SNP molecular marker combination and application thereof in identifying persimmon da cha 30

Abstract

The application discloses a SNP molecular marker combination and application thereof in identifying persimmon Da Chayi No. 30, and belongs to the technical field of molecular biology. The SNP molecular marker combination comprises three KASP markers, namely Cs-KASP1-3, and takes Zhongcha 102 as a reference genome, and specific information is shown in Table 1. The application adopts the KASP technology, which is an applied genetic germplasm analysis method developed in recent years along with the development of molecular biology, and has the characteristics of good stability, simple operation and high accuracy, and provides a precise, rapid and simple method for identifying the persimmon Da Chayi No. 30 excellent strain.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a combination of SNP molecular markers and its application in the identification of Shida Tea No. 30. Background Technology

[0002] Shida Tea No. 30 is a robust tea seedling discovered by local tea farmers in Chaoyang Village, Taipinghu Town, Huangshan District, Huangshan City, Anhui Province, exhibiting strong resistance and vigorous growth. When harvested from this superior single plant to produce Taiping Houkui tea, the dry tea has a prominent aroma with a distinct orchid fragrance, a fresh and mellow taste, and a bright green liquor. Asexual propagation through cuttings was conducted in 2009, employing a systematic selection method. Using Shuchazao as a control, comparative trials were conducted from 2008 to 2011, followed by varietal comparison trials from 2011 to 2016. Regional trials were carried out from 2018 to 2024, resulting in the selection of this varietal, named "Shida Tea No. 30," which belongs to the tea variety category. The "Shida Tea No. 30" variety was publicly announced on the National Agricultural Technology Extension Network on August 19, 2025, at the following URL: https: / / www.natesc.org.cn / news / des?id=24f9101a-aeb1-4f90-8185-a3f557db6e46&kind=TZGG&Category=%E9%80%9A%E7%9F%A5%E5%85%AC%E5%91%8A&CategoryId=d00be10c-6b4f-478b-be40-39fab99f9710.

[0003] Shida Tea No. 30 is an asexual propagation, a shrub type, and a medium-leaved variety. It exhibits strong growth, a semi-erect growth habit, and medium branching density. The leaves are 10.4 cm long and 4.5 cm wide, medium green in color, obliquely upward-pointing, and medium-sized elliptical in shape. Each leaf has 8 pairs of veins, a slightly raised surface, a flat body, and a firm texture. The leaves have sharp, dense, and shallow serrations, a wedge-shaped base, a pointed tip, and a flat margin. The leaves are medium green and glossy. Young buds are light green with abundant pubescence. One hundred buds (one bud with two leaves) weigh 66g. Peak flowering occurs at the end of October, with relatively few flowers. Each flower has 5 green, hairless sepals and 6 white, soft petals. The corolla diameter is 3.0 cm. The ovary is pubescent, and the style length is 1.6 cm. The stigma is centrally lobed, and the style has 3 lobes. The pistil is longer than the stamen. The fruit has a low fruit set rate, with 1-4 locules. The fruit is quadrilateral, triangular, kidney-shaped, or spherical, with an average diameter of 1.8 cm and a relatively thin pericarp, averaging 0.09 cm thick. The seeds are spherical, with an average diameter of 1.2 cm, a brown seed coat, and a weight of 36.12 g per 100 seeds.

[0004] Shida Tea No. 30 is suitable for making green tea. When processed into Taiping Houkui pointed shape, it is flat and straight, with a light and floral aroma; bright green liquor; fresh and mellow taste; uniform new shoot growth; stable yield under suitable conditions; and beneficial for tea garden and harvesting management. It also has high resistance to common diseases and pests such as tea anthracnose and tea false eye green leafhopper, making it highly valuable for promotion.

[0005] The introduction and exchange of tea varietal resources can easily lead to unclear germplasm sources, posing numerous difficulties for the protection and utilization of tea varietal resources. To better protect and promote the new tea varietal variety Shida Tea No. 30 and effectively distinguish the authenticity of fresh leaves and dried tea, it is urgent to establish a rapid and simple method for accurate identification of the Shida Tea No. 30 strain.

[0006] Chinese patent application CN108841981A discloses a method for identifying tea varieties with large and small leaves using InDel molecular markers. The upstream nucleotide sequence of the InDel molecular marker is shown in SEQ ID NO. 1, and the downstream nucleotide sequence is shown in SEQ ID NO. 2. The detection method includes the following sequential steps: S1: Extracting total DNA from the tea sample to obtain the DNA to be amplified; S2: Performing PCR amplification on the DNA to be amplified using F1 and R1 to obtain the amplification product; S3: Detecting the amplification product by electrophoresis. This patent, in the field of molecular biology, identifies large-leaf and small-leaf tea varieties, solving the technical problems of cumbersome and inaccurate traditional methods for identifying large-leaf and small-leaf tea varieties, and has broad application prospects. However, the InDel molecular marker in this patent cannot accurately identify Shida Tea No. 30, therefore further research and improvement are needed. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to propose an SNP molecular marker combination and its application in the identification of Shida Tea No. 30. This invention solves the above-mentioned technical problem through the following technical means: The first aspect of this invention proposes an SNP molecular marker combination for identifying Shida Tea 30 and other tea varieties, comprising three KASP markers, namely Cs-KASP1~3, using Zhongcha 102 as the reference genome, with the specific information as follows:

[0008] Preferably, the other tea tree varieties include, but are not limited to, one or more of the following: Shida Tea No. 3, Shida Tea No. 6, Shida Tea No. 2, Shida Tea Puzhong, Shida Tea Huangzhong, Shida Tea Xiangzao, Shida Tea Xiaoqingye, Fuding Dabai Tea, and Shucha Zao.

[0009] A second aspect of the present invention provides a KASP primer set for amplifying the above-mentioned SNP molecular marker combinations, the primer set comprising any one of the following primer sets (1)-(3): The primer set (1) includes the forward primers shown in SEQ ID NO.1-2 and the reverse universal primer shown in SEQ ID NO.3; The primer set (2) includes the forward primers shown in SEQ ID NO.4-5 and the reverse universal primers shown in SEQ ID NO.6; The primer set (3) includes the forward primers shown in SEQ ID NO.7-8 and the reverse universal primers shown in SEQ ID NO.9.

[0010] The third aspect of this invention proposes the application of the above-mentioned SNP molecular marker combination and KASP primer set in the identification of Shida Tea No. 30 and other tea trees.

[0011] The fourth aspect of this invention proposes the application of the above-mentioned SNP molecular marker combination and KASP primer set in molecular marker-assisted breeding of the Shida Tea No. 30 tea variety. The fifth aspect of this invention provides a method for identifying Shida Tea No. 30 from other tea varieties, comprising the following steps: (1) Using the genomic DNA of the tea plant to be tested as a template; (2) PCR amplification reaction was performed using the above KASP primer set to obtain amplification products; (3) The amplification products were then scanned for fluorescence signals and genotyped using a fluorescence detection platform; (4) Read the genotyping data and obtain the genotyping map.

[0012] Preferably, if the tea tree to be tested simultaneously shows a C / C genotype in the Cs-KASP1 primer, a G / G genotype in the Cs-KASP2 primer, and a C / C genotype in the Cs-KASP3 primer, then the tea tree variety is Shida Tea No. 30.

[0013] Preferably, the PCR amplification system is 10 μL: 5 μL 2×KASP Master Mix, 4.8 μL 20 ng / μL DNA template, 0.024 μL 100 μM one forward primer, 0.024 μL 100 μM another forward primer, 0.06 μL 100 μM reverse primer, and the volume is made up to 10 μL with ddH2O. Preferably, the PCR amplification program is as follows: Pre-denaturation: 94℃, 15 min, 1 cycle; Denaturation: 94℃, 20 sec; Annealing / Extension: 61-55℃, 60 sec, temperature decreasing by 0.6℃ per cycle; Step 2: 10 cycles; Denaturation: 94℃, 20 sec; Annealing / Extension: 55℃, 60 sec; Step 3: 26 cycles. Finally, genotyping data is read at 25℃ for 30 sec.

[0014] The beneficial effects of this invention are as follows: 1. The primers provided in this invention are based on tea tree genome analysis and, compared with EST-SSR, exhibit high polymorphism and a large number of primers. Through extensive primer screening, three core primer pairs were ultimately identified for the identification of the new Shida Tea 30 variety. 2. This invention employs KASP technology, an applied genetic germplasm analysis method that has developed in recent years with the advancement of molecular biology. It features good stability, simple operation, and high accuracy, providing an accurate, rapid, and simple method for identifying superior strains of Shida Tea No. 30. 3. The materials used in this invention are not limited by season, environment and testing time. DNA can be extracted from any leaf of the Shida Tea No. 30 variety at any growth stage without affecting the identification results. 4. The identification method of this invention can replace the identification method using capillary electrophoresis to screen for specific SSR markers, enabling rapid, accurate, and effective identification of tea hybrid progeny while reducing sequencing costs. It provides important basis and support for the efficient creation of tea germplasm through molecular breeding.

[0015] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0016] Figure 1 The following is a genotyping diagram of the KASP marker Cs-KASP1 in 10 tea cultivars in Example 2: the scatter plot on the X and Y axes represents the marker identification of the 10 tea cultivars; the blue and orange dots represent different homozygotes. The red dot is the location of the specially marked Shida Tea No. 30.

[0017] Figure 2 The following is a genotyping diagram of the KASP marker Cs-KASP2 in 10 tea cultivars in Example 2: the scatter plot on the X and Y axes represents the marker identification of the 10 tea cultivars; the blue and orange dots represent different homozygotes. The red dot is the location of the specially marked Shida Tea No. 30.

[0018] Figure 3The following is a typographical diagram of the KASP marker Cs-KASP3 in 10 tea cultivars from Example 2: the scatter plot on the X and Y axes represents the marker identification of the 10 tea cultivars; the blue and green dots represent homozygotes and heterozygotes, respectively. The red dot marks the location of the specially marked Shida Tea No. 30. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0020] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0021] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0022] Example 1: A method based on KASP technology for identifying the Shida Tea No. 30 tea tree variety is developed, which can more accurately identify the Shida Tea No. 30 tea tree variety resources at the molecular level of plant genes.

[0023] The specific steps are as follows: 1. Determination of core SNP sites: Four tea varieties—Shida Cha Puzhong, Shida Cha No. 30, Shida Cha Xiangzao, and Shucha Zao—were selected for resequencing. Based on the VCF files in the resequencing data, SNPs were screened using a Linux server and the vcftools command was used for filtering. Specific parameters were set as follows: (1) Preserve biallelic loci; (2) Set the genotype deletion rate to be less than 1; (3) Set the minimum allele frequency to less than 0.05; (4) Set the average sequencing depth to 5×.

[0024] The bcftools command was used to extract SNP information that distinguishes the Shida tea variety from other tea varieties from the filtered VCF file. The specific command requires outputting the chromosome name, SNP physical location, reference allele, alternative allele, and genotype. The principle was that there should be no other variable sites within 100 bp before and after the SNP site. Finally, three core SNP markers were selected, with the reference genome being "Zhongcha 102". Specific information is shown in Table 1 below. Table 1:

[0025] 2. Synthesis of KASP primer pair combinations: Based on the developed SNP sites, KASP primers were converted. By comparing with the reference genome, conserved flanking sequences of 100 bp before and after each of the three SNP sites were extracted. For each SNP site, two forward primers were designed upstream and one reverse primer was designed downstream. Each KASP primer pair was used to amplify the corresponding SNP marker. A total of nine primers were created, and their nucleotide sequences are shown in Table 2. Table 2:

[0026] Example 2: This embodiment utilizes the Cs-KASP1~3 primer pairs designed in Example 1, and uses genomic DNA from seedling tissues of 10 tea varieties—Shida Tea No. 6, Shida Tea No. 3, Shida Tea No. 2, Shida Tea Puzhong, Shida Tea No. 30, Shida Tea Huangzhong, Shida Tea Xiangzao, Shida Tea Xiaoqingye, Fuding Dabai Tea, and Shuchazao—as templates, with double-distilled water as a control, to perform KASP genotyping identification of the new variety Shida Tea No. 30. The steps include: 1. DNA sample detection and dilution: The quality and concentration of DNA were detected and confirmed using a NanoDrop 2000 (ThermoScientific) nucleic acid analyzer, and then uniformly diluted to 10-20 ng / μL.

[0027] 2. PCR amplification: PCR amplification was performed on a BIO-RAD real-time quantitative instrument using the KASP primer pair designed in Example 1, and the products were scanned for fluorescence signals and genotyped. (1) The PCR amplification system is as follows:

[0028] (2) The PCR amplification procedure is as follows:

[0029] (3) Fluorescence data reading and analysis After the PCR reaction, fluorescence data were read and analyzed using a BIO-RAD real-time quantitative instrument, and different tea varieties were identified based on the genotyping results. If the tea plant being tested simultaneously showed a C / C genotype in the Cs-KASP1 primer, a G / G genotype in the Cs-KASP2 primer, and a C / C genotype in the Cs-KASP3 primer, then the tea variety was identified as Shida Tea 30. The genotyping fingerprints of the three KASP genes for Shida Tea 30 and the control variety (germplasm) are shown in Table 5.

[0030] Table 5:

[0031] If a sufficiently defined genotype cluster is not obtained after the initial KASP thermal cycling procedure, the thermal cycle should be repeated 3 times using the conditions detailed in Table 6. Then, the reaction plate should be reread and the results analyzed.

[0032]

[0033] The results are as follows Figures 1-3 As shown, the results indicate that PCR primers designed using the SNP combination of this invention, combined with competitive allele-specific PCR (KASP), can distinguish tea plants with different genotypes. Points closer to the Y-axis and X-axis represent two different homozygous genotypes, while points on the diagonal of the coordinate axes represent heterozygous genotypes. The KASP genotyping technology of this invention performs precise bicelestem typing of target SNPs. Based on the developed SNP sites and designed primers, it can be used to identify the tea variety Shida Tea 30.

[0034] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A KASP primer set for differentiating Shida Tea No. 30 from other tea varieties, characterized in that, The primer set includes the following primer sets (1)-(3): The primer set (1) includes the forward primer shown in SEQ ID NO.1-2 and the reverse universal primer shown in SEQ ID NO.3; the primer set (2) includes the forward primer shown in SEQ ID NO.4-5 and the reverse universal primer shown in SEQ ID NO.6; the primer set (3) includes the forward primer shown in SEQ ID NO.7-8 and the reverse universal primer shown in SEQ ID NO.9; the other tea varieties are one or more of the following: Shida Tea No.3, Shida Tea No.6, Shida Tea No.2, Shida Tea Puzhong, Shida Tea Huangzhong, Shida Tea Xiangzao, Shida Tea Xiaoqingye, Fuding Dabai Tea, and Shucha Zao.

2. The application of the KASP primer set according to claim 1 in distinguishing Shida Tea No. 30 from other tea varieties, characterized in that, In the genotyping results, if the tea tree to be tested simultaneously meets the following conditions: the amplification result of primer group (1) is C / C genotype, the amplification result of primer group (2) is G / G genotype, and the amplification result of primer group (3) is C / C genotype, then the tea tree to be tested is Shida Tea No. 30; the other tea tree varieties are one or more of Shida Tea No. 3, Shida Tea No. 6, Shida Tea No. 2, Shida Tea Puzhong, Shida Tea Huangzhong, Shida Tea Xiangzao, Shida Tea Xiaoqingye, Fuding Dabai Tea, and Shuchazao.

3. A method for distinguishing Shida Tea No. 30 from other tea varieties, characterized in that, Includes the following steps: (1) Using the genomic DNA of the tea plant to be tested as a template; (2) PCR amplification reaction was performed using the KASP primer set described in claim 1 to obtain amplification products; (3) The amplification products were then scanned for fluorescence signals and genotyped using a fluorescence detection platform; (4) Read the genotyping data and obtain the genotyping map; In the genotyping results, if the tea tree to be tested simultaneously meets the following conditions: the amplification result of primer group (1) is C / C genotype, the amplification result of primer group (2) is G / G genotype, and the amplification result of primer group (3) is C / C genotype, then the tea tree to be tested is Shida Tea No. 30; the other tea tree varieties are one or more of Shida Tea No. 3, Shida Tea No. 6, Shida Tea No. 2, Shida Tea Puzhong, Shida Tea Huangzhong, Shida Tea Xiangzao, Shida Tea Xiaoqingye, Fuding Dabai Tea, and Shuchazao.

4. The method according to claim 3, characterized in that, The PCR amplification system consisted of 10 μL: 5 μL 2×KASPMaster Mix, 4.8 μL 20 ng / μL DNA template, 0.024 μL 100 μM one forward primer, 0.024 μL 100 μM another forward primer, 0.06 μL 100 μM reverse primer, and ddH2O was added to bring the volume to 10 μL.

5. The method according to claim 3, characterized in that, The other tea tree variety mentioned is Shida Tea No.

3.

6. The method according to claim 3, characterized in that, The other tea tree variety mentioned is Shida Tea No.

6.

7. The method according to claim 3, characterized in that, The other tea tree variety mentioned is Shida Tea No.

2.

8. The method according to claim 3, characterized in that, The other tea tree varieties mentioned are Shida Chahuangzhong.

9. The method according to claim 3, characterized in that, The other tea tree varieties mentioned are Shidacha Xiangzao.

10. The method according to claim 3, characterized in that, The other tea tree varieties mentioned are Shida Tea and Xiaoqingye Tea.

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