Method for identifying persimmon da cha 6 based on KASP molecular marker

Abstract

The application discloses a method for identifying persimmon large tea No. 6 based on KASP molecular markers 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, has the characteristics of good stability, simple operation and high accuracy, and provides a precise, rapid and simple method for identifying the persimmon large tea No. 6 excellent strain.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a method for identifying Shida Tea No. 6 based on KASP molecular markers. Background Technology

[0002] Shida Tea No. 6 is a tea seedling with strong resistance and robust growth discovered by local tea farmers in Huangshan District, Huangshan City, Anhui Province, within the Shida Tea population of the Shanggao Kangcha area. Subsequently, asexual propagation was carried out, and a systematic selection method was employed, using Shuchazao as a control. From 2009 to 2012, comparative trials were conducted on different strains; from 2012 to 2018, comparative trials on different varieties were conducted; and from 2018 to 2024, regional trials were carried out, resulting in the selection of this strain, named "Shida Tea No. 6," which belongs to the tea variety category. Shida Tea No. 6 is suitable for making green tea. When processed into Taiping Houkui, it has a dark green color, a fragrant aroma with floral notes, and a mellow taste. New shoots sprout uniformly, and yields are stable under suitable conditions, facilitating tea garden management. It also exhibits some resistance to common pests such as the tea false eye green cicada and has strong drought and cold resistance. The "Shida Tea No. 6" 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. 6 is an asexual propagation, a shrub type, and a large-leaved variety. It exhibits strong growth, a semi-upright growth habit, and dense branching. Leaves are 12.8cm long and 5.0cm wide, growing obliquely upwards. The leaves are elliptical in shape, with 11 pairs of veins, a slightly raised surface, a flat body, and a firm texture. The leaves have sharp, dense, and deep serrations, a wedge-shaped base, an acuminate tip, and slightly wavy margins. The leaves are medium green and glossy. Young buds are light green with abundant pubescence. One hundred buds (one bud with two leaves) weigh 60.13g. Peak flowering occurs at the end of October, with relatively few flowers. Each flower has 5 sepals, a green, pubescent calyx, and 5 white, soft petals. The corolla diameter is 3.8cm. The ovary is pubescent, and the style is 1.0cm long with a shallowly lobed stigma. The style has 3 lobes, and 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 1.7 cm and a relatively thin pericarp, averaging 0.1 cm thick. The seeds are spherical, with an average diameter of 1.1 cm, and a brown seed coat. The weight of 100 seeds is 35.21 g.

[0004] The initial unfolding period of one bud and one leaf is generally in mid-March, and the peak period of one bud and two leaves is generally in early April. Regional trials in 2023 and 2024 showed that the initial emergence of one bud and one leaf in spring was on March 27th. The buds and leaves exhibit strong growth, with a bud density of 105 buds / 1109 square centimeters. The buds are dense, sprout uniformly, retain their tenderness well, and have abundant downy hairs. The weight of 100 buds with one bud and three leaves is 73.4 grams. It has strong propagation ability through cuttings and a high transplant survival rate. The dry tea sample contains 24.57% tea polyphenols, 3.61% amino acids, 4.35% caffeine, and 41.3% water extract. Taiping Houkui tea made from this superior single plant has a prominent aroma with a distinct orchid fragrance, a fresh and mellow taste, and a clear liquor. Taiping Houkui tea made from Shida Tea No. 6 is highly favored in the market due to its prominent aroma, distinct orchid fragrance, and superior quality, commanding a higher price than similar products on the market, making it highly valuable for promotion. 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 variety Shida Tea No. 6 and effectively distinguish the authenticity of fresh leaves and dried tea, it is urgent to establish a rapid and simple method for the accurate identification of the Shida Tea No. 6 strain.

[0005] 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 the Shida Tea No. 6 variety, therefore further research and improvement are needed. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to identify the Shida Tea No. 6 tea variety using KASP molecular markers.

[0007] The present invention solves the above-mentioned technical problems through the following technical means: The first aspect of this invention proposes an SNP molecular marker combination for identifying Shida Tea No. 6 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. 2, Shida Tea Puzhong, Shida Tea No. 30, 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. 6 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. 6 tea cultivar. The fifth aspect of this invention provides a method for identifying Shida Tea No. 6 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 T / T genotype in the Cs-KASP1 primer, a T / C genotype in the Cs-KASP2 primer, and an A / A genotype in the Cs-KASP3 primer, then the tea tree variety is Shida Tea No. 6.

[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: (I) The primers provided in this invention are based on tea tree genome analysis and, compared with EST-SSR, have the characteristics of high polymorphism and large quantity. Through extensive primer screening, three pairs of core primers were finally determined for the identification of the new Shida Tea No. 6 variety. (II) This invention uses KASP technology, which is an applied genetic germplasm analysis method that has been developed in recent years with the development of molecular biology. It has the characteristics of good stability, simple operation and high accuracy, and provides an accurate, fast and simple method for identifying the superior strain of Shida Tea No. 6. (III) 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. 6 variety at any growth stage without affecting the identification results. (iv) 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. This 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. 6.

[0017] Figure 2The following is a typographical 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 green dots represent homozygotes and heterozygotes, respectively. The red dot is the location of the specially marked Shida Tea No. 6.

[0018] Figure 3 The 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 is the location of the specially marked Shida Tea No. 6. 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 new tea varieties of Shida Tea No. 6 can more accurately identify Shida Tea No. 6 resources at the molecular level of plant genes.

[0023] The specific steps are as follows: 1. Determination of core SNP sites: Five tea varieties—Shida Tea No. 6, Shida Tea Puzhong, Shida Tea Xiangzao, Fuding Dabai Tea, and Shuchazao—were selected for resequencing. Based on the VCF files in the resequencing data, SNPs were screened using a Linux server, employing the vcftools command 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 Shida Tea No. 6 variety. 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 T / T genotype in the Cs-KASP1 primer, a T / C genotype in the Cs-KASP2 primer, and an A / A genotype in the Cs-KASP3 primer, then the tea variety was identified as Shida Tea No. 6. The genotyping fingerprints of the three KASP genes for Shida Tea No. 6 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, and can be used to identify the tea variety Shida Tea No. 6 based on the developed SNP sites and designed primers.

[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 combination of SNP molecular markers for identifying Shida Tea No. 6 from other tea varieties, characterized in that, It consists of three KASP markers, namely Cs-KASP1~3, with Zhongcha 102 as the reference genome. The specific information is as follows: The other tea tree varieties mentioned are one or more of the following: 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 Shucha Zao.

2. The SNP molecular marker combination according to claim 1, characterized in that, The other tea tree variety mentioned is Shida Tea No.

2.

3. The SNP molecular marker combination according to claim 1, characterized in that, The other tea tree variety mentioned is Shida Tea No.

3.

4. The SNP molecular marker combination according to claim 1, characterized in that, The other tea tree varieties mentioned are the Persimmon Tea Species.

5. A KASP primer set for amplifying the SNP molecular marker combination of claim 1, characterized in that, The primer set includes any one of 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.

6. The application of the SNP molecular marker combination of claim 1 and the KASP primer set of claim 5 in the identification of Shida Tea No. 6 from other tea varieties; wherein the other tea varieties are one or more of 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.

7. A method for distinguishing Shida Tea No. 6 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 5 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; The other tea tree varieties mentioned are one or more of the following: 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 Shucha Zao.

8. The method according to claim 7, characterized in that, If the tea tree to be tested simultaneously shows a T / T genotype in the Cs-KASP1 primer, a T / C genotype in the Cs-KASP2 primer, and an A / A genotype in the Cs-KASP3 primer, then the tea tree variety is Shida Tea No.

6.

9. The method according to claim 7, 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.

10. The method according to claim 7, characterized in that, 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 decreases by 0.6℃ per cycle; Step 2: 10 cycles; Denaturation: 94℃, 20 sec; Annealing / Extension: 55℃, 60 sec; Step 3: 26 cycles.

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