A high-quality theacrine camellia single-plant screening method based on "resistance trait-chemical fingerprint-SSR molecular marker"

Abstract

The application discloses a method for screening high-quality high-theophylline camellia sinensis single plant based on a resistance character-chemical fingerprint-SSR molecular marker, which comprises the following steps: (1) field resistance character comprehensive evaluation is carried out on camellia sinensis natural hybridization offspring seed seedlings, and single plants with strong disease resistance, insect resistance, cold resistance and drought resistance are screened; (2) the theophylline content of the screened single plants is determined, and the theophylline content greater than or equal to 1.0% is taken as a screening standard; (3) SSR fluorescent marker primers are used for PCR amplification, capillary electrophoresis detection is carried out on the PCR products, the genetic similarity coefficients and the genetic relationship among the single plants are analyzed, and the single plants with rich genetic diversity are reserved. The application firstly constructs a three-level screening system of "resistance character primary screening-chemical secondary screening-SSR genetic diversity analysis", evaluates the genetic background difference of candidate single plants through the SSR marker, and screens the single plants with rich genetic diversity, so as to provide high-quality germplasm resources with rich genetic foundation for subsequent camellia sinensis variety breeding.

Description

Technical Field

[0001] This invention belongs to the field of tea tree breeding and germplasm resource screening technology, specifically involving a method for screening high-quality bitter tea single plants with high theophylline content based on "resistance traits-chemical fingerprint-SSR molecular markers". Background Technology

[0002] bitter tea ( Camellia sinensis var. kuch Theophylline (1,3,7,9-tetramethyluric acid) is a unique wild tea germplasm resource in my country, and its leaves contain significantly higher levels of theophylline (1,3,7,9-tetramethyluric acid) than those of common tea trees. Theophylline possesses various pharmacological activities, including sedation, hypnosis, antidepressant effects, and cognitive enhancement. Compared to traditional caffeine, it has significant advantages in terms of lower neurostimulation and higher biocompatibility, demonstrating broad development prospects and market potential in high-value-added industries such as functional health teas, natural plant medicines, and health and wellness derivatives. Therefore, targeted breeding of theophylline-rich bitter tea varieties is crucial for the effective utilization, industrial development, and germplasm innovation of wild bitter tea resources. Natural hybridization is a commonly used and effective method in the breeding of new tea varieties, but natural hybridization populations are characterized by intense phenotypic segregation, complex genetic backgrounds, and strong randomness in gene recombination. Currently, the industry's single-plant screening technology system for bitter tea is not yet perfect. There is a lack of a comprehensive screening system that can take into account multiple dimensions, systematization, and hierarchy. It is difficult to take into account the field adaptability, target quality traits, and genetic diversity of single plants. This leads to the easy screening of breeding materials with highly homogeneous genetic backgrounds in batches, resulting in a narrow genetic base of breeding populations and seriously restricting the innovation of bitter tea germplasm and the exploration of breeding potential.

[0003] In breeding practice, tea tree resistance traits are often comprehensively evaluated through natural field assessment or artificial stress to select individual plants with superior overall traits. However, relying solely on phenotypic evaluation cannot determine the content of target components in individual plants, nor can it assess differences in their genetic background. This can easily lead to the selection of individuals with overly close kinship, resulting in insufficient genetic diversity in the breeding population. High-performance liquid chromatography (HPLC) can directly determine the theophylline content in tea leaves. This method can quickly identify individual plants with high theophylline content, but its shortcomings are also prominent: First, it only focuses on metabolite levels and does not incorporate field adaptability indicators such as disease resistance, drought resistance, and cold resistance into the evaluation system. This results in selected individual plants often exhibiting weak resistance traits, low survival rates, and unstable yields under cultivation conditions. Second, relying solely on chemical data cannot determine the genetic relationship between different individual plants, potentially selecting individuals with high content but overly close kinship, which is detrimental to subsequent breeding. Simple sequence repeat (SSR) molecular markers have advantages such as high co-dominance, good repeatability, and rich polymorphism, and have been widely used in the assessment of genetic diversity, kinship analysis, and variety identification of tea germplasm resources. Examples include the Penta SSR marker (CN 119332006 A) used for parent identification and the SSR marker in the promoter region of the tea S-adenosylmethionine synthase gene (CN 114480695 B). Some studies have used SSR markers to perform cluster analysis on tea resources, screening individuals with significant genetic differences for hybridization breeding, which can effectively avoid the problem of a narrow genetic base caused by long-term asexual reproduction or artificial selection. However, SSR analysis is usually independent of chemical screening and resistance trait evaluation, lacking "genotype-trait-quality" association analysis. This means that the genetically diverse individual plants selected may not simultaneously possess high theophylline content and good field adaptability.

[0004] In summary, while existing technologies have applied resistance trait evaluation, chemical fingerprinting, and SSR molecular markers to tea plant resource screening, a comprehensive "three-in-one" hierarchical screening system integrating field resistance, quality chemical characteristics, and genetic diversity has not yet been established. This makes it difficult to achieve simultaneous and precise screening of bitter tea plants in terms of quality, high resistance, and genetic heterogeneity. Therefore, developing a screening method for high-theophylline tea plants that balances resistance and rich genetic diversity is of great significance for the innovation of bitter tea germplasm resources and the breeding of superior varieties. Summary of the Invention

[0005] This invention provides a method for screening high-quality bitter tea plants with high theophylline content based on "resistance traits-chemical fingerprint-SSR molecular markers", aiming to solve the technical problems of poor agronomic traits, single genetic background, and imperfect screening system of high theophylline single plants in the prior art.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: A method for screening high-quality bitter tea plants with high theophylline content based on "resistance traits-chemical fingerprint-SSR molecular markers" includes the following steps: (1) Initial screening of resistance traits: A comprehensive evaluation of the resistance traits of seedlings from natural hybridization of bitter tea was conducted in the field to screen out individual plants with excellent resistance to disease, insects, cold and drought. (2) Chemical fingerprint rescreening: The content of theophylline in the single plant selected in step (1) was determined by high performance liquid chromatography (HPLC), with the content of theophylline ≥1.0% as the screening standard; (3) SSR molecular marker genetic diversity analysis: The DNA of the single plants screened in step (2) was extracted and PCR amplified using high polymorphic SSR primers. The SSR fingerprint of each single plant was obtained by electrophoresis. The genetic similarity coefficient between each single plant was calculated, the kinship between each single plant was analyzed, and single plants with genetic similarity coefficients higher than the preset threshold were removed, while single plants with rich genetic diversity were retained. (4) Target plant identification: Plants that meet the conditions of steps (1), (2) and (3) are identified as high-quality bitter tea plants with high theophylline content.

[0007] Furthermore, the comprehensive evaluation of resistance traits in step (1) includes the investigation and rating of disease resistance, insect resistance, drought resistance and cold resistance.

[0008] Furthermore, in step (3), the PCR amplification system includes forward primers with adapters, reverse primers, and adapter primers with FAM fluorescent labels.

[0009] Furthermore, the total PCR volume is 10 μL, containing 1.0 μL of 20.0 ng / μL template, 5.0 μL of 2×Taq PCRMaster Mix, 1 μL of 10 μmol / L tailed forward primer, reverse primer and fluorescently labeled primer, and 3 μL of ddH2O to 10 μL.

[0010] Furthermore, the PCR reaction procedure is as follows: 95 °C pre-denaturation for 5 min, 95 °C denaturation for 30 s; annealing at a temperature gradient of 62 °C-52 °C for 30 s, extension at 72 °C for 30 s, for 10 cycles, with each cycle decreasing by 1 °C; after reaching the 52 °C annealing temperature, run for another 25 cycles, for a total of 35 cycles, then extend at 72 °C for 20 min, and finally store at 4 °C.

[0011] Furthermore, in step (3), the electrophoretic identification of the fluorescent PCR products is carried out in order to ensure the specificity of the fluorescent PCR amplification and the uniformity of the concentration of the samples to be detected. After the fluorescent PCR amplification is completed, 2 μL of PCR products are taken for agarose gel electrophoresis detection. The band pattern of the PCR products is used to determine the amplification specificity of each SSR primer, and the brightness of the PCR product bands is used to determine the amplification efficiency of each SSR primer. According to the concentration requirements of the samples to be detected, each fluorescent PCR product is diluted to obtain fluorescent PCR products with uniform concentration.

[0012] Furthermore, the mass concentration of the agarose is 1%.

[0013] Furthermore, in step (3), PCR amplification was performed using 11 pairs of SSR fluorescently labeled primers, and allelic variation data were collected by capillary electrophoresis. The collected data were then clustered to construct a barley variety database.

[0014] Furthermore, the preset threshold mentioned in step (3) is a genetic similarity coefficient ≥ 0.80.

[0015] Furthermore, in step (3), the kinship analysis is performed using the UPGMA method for cluster analysis, a kinship tree diagram is constructed, and representative strains are selected from different subclasses.

[0016] (1) This invention is the first to construct a three-level screening system of “preliminary screening of resistance traits - chemical secondary screening - SSR genetic diversity analysis”, which overcomes the limitations of single-dimensional screening and realizes the full-dimensional evaluation from field phenotype to chemical quality to genetic background, significantly improving the screening efficiency of superior single plants with high theophylline content.

[0017] (2) The single plants screened by this invention have both high theophylline content and good field adaptability (disease resistance, insect resistance, drought resistance, and cold resistance), and can be directly used for production and promotion.

[0018] (3) This invention uses SSR molecular markers to evaluate the kinship and genetic diversity of candidate plants, eliminate plants with highly similar genetic backgrounds, avoid the narrow genetic base of offspring due to close kinship, and provide germplasm rich in genetic resources for subsequent bitter tea variety breeding. Attached Figure Description

[0019] Figure 1 This is a flowchart of the technical route of the present invention; Figure 2 This is a flowchart of the cultivation process for seedlings of naturally hybridized offspring of bitter tea. Figure 3 These are HPLC chromatograms of theophylline standard and high-theophylline single-plant samples. Figure 4This is a DNA extraction gel image (M: Marker; 1-32: 32 is the maternal parent, the rest are different individual plants). Figure 5 It is an amplification electrophoresis gel image; Figure 6 It is a genotype PDF typing peak diagram; Figure 7 It is a UPGMA clustering dendrogram of candidate single plants based on SSR tags. Detailed Implementation

[0020] like Figure 1 As shown, a method for screening high-quality bitter tea plants with high theophylline content based on "resistance traits-chemical fingerprint-SSR molecular markers" includes the following steps: Step 1: Cultivation of seedlings from natural hybridization offspring of bitter tea (see...) Figure 2 ) The seeds of naturally hybridized bitter tea were sown and raised to obtain the F1 generation segregating population, which was then transplanted to the field and managed in a conventional manner.

[0021] Step Two: Initial Screening of Resistance Traits – Comprehensive Evaluation of Resistance Traits in the Field Field observations of the F1 generation population were conducted for 18-24 months, with evaluation indicators including: (1) Evaluation of disease resistance The natural disease occurrence method in the field was used to classify the main leaf diseases into grades of 0-5 (see Table 1).

[0022]

[0023] The damage rates of major pests such as the tea green leafhopper and the tea geometrid moth were investigated. For the tea green leafhopper, the population density per 100 shoots was used, with a density of ≤5 leafhoppers per 100 shoots indicating excellent resistance. For the tea geometrid moth, the damage rate was assessed, with a damage rate of ≤10% indicating excellent resistance. The overall disease resistance score was calculated as (anthracnose score + tea leaf blight score) / 2, with a score of ≤2 indicating excellent disease resistance.

[0024] (2) Evaluation of cold resistance The field natural identification method was adopted: when the tea trees were damaged by freezing in winter, the degree of freezing damage was investigated on a per-tree basis after the winter. Any leaves with more than 1 / 3 of the middle and upper parts of the leaves turning red or green were considered to be frozen leaves and were graded according to the standards in Table 2.

[0025]

[0026] (3) Drought resistance evaluation The field natural drought scoring method was adopted: after a period of high temperature and drought (15 consecutive days without rainfall), the damage to the leaves was investigated and rated according to the standards in Table 3.

[0027] Screening criteria: First, eliminate individual categories (average disease resistance > 2 levels → elimination; drought resistance > 2 levels → elimination; cold resistance > 2 levels → elimination; tea green leafhopper > 5 per 100 shoots and tea geometrid moth > 10% → elimination). The remaining individual plants are then ranked according to their scores, and the top 30% based on the overall score advance to the next step.

[0028] Step 3: Chemical fingerprint rescreening – HPLC content determination For the superior individual plants selected in step two, one bud and two leaves of new shoots were picked in spring, immediately put into liquid nitrogen for quick freezing, and then thoroughly dried in a freeze dryer. The shoots were then passed through a 40-mesh sieve, and the content of theophylline was determined by high performance liquid chromatography.

[0029] (1) Sample pretreatment Accurately weigh 0.1000 g of tea powder into a 15 mL centrifuge tube, add 10 mL of 70% methanol aqueous solution, vortex to mix, and then sonicate at 40℃ for 30 min, shaking up and down 5 times every 5 min during extraction. After extraction, let the extract stand at 4℃ for 2 h, then aspirate the supernatant, filter it through a 0.22 μm organic phase filter membrane, and transfer it to a liquid chromatography vial for analysis.

[0030] (2) Chromatographic conditions UltiMate 3000 high-performance liquid chromatograph; Acciaim™ 120 C18 column (4.6 mm × 250 mm, 5 μm); flow rate 0.5 mL·min -1 Column temperature 35 ℃; detection wavelength 280 nm; mobile phase A (water: acetonitrile: acetic acid = 96.5: 3: 0.5); mobile phase B (water: acetonitrile: acetic acid = 69.5: 30: 0.5), gradient elution program is shown in Table 4.

[0031]

[0032] (3) Qualitative and quantitative methods Theophylline was used as the standard, and retention time was used for qualitative analysis, while external standard method was used for quantitative analysis.

[0033] (4) Screening criteria Individual plants meeting the following criteria will proceed to the next screening step: Theophylline content ≥ 1.0% (dry weight). Step 4: SSR Molecular Marker Genetic Diversity Analysis For the high-theophylline single plants selected in step three, young leaves were collected, genomic DNA was extracted, and SSR molecular marker genetic diversity analysis was performed.

[0034] DNA extraction Genomic DNA was extracted from young tea leaves using a modified CTAB method. The integrity was assessed by 1% agarose gel electrophoresis, and the purity (OD260 / 280 = 1.8-2.0) and concentration were determined by UV spectrophotometry. The DNA was then diluted to 50 ng / μL for later use.

[0035] (2) SSR primer screening Twenty-eight publicly published tea plant SSR primer pairs were collected (Liu Benying. Research on the application of EST-SSR and ISSR molecular markers in Yunnan tea plant resources [J]. Beijing: Tea Research Institute, Chinese Academy of Agricultural Sciences. 2009.), and 11 pairs of SSR marker primers with good polymorphism were screened out. The SSR primer sequences selected in this invention are shown in Table 5.

[0036]

[0037] (3) PCR amplification system Eleven primer pairs were used to amplify 32 samples with significant differences in genetic background, as shown in Table 6.

[0038]

[0039] (4) Genetic diversity analysis In software such as GenAlEx version 6.501, various genetic diversity indicators of SSR loci and populations are calculated, including observed alleles ( Na ), effective alleles ( Ne Shannon Index () I ), Polymorphic Information Index ( PIC ), observed heterozygosity ( Ho ), expected heterozygosity ( He (6) Genetic similarity coefficient clustering Genetic similarity coefficients among samples were calculated using NTsys 2.10e software. The genetic similarity coefficients were calculated using the J function and clustered using the UPGMA method, and a genetic similarity coefficient cluster diagram was plotted.

[0040] Screening criteria: Individual plants with highly similar genetic backgrounds (genetic similarity coefficient ≥ 0.80) were removed; individual plants with lower genetic similarity coefficients and rich genetic diversity were retained; representative individual plants were selected from different subclasses based on the cluster analysis results.

[0041] Step 5: Identification of target individual plants Based on the screening results of steps two, three, and four, the final target individual plants are determined according to the following criteria: (i) The initial screening of resistance traits was qualified: the scores for disease resistance, cold resistance, and drought resistance all reached the excellent level; (ii) Chemical fingerprint re-screening passed: theophylline content ≥1.0%; (iii) SSR genetic diversity analysis is qualified: the genetic similarity coefficient with other individual plants in the population is ≤0.80 and they come from different groups.

[0042] Individual plants that meet the above conditions are identified as target individual plants of high-quality, high-theophylline tea trees for subsequent germplasm resource preservation, variety selection, or production promotion.

[0043] To make the present invention more fully disclosed, more specific embodiments are described below.

[0044] Example 1: Screening of single-plant bitter tea with high theophylline content 1). Experimental Materials Using the natural hybrid seeds of the bitter tea variety 'Zimai Longyun' (GPD Tea Tree (2024) 450008) as material, 193 F1 generation plants were obtained by sowing and raising seedlings, and then transplanted to the experimental base of the Guangxi South Subtropical Agricultural Research Institute (22°19'57''N, 106°47'17''E, altitude 101.9 m).

[0045] 2). Initial screening of resistance traits Field observations were conducted on 193 F1 generation plants: Disease resistance evaluation: During the peak season for tea anthracnose (July-August), the field natural disease incidence method was used for scoring. The results showed that 67 individual plants (34.72%) were rated as level 1-2 (excellent disease resistance).

[0046] Insect resistance evaluation: During the peak season of tea green leafhopper (May-June and September-October), the number of insects per 100 shoots was investigated: 80 individual plants had ≤5 insects per 100 shoots (accounting for 41.45%).

[0047] Drought resistance evaluation: During the spring drought period (March-April), after 15 consecutive days without rainfall, the field natural drought rating was conducted: 65 individual plants (33.68%) were rated as level 1-2 (excellent drought resistance).

[0048] Cold resistance evaluation: After overwintering, the field natural identification method was used when the plant was damaged by frost in winter: 73 plants (accounting for 37.82%) were rated as level 1-2 (excellent cold resistance).

[0049] Based on the above four categories of indicators, 58 individual plants that ranked in the top 30% of the overall scores were selected to proceed to the next step.

[0050] 3). Chemical fingerprint rescreening HPLC analysis was performed on 58 candidate individual plants: new shoots with one bud and two leaves were harvested in the spring of 2025 (March 25 - April 10) and processed and analyzed according to the method in step three.

[0051] Results (see) Figure 3 ): 31 individual plants (53.45% of the 58 plants) meeting the requirement of theophylline ≥1.0% were selected and proceeded to the next step. Among them, the plant with the highest theophylline content (Z15) reached 1.72%.

[0052] 4. SSR molecular marker genetic diversity analysis (1) DNA extraction: Young leaves of 31 candidate plants were collected, and genomic DNA was extracted. The quality of the DNA was qualified (see [link to relevant documentation]). Figure 4 ).

[0053] (2) SSR amplification: 11 pairs of primers with good polymorphism and clear bands were selected, and on average, each pair of primers amplified 4 alleles. Na ), of which SSR30# and SSR21 amplified 7 and 6 respectively (Table 7). Effective alleles ( Ne The Shannon Information Index (SII) ranges from 1.01 to 3.97, with an average of 2.50. I The polymorphism information content of the 11 primer pairs ranged from 0.08 to 1.48, with an average of 0.91. The observed heterozygosity of each primer ranged from 0.00 to 0.97, with SSR18 having the highest heterozygosity (0.97) and SSR29 the lowest (0.00). The expected heterozygosity ranged from 0.03 to 0.75, with an average of 0.48, with SSR18 and SSR21 having the highest heterozygosity and SSR48 the lowest. PIC The values ​​ranged from 0.03 to 0.70, with an average of 0.44. Seven pairs (63.64%) had values ​​higher than the average, and six pairs of primers were highly polymorphic. PIC >0.50), 3 pairs of primers with moderate polymorphism (0.25 < PIC <0.50), low polymorphism primer pair 2 ( PIC <0.25), indicating that the 11 primer pairs have relatively high polymorphism.

[0054]

[0055] Note: N a: Observing alleles; N e: effective alleles; I Shannon Index; H o: Observed heterozygosity; H e: Expected heterozygosity; Hs : Group aggregation heterozygosity; PIC Polymorphic Information Index (3) Electrophoretic identification of fluorescent PCR products by dilution To ensure the specificity of fluorescent PCR amplification and the uniformity of sample concentration for instrumental analysis, after fluorescent PCR amplification, 2 μL of PCR product was subjected to agarose gel electrophoresis (1% concentration). The band pattern of the PCR product was used to determine the amplification specificity of each SSR primer, and the brightness of the PCR product bands was used to determine the amplification efficiency of each SSR primer. The fluorescent PCR products were diluted according to the required sample concentration for instrumental analysis to obtain fluorescent PCR products of uniform concentration (see [link to sample analysis]). Figure 5 ).

[0056] (4) Fluorescent capillary electrophoresis detection Add the fluorescent PCR product diluted to a uniform concentration to the test plate, and add the test reagents according to the following system:

[0057] Centrifuge the sample and reagent-filled test plate and place it on a PCR instrument to run the denaturation program (95℃, 3 min). Cool immediately after denaturation. Following the ABI 3730xl operating procedure, select the test file corresponding to the test plate name and run the SSR sample analysis test program. The test results are shown below. Figure 6 .

[0058] (5) Calculation of genetic similarity coefficient: Based on the amplification results of 11 SSR loci, the genetic similarity coefficient (GS) between each pair of 31 individual plants was calculated, and the results are shown in Table 8.

[0059]

[0060] 5. Target plant identification (1) Cluster analysis: Based on the genetic similarity coefficient matrix, the UPGMA method was used for cluster analysis to construct a kinship tree diagram. Figure 7 Clustering results showed that the 31 candidate plants could be divided into four main groups at a genetic similarity coefficient of approximately 0.44: Group I: Z07, Z19 (2 plants in total); Group II: Z31, Z14, Z23, Z22 (4 plants in total); Group III: Z10 (1 plant in total); Group IV: Z02, Z04, Z27, Z25, Z20, Z03, Z05, Z11, Z18, Z24, Z08, Z12, Z21, Z16, Z26, Z17, Z09, Z01, Z13, Z28, Z06, Z29, Z15, Z30 (24 plants in total).

[0061] (2) Application of screening criteria: Individual plants with highly similar genetic backgrounds and a genetic similarity coefficient ≥0.80 were removed: The similarity coefficients of Z08 and Z24, and Z17 and Z26 were both 0.83, which are highly similar. The plants with higher theophylline content were selected, and Z24 and Z17 were removed, while Z08 and Z26 were retained. Representative individual plants were selected from the four main groups to ensure genetic diversity: Z07 and Z19 were selected from group I; Z22 and Z31 were selected from group II; Z10 was selected from group III; and Z15, Z20, Z26, Z13, Z08, Z22, Z03, and Z09 were selected from group IV.

[0062] Individual plants with high theophylline content were prioritized for retention: Z07 (1.12%) and Z19 (1.23%) were selected for Group I; Z22 (1.24%) and Z31 (1.26%) were selected for Group II; Z10 (1.31%) was retained as a representative of Group III; and Z15 (1.53%), Z13 (1.48%), Z08 (1.42%), and Z22 (1.42%) were selected for Group IV.

[0063] (3) Based on the results of the three-level screening, the final target individual plants were determined (see Table 9), and a total of 9 plants were retained: Z7, Z19, Z22, Z31, Z10, Z15, Z13, Z08 and Z22.

[0064]

[0065] Nine tea trees were ultimately selected as high-quality, high-theophylline tea trees as target individual plants. Among them, the best overall performance was: theophylline content 1.53%, disease resistance score 1.2, drought resistance score 1, and it came from genetic group IV. It was not closely related to the individual plants from groups I, II, and III (GS≤0.63) and had a rich genetic background.

[0066] The aforementioned nine target plants have been transplanted to the germplasm resource nursery for preservation and can be used for subsequent variety selection and development of functional tea products.

[0067] Comparative Example 1: Screening using HPLC only Screening was conducted solely using HPLC to determine the theophylline content, and 70 plants were selected based on a content ≥1.0%. Field resistance trait surveys and SSR genetic diversity analyses were performed on these 70 plants, and the results are shown in Table 10. Most of the high-theophylline plants exhibited poor resistance traits and had highly similar genetic backgrounds (mainly concentrated in two groups), which is unfavorable for subsequent production promotion and breeding needs.

[0068]

[0069] Comparative Example 2: Screening for resistance traits only Screening was conducted only for disease resistance, insect resistance, drought resistance, and cold resistance, resulting in 58 individual plants ranking in the top 30% based on their comprehensive scores. These 58 plants underwent theophylline content testing and SSR genetic diversity analysis, the results of which are shown in Table 11. Among them, there were still many low-theophylline plants, and their genetic backgrounds were highly similar (mainly concentrated in three groups), which could not meet the breeding requirements for high-theophylline plants.

[0070]

[0071] Comparative Example 3: Resistance Traits + HPLC Screening Thirty-one strains were selected from the same population using a combination of initial screening for resistance traits and HPLC secondary screening. Since further regional trials and widespread application are still needed, SSR genetic diversity analysis was performed on these 31 strains, and the results are shown in Table 12.

[0072]

[0073] Table 13 shows the comparison of the effects of different screening methods. The three-level screening system constructed in this invention finally screened 9 excellent individual plants. The number of selected individual plants is concise and conducive to practical promotion and application. At the same time, the selected individual plants cover all 4 genetic groups, and the average genetic similarity coefficient is as low as 0.44, which proves that the individual plants screened by this method have higher genetic diversity and can provide germplasm materials with a broader genetic basis for subsequent bitter tea variety breeding.

[0074]

[0075] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for screening high-quality bitter tea plants with high theophylline content based on "resistance traits-chemical fingerprint-SSR molecular markers", characterized in that, Includes the following steps: (1) Initial screening of resistance traits: The seedlings of the natural hybrid offspring of bitter tea were comprehensively evaluated in the field to screen out individual plants with excellent resistance to disease, insects, cold and drought. (2) Chemical fingerprint rescreening: The content of theophylline in the single plant screened in step (1) was determined by high performance liquid chromatography, with the content of theophylline ≥1.0% as the screening standard; (3) SSR molecular marker genetic diversity analysis: Extract DNA from the single plants screened in step (2), perform PCR amplification using highly polymorphic SSR primers, obtain the SSR fingerprint of each single plant by electrophoresis, calculate the genetic similarity coefficient between single plants, analyze the kinship between single plants, remove single plants with genetic similarity coefficients higher than the preset threshold, and retain single plants with rich genetic diversity. (4) Target plant identification: Plants that meet the conditions of steps (1), (2) and (3) are identified as high-quality bitter tea plants with high theophylline content.

2. The method for screening high-quality, high-theophylline bitter tea single plants based on "resistance traits-chemical fingerprints-SSR molecular markers" according to claim 1, characterized in that, The comprehensive evaluation of resistance traits in step (1) includes the investigation and rating of disease resistance, insect resistance, cold resistance and drought resistance.

3. The method for screening high-quality, high-theophylline bitter tea single plants based on "resistance traits-chemical fingerprints-SSR molecular markers" according to claim 1, characterized in that, In step (3), the PCR amplification system contains forward primers with adapters, reverse primers, and adapter primers with FAM fluorescent labels.

4. The method for screening high-quality, high-theophylline bitter tea single plants based on "resistance traits-chemical fingerprints-SSR molecular markers" according to claim 3, characterized in that, The total PCR volume was 10 μL, containing 1.0 μL of 20.0 ng / μL template, 5.0 μL of 2×TaqPCR Master Mix, 1 μL of 10 μmol / L tailed forward primer, reverse primer, and fluorescently labeled primer, and 3 μL of ddH2O to 10 μL.

5. The method for screening high-quality, high-theophylline bitter tea single plants based on "resistance traits-chemical fingerprints-SSR molecular markers" according to claim 4, characterized in that, The PCR reaction procedure is as follows: 95 °C pre-denaturation for 5 min, 95 °C denaturation for 30 s; annealing at a temperature gradient of 62 °C-52 °C for 30 s, extension at 72 °C for 30 s, for 10 cycles, decreasing by 1 °C in each cycle; run for another 25 cycles after reaching the 52 °C annealing temperature, for a total of 35 cycles, then extend at 72 °C for 20 min, and finally store at 4 °C.

6. The method for screening high-quality, high-theophylline bitter tea single plants based on "resistance traits-chemical fingerprints-SSR molecular markers" according to claim 1, characterized in that, In step (3), the fluorescent PCR product is identified by electrophoresis. In order to ensure the specificity of fluorescent PCR amplification and the uniformity of the concentration of the sample to be detected, after the fluorescent PCR amplification is completed, 2 μL of PCR product is taken for agarose gel electrophoresis detection. The band pattern of the PCR product is used to determine the specificity of each SSR primer amplification, and the brightness of the PCR product band is used to determine the amplification efficiency of each SSR primer.

7. The method for screening high-quality, high-theophylline bitter tea single plants based on "resistance traits-chemical fingerprints-SSR molecular markers" according to claim 6, characterized in that, The agarose concentration is 1%.

8. The method for screening high-quality, high-theophylline bitter tea single plants based on "resistance traits-chemical fingerprints-SSR molecular markers" according to claim 1, characterized in that, In step (3), PCR amplification was performed using 11 pairs of SSR fluorescently labeled primers. Allelic variation data were collected by capillary electrophoresis and then clustered to construct a barley variety database.

9. The method for screening high-quality, high-theophylline bitter tea single plants based on "resistance traits-chemical fingerprints-SSR molecular markers" according to claim 1, characterized in that, The preset threshold mentioned in step (3) is a genetic similarity coefficient ≥ 0.

80.

10. The method for screening high-quality, high-theophylline bitter tea single plants based on "resistance traits-chemical fingerprints-SSR molecular markers" according to claim 1, characterized in that, The kinship analysis in step (3) uses the UPGMA method for cluster analysis to construct a kinship tree diagram and select representative strains from different subclasses.

Images