A set of SSR primer combinations for rapid identification of tea varieties and their applications

Abstract

This invention provides a set of SSR primer combinations for rapid identification of tea plant varieties and their applications. Sixteen SSR molecular marker combinations obtained through screening can be used for rapid identification of tea plant varieties using PCR and electrophoresis detection techniques. This allows for intuitive, efficient, and accurate identification of different tea plant varieties. This invention effectively solves the problems of homonymous and synonymous tea plant germplasm resources, avoids redundant collection and preservation, and facilitates the identification, evaluation, and shared utilization of tea plant germplasm resources.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a set of SSR primer combinations for rapid identification of tea varieties and their applications. Background Technology

[0002] Tea (Camellia sinensis L.) is a perennial woody plant and an important leaf crop in my country. As a major origin of tea, my country has a wide tea-growing region with a cultivation history of over 3000 years. Propagation is primarily by seed. The tea plant's cross-pollination and self-incompatibility contribute to its rich genetic diversity, providing a crucial physiological basis for cultivating superior tea varieties. Traditional identification methods rely heavily on plant morphology, which is susceptible to influences from plant form and external factors, and requires experienced tea breeders for accurate differentiation. However, the development of molecular biology identification techniques offers a new approach for rapid, accurate, and objective identification of tea varieties, effectively addressing the shortcomings of traditional phenotypic methods.

[0003] Molecular markers can directly reflect variations at the DNA level, are less affected by limiting factors, are unaffected by environmental factors, and can quickly and intuitively reflect differences between similar individuals. SSR markers (simple repeat sequences, also known as microsatellites) typically refer to DNA sequences with multiple tandem repeats of 2–5 nucleotides, although a few use 1–6 nucleotides as tandem repeat units. They are characterized by codominance, stability, good reproducibility, and simple and rapid operation. Furthermore, SSR markers exhibit good universality and specificity across species; a small number of marker combinations can provide high detection efficiency, and the identification results are stable, highly reproducible, and unaffected by experimental environmental conditions. This makes them an ideal technique widely used for the identification of plant and animal germplasm resources, playing a crucial role in genetic diversity research, germplasm resource assessment, variety identification, and genetic map construction.

[0004] Therefore, by utilizing SSR locus combinations, a set of primer combinations that can effectively, quickly, and accurately identify tea varieties can be developed for application in tea germplasm resources. This can effectively solve the problems of homonymous and synonymous tea germplasm resources, avoid duplicate collection and preservation, facilitate the identification, evaluation, and sharing of tea germplasm resources, and provide a technical foundation for the data-driven and intelligent management of tea germplasm resources. Summary of the Invention

[0005] One of the objectives of this invention is to provide a set of SSR primer combinations for rapid identification of tea varieties, the sequences of which are shown in SEQ ID NO:1-SEQ ID NO:32.

[0006] Furthermore, the tea varieties include Yunkang No. 10, Yunkang No. 14, Fengqing No. 1, Chuancha No. 3, Mengshan No. 9, Tianfu No. 36, Siming Xueya, Xiangfei Cui, Xiangbo Lv No. 2, Huangjincha No. 1, Qianmei 601, Qiancha No. 1, Qianmei 0310, Huangkui, Huangshan Baicha, Cui Lv No. 1, Shucha Zao, Gui Lv No. 1, Mabian Lv No. 1, Guixiang, Yaoshan Xiulu, Fuding Dabai, Lvya Foshou, Huang Guanyin, Fuyun No. 6, Fu'an Dabai, Fuding Dahao, Jin Guanyin, Baijiguan, Taicha No. 12, Gancha No. 4, Sucha 120, Suyuhuang, Huafeng No. 1, Echa No. 1, Feng Phoenix Dancong, Yinghong No. 9, Danxia No. 1, Qingxin No. 1, Yueming No. 6, Dingnan Daye No. 1, Dingnan Daye No. 6, Zaobaijian No. 5, Longjing No. 43, Yingshuang, Zhenong No. 21, Baiye No. 1, Qiannianxue, Jiaming No. 1, Chunyu No. 1, Chunyu No. 2, Wanghaicha No. 1, Zhonghuang No. 1, Zhonghuang No. 3, Yunbai No. 1, Zhonghuang No. 2, Zisongzhong, Yawang, Zidantou, Jingbai No. 1, Jingbai No. 2, Lizi No. 1, Lizi No. 2, Lizaoxiang, Lihuang No. 2, Lihuang No. 3, JN-1, Yujinxiang, Shuijingbai, Titianbai No. 1, Titianbai No. 2, Yinhou, Zhexiao No. 1 and HM8.

[0007] The second objective of this invention is to provide a method for identifying tea tree varieties, comprising the following steps:

[0008] (1) Extract genomic DNA from the tea variety to be tested;

[0009] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed on it using the above primer combination;

[0010] (3) The PCR product obtained in step (2) is subjected to gel electrophoresis to obtain the fingerprint pattern of the tea variety to be tested. The fingerprint pattern is compared with the constructed standard fingerprint pattern to identify the tea variety.

[0011] Furthermore, the PCR amplification reaction system included: 10 μL reaction volume, 5 μL of 2×T5 Super PCRMix for PAGE, 1 μL of DNA template and 3 μL of ddH2O, and 0.5 μL each of forward and reverse primers; the PCR amplification reaction program was: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 5 min, for a total of 30 cycles; and a final extension at 72℃ for 2 min, followed by storage at 4℃.

[0012] The beneficial effects of this invention are:

[0013] Using SSR molecular markers, 74 tea plant germplasm resources can be rapidly identified with high accuracy and stability, significantly improving the efficiency of tea germplasm resource identification. This lays the theoretical and technical foundation for constructing molecular identity cards for tea varieties and is of great significance for the protection of intellectual property rights of germplasm resources. Attached Figure Description

[0014] Figure 1 The results of amplification of 74 tea varieties using primer Cs.5-47 are shown.

[0015] Figure 2 The results of amplification of 74 tea varieties using primers Cs.6-8 are shown.

[0016] Figure 3 The results of amplification of 74 tea varieties using primer Cs.6-30 are shown.

[0017] Figure 4 The results of amplification of 74 tea varieties using primer Cs.5-48 are shown.

[0018] Figure 5 The results of amplification of 74 tea varieties using primer Cs.6-25 are shown.

[0019] Figure 6 The results of amplification of 74 tea varieties using primer Cs.6-24 are shown.

[0020] Figure 7 The results of amplification of 74 tea varieties using primers Cs.5-9 are shown.

[0021] Figure 8 The results of amplification of 74 tea varieties using primer Cs.6-36 are shown.

[0022] Figure 9 The results of amplification of 74 tea varieties using primer Cs.5-30 are shown.

[0023] Figure 10 The results of amplification of 74 tea varieties using primer Cs.5-29 are shown.

[0024] Figure 11 The results of amplification of 74 tea varieties using primer Cs.5-44 are shown.

[0025] Figure 12 The results of amplification of 74 tea varieties using primer Cs.5-45 are shown.

[0026] Figure 13 The results of amplification of 74 tea varieties using primer Cs.6-29 are shown.

[0027] Figure 14 The results of amplification of 74 tea varieties using primer Cs.6-28 are shown.

[0028] Figure 15 The results of amplification of 74 tea varieties using primer Cs.5-42 are shown.

[0029] Figure 16The results of amplification of 74 tea varieties using primers Cs.6-78 are shown.

[0030] Figure 17 and Figure 18 The fingerprint maps of 74 tea tree varieties constructed for this invention.

[0031] above Figures 1-16 In the diagram, M stands for marker, numbers 1-74 represent the labels for 74 tea varieties, and N is null, indicating an invalid sample. Detailed Implementation

[0032] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Example 1

[0036] I. Design of SSR Primers and Screening of SSR Loci in Tea Trees

[0037] SSR repeat sequences were selected from the FASTA file (Asp.misa) of the 'Longjing 43' genome and Primer Premier 6.0 software was used. Primers were designed based on the flanking regions (approximately 200 bp) of each SSR locus. The primer parameters were: oligonucleotide primer length 18–23 bp, annealing temperature 55–60℃, GC content 35%–60%, and primer score higher than 80.

[0038] PCR amplification was performed on 163 pairs of SSR primers in 8 randomly selected tea varieties. 117 SSRs were found to be polymorphic. Based on the presence or absence of amplified fragments, 16 pairs of SSR primers were selected to distinguish and identify 74 tea varieties. These 16 pairs of SSR primers are as follows:

[0039]

[0040]

[0041] Each SSR's encoding represents: tea plant SSR markers, repetitive motif type, and chromosome location in the genome.

[0042] II. SSR Primer PCR Detection

[0043] PCR was performed on 74 tea varieties using the primers described above. The 74 tea varieties are: Yunkang 10 (1), Yunkang 14 (2), Fengqing 1 (3), Chuancha 3 (4), Mengshan 9 (5), Tianfu 36 (6), Siming Xueya (7), Xiangfei Cui (8), Xiangbo Lv 2 (9), Huangjincha 1 (10), Qianmei 601 (11), Qiancha 1 (12), Qianmei 0310 (13), Huangkui (14), Huangshan Baicha (15), and Cui Lv 1 (16). Shucha Zao (17), Guilu No. 1 (18), Mabianlu No. 1 (19), Guixiang (20), Yaoshan Xiulu (21), Fuding Dabai (22), Lvya Foshou (23), Huang Guanyin (24), Fuyun No. 6 (25), Fu'an Dabai (26), Fuding Dahao (27), Jin Guanyin (28), Baijiguan (29), Taiwan Tea 12 (30), Gan Tea No. 4 (31), Su Tea 120 (32), Suyuhuang (33), Huafeng No. 1 (34), E Tea No. 1 (35), Fenghuang Dancong (36), Yinghong No. 9 (37), Danxia No. 1 (38), Qingxin No. 1 (39), Yueming No. 6 (40), Dingnan Daye No. 1 (41), Dingnan Daye No. 6 (42), Zaobaijian No. 5 (43), Longjing No. 43 (44), Yingshuang (45), Zhenong No. 21 (46), Baiye No. 1 (47), Qiannianxue (48), Jiaming No. 1 (49), Chunyu No. 1 (50), Chunyu No. 2 (51), Wanghaicha No. 1 (52), Zhonghuang No. 1 (53), Zhonghuang No. 3 (54), Yun White No. 1 (55), Zhonghuang No. 2 (56), Zisongzhong (57), Yawang (58), Zidantou (59), Jingbai No. 1 (60), Jingbai No. 2 (61), Lizi No. 1 (62), Lizi No. 2 (63), Lizaoxiang (64), Lihuang No. 2 (65), Lihuang No. 3 (66), JN-1 (67), Yujinxiang (68), Shuijingbai (69), Titianbai No. 1 (70), Titianbai No. 2 (71), Yinhou (72), Zhexiao No. 1 (73) and HM8 (74).

[0044] The steps are as follows:

[0045] (1) Extraction of genomic DNA from the tea variety to be tested;

[0046] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the above 16 pairs of primers to obtain PCR products;

[0047] ①The PCR reaction system is 10μL, and the components are as follows: 5μL of 2×T5 Super PCR Mix for PAGE, 3μL of ddH2O, 0.5μL of forward and reverse primers, and 1μL of DNA template.

[0048] ②The reaction procedure for PCR amplification is as follows:

[0049] a. Pre-denaturation at 98℃ for 5 minutes

[0050] b. Denaturation at 98℃ for 30 seconds

[0051] c. Anneal at 58℃ for 30 seconds

[0052] d.72℃ extended for 40s

[0053] bd. 30 loops

[0054] e. Extend at 72℃ for 5 minutes

[0055] (3) Perform gel electrophoresis on the PCR products obtained in step (2) to observe the band specificity.

[0056] PCR products were separated on an 8.0% (g / mL) polyacrylamide gel at 200V for 80 min. The electrophoresis apparatus used was a Beijing Junyi JY600C universal electrophoresis apparatus, and the electrophoresis tank was a JY-CZ-B vertical electrophoresis tank. After electrophoresis, the bands were washed and developed using silver staining. Finally, the images were photographed under a viewing light and saved.

[0057] like Figures 1-16 The image shows the amplification results of the above 16 primer pairs for 74 tea varieties.

[0058] (4) Analysis and annotation of band data: For each SSR molecular marker PCR amplification band among different tea varieties, values ​​were assigned. Clear and reliable bands on the electrophoresis pattern were calculated based on the SSR amplification products. Bands appearing at the same position were marked as "1", and no bands were marked as "0". Fingerprint patterns of 74 tea varieties were obtained, as shown below. Figure 17 and Figure 18 As shown, the tea tree varieties to be tested can be identified based on the constructed standard fingerprint spectrum.

Claims

1. A set of SSR primers for identifying tea varieties, characterized in that, The sequences of the primer combination are shown in SEQ ID NO:1-SEQ ID NO:

32.

2. The primer combination according to claim 1, characterized in that, The tea varieties mentioned are: Yunkang No. 10, Yunkang No. 14, Fengqing No. 1, Chuancha No. 3, Mengshan No. 9, Tianfu No. 36, Siming Xueya, Xiangfei Cui, Xiangbo Lv No. 2, Huangjin Cha No. 1, Qianmei 601, Qiancha No. 1, Qianmei 0310, Huang Kui, Huangshan Baicha, Cui Lv No. 1, Shucha Zao, Gui Lv No. 1, Mabian Lv No. 1, Guixiang, Yaoshan Xiulu, Fuding Dabai, Lvya Foshou, Huang Guanyin, Fuyun No. 6, Fu'an Dabai, Fuding Dahao, Jin Guanyin, Baijiguan, Taicha No. 12, Gancha No. 4, Sucha No. 120, Suyuhuang, Huafeng No. 1, Echa No. 1, and Fenghuang Dancong. Yinghong No. 9, Danxia No. 1, Qingxin No. 1, Yueming No. 6, Dingnan Daye No. 1, Dingnan Daye No. 6, Zaobaijian No. 5, Longjing No. 43, Yingshuang, Zhenong No. 21, Baiye No. 1, Qiannianxue, Jiaming No. 1, Chunyu No. 1, Chunyu No. 2, Wanghaicha No. 1, Zhonghuang No. 1, Zhonghuang No. 3, Yunbai No. 1, Zhonghuang No. 2, Zisongzhong, Yawang, Zidantou, Jingbai No. 1, Jingbai No. 2, Lizi No. 1, Lizi No. 2, Lizaoxiang, Lihuang No. 2, Lihuang No. 3, JN-1, Yujinxiang, Shuijingbai, Titianbai No. 1, Titianbai No. 2, Yinhou, Zhexiao No. 1 or HM8.

3. A method for identifying tea tree varieties, characterized in that, Includes the following steps: (1) Extract genomic DNA from the tea variety to be tested; (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed on it using the primer combination described in claim 1; (3) The PCR products obtained in step (2) are subjected to gel electrophoresis to obtain the fingerprint pattern of the tea variety to be tested. The fingerprint pattern is compared with the constructed standard fingerprint pattern to identify the tea variety. The tea varieties mentioned are: Yunkang No. 10, Yunkang No. 14, Fengqing No. 1, Chuancha No. 3, Mengshan No. 9, Tianfu No. 36, Siming Xueya, Xiangfei Cui, Xiangbo Lv No. 2, Huangjin Cha No. 1, Qianmei 601, Qiancha No. 1, Qianmei 0310, Huang Kui, Huangshan Baicha, Cui Lv No. 1, Shucha Zao, Gui Lv No. 1, Mabian Lv No. 1, Guixiang, Yaoshan Xiulu, Fuding Dabai, Lvya Foshou, Huang Guanyin, Fuyun No. 6, Fu'an Dabai, Fuding Dahao, Jin Guanyin, Baijiguan, Taicha No. 12, Gancha No. 4, Sucha No. 120, Suyuhuang, Huafeng No. 1, Echa No. 1, and Fenghuang Dancong. Yinghong No. 9, Danxia No. 1, Qingxin No. 1, Yueming No. 6, Dingnan Daye No. 1, Dingnan Daye No. 6, Zaobaijian No. 5, Longjing No. 43, Yingshuang, Zhenong No. 21, Baiye No. 1, Qiannianxue, Jiaming No. 1, Chunyu No. 1, Chunyu No. 2, Wanghaicha No. 1, Zhonghuang No. 1, Zhonghuang No. 3, Yunbai No. 1, Zhonghuang No. 2, Zisongzhong, Yawang, Zidantou, Jingbai No. 1, Jingbai No. 2, Lizi No. 1, Lizi No. 2, Lizaoxiang, Lihuang No. 2, Lihuang No. 3, JN-1, Yujinxiang, Shuijingbai, Titianbai No. 1, Titianbai No. 2, Yinhou, Zhexiao No. 1 or HM8.

4. The identification method according to claim 3, characterized in that, The PCR amplification reaction system included: 10 μL reaction volume, 5 μL of 2×T5 Super PCR Mix for PAGE, 1 μL of DNA template and 3 μL of ddH2O, and 0.5 μL each of forward and reverse primers. The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 5 min, for a total of 30 cycles; and a final extension at 72℃ for 2 min. After the amplification, the product was stored at 4℃.

5. A reagent for identifying tea tree varieties, characterized in that, Includes the primer combination described in claim 1.

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