GTAGACTACGCTTGGTTGCCAT; the primer sequence of DX8.SSR-1-R is CTTGTGCGAATCTTGAGACTCTAG; the primer sequence of DX8.SSR-2-F is TCCACAACGACAACACCAACA; the primer sequence of DX8.SSR-2-R is GCAGATACAGAAACGACCAGGTC; the primer sequence of DX8.SSR-3-F is TCATGAATATCACAAACCTGGTAACT; the primer sequence of DX8.SSR-3-R is CCTCAGATTGACCTGCATGAGTT.

2. The SSR core primer combination according to claim 1, characterized in that The 5' end of the SSR core primer shown in the nucleotide sequence DX8.SSR-1-F, the 5' end of the SSR core primer shown in the nucleotide sequence DX8.SSR-2-F and / or the 5' end of the SSR core primer shown in the nucleotide sequence DX8.SSR-3-F is labeled with a fluorescent reporter group.

3. The SSR core primer combination according to claim 2, characterized in that The fluorescent reporter group is FAM, HEX, TAMRA or ROX.

4. Use of the SSR core primer combination described in any one of claims 1 to 3 in preparing an identification product of the tea variety "Danxia No. 8".

5. Use of a product comprising the SSR core primer combination described in any one of claims 1 to 3 in identifying the tea variety "Danxia No. 8".

6. A kit for identifying the tea variety "Danxia No. 8", characterized in that: Comprising the SSR core primer combination described in any one of claims 1 to 3.

7. The kit according to claim 6, characterized in that The kit also includes HSTaq DNA polymerase, dNTPs, 10×Buffer, a positive control reference substance, a negative control reference substance, and ultrapure water.

8. A method for identifying the tea variety "Danxia No. 8", characterized in that: The identification is performed using the SSR core primer combination described in any one of claims 1 to 3 or the kit described in any one of claims 6 to 7.

9. The method according to claim 8, characterized in that The following steps are involved: S1. Extracting genomic DNA from the tea tree sample to be tested; S2. Using the genomic DNA extracted in step S1 as a template, PCR amplification is performed using the SSR core primer combination described in any one of claims 1 to 3; S3. Typing the PCR amplification product obtained in step S2, and performing band discrimination on the typing result: If the SSR core primers represented by the nucleotide sequences of DX8.SSR-1-F and DX8.SSR-1-R amplify two characteristic bands of 260bp and 264bp, the SSR core primers represented by the nucleotide sequences of DX8.SSR-2-F and DX8.SSR-2-R amplify two characteristic bands of 420bp and 423bp, and the SSR core primers represented by the nucleotide sequences of DX8.SSR-3-F and DX8.SSR-3-R amplify two characteristic bands of 272bp and 287bp, it indicates that the tea tree sample to be tested is "Danxia No. 8"; the appearance of other bands indicates that the tea tree sample to be tested is not "Danxia No. 8".

10. The method according to claim 9, characterized in that The typing method in step S3 is to perform capillary electrophoresis on the PCR amplification product; For PCR amplification, the total reaction system was 10 μL, including: 10×Buffer I 1 μL, 2.5 mM dNTPs 0.8 μL, 2.5 μM upstream primer 0.6 μL, 2.5 μM downstream primer 0.6 μL, TAKARA HSTaq 0.1 μL, DNA 1 μL, and ddH2O 10 μL; the PCR amplification reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s; annealing at 60°C for 30 s for 35 cycles; extension at 72°C for 30 s; and finally extension at 60°C for 30 min.

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