KASP molecular marker primer group and kit for detecting genetic loci related to environmental adaptation key traits of tea trees and application of KASP molecular marker primer group and kit

Abstract

The invention belongs to the field of molecular markers, and particularly relates to a KASP molecular marker primer group for detecting genetic loci related to environmental adaptation key traits of tea trees, a kit and application of the KASP molecular marker primer group. The invention provides two InDel sites which are identified on the basis of whole genome association analysis (GWAS) and are obviously related to key agronomic characters and characteristic flavor of a tea tree, and the sites relate to frost resistance (FDI) of the tea tree. Based on the loci, the invention develops a corresponding KASP primer group which is used for detecting tea tree environment adaptation key character genotyping and assisting in selective breeding. The system has the advantages of high throughput, high accuracy, low cost and the like, not only enriches tea tree molecular marker resources, but also can be used for early selection and excellent single plant screening in tea tree molecular breeding, and significantly improves the breeding efficiency.

Description

[0001] This application is a divisional application. The original application number is CN2025118212953, the application date is December 5, 2025, and the invention title is KASP molecular marker primer set, kit and application thereof for detecting genetic loci related to key traits of tea plants. Technical Field

[0002] This invention belongs to the field of molecular markers, specifically relating to KASP molecular marker primer sets, kits, and their applications for detecting genetic loci related to key traits of environmental adaptation in tea plants. Background Technology

[0003] tea tree( Camellia sinensis As an important economic crop, tea production is a highly globalized agricultural industry, with an annual output value exceeding US$200 billion and a yield of over 663 tons. Tea is not only a widely consumed beverage worldwide, but also possesses significant health benefits due to its various bioactive components (such as tea polyphenols, theanine, and caffeine), including antioxidant, anti-inflammatory, lipid-lowering, and chronic disease prevention properties. With increasing global consumer health awareness and diversified market demands, the need for higher quality and distinctive flavors in tea is growing, placing higher demands on the improvement of tea varieties.

[0004] Key agronomic traits of tea plants, such as high and stable yields, disease and stress resistance (including resistance to cold, drought, and pests), and environmental adaptability, directly affect the stability of tea production and the economic benefits of tea cultivation. Meanwhile, secondary metabolites of tea, such as catechins (especially epigallocatechin gallate, EGCG), theanine, caffeine, and aroma compounds, are core factors determining tea quality, flavor characteristics, and health benefits. Most of these traits are quantitative traits, controlled by multiple genes, and are easily influenced by environmental factors (such as climate, soil, and cultivation practices), exhibiting complex genetic mechanisms.

[0005] Traditional tea breeding primarily relies on phenotypic selection, employing methods such as hybridization, mutation breeding, and systematic selection, combined with long-term field observation and evaluation, to screen individuals with superior traits. However, this method has significant limitations, including a lengthy process, low efficiency, low accuracy in phenotypic selection, and unclear objectives. These factors severely restrict the progress of tea breeding work and the efficiency of new variety selection.

[0006] In recent years, with the accumulation of tea plant genome data, marker-assisted selection breeding based on molecular markers has become an effective means to improve breeding efficiency. However, commonly used markers such as SSR and RFLP have limited numbers, low throughput, and high costs, and the association between most markers and traits has not been verified in multiple environments, limiting their large-scale application. KASP (Kompetitive Allele Specific PCR) technology is a PCR-based SNP genotyping technique that can achieve high-throughput, low-cost, and automated large-scale sample genotyping through allele-specific primers and fluorescence signal detection. Developing a molecular marker system based on KASP technology will help overcome the bottlenecks in tea plant breeding and promote the development of tea plant breeding towards precision and efficiency. Summary of the Invention

[0007] To address the problems of limited number of molecular markers for tea plants, low detection efficiency, and poor versatility in existing technologies, this invention aims to provide a molecular marker system based on GWAS identification and KASP technology, which can be used for rapid and accurate typing of key traits in tea plants and can be applied to the rapid screening of highly resistant superior varieties in tea plant molecular breeding.

[0008] This invention is achieved through the following technical solutions: The first aspect of this invention provides a KASP molecular marker primer set for detecting genetic loci related to key traits of environmental adaptation in tea plants, wherein the key traits of environmental adaptation in tea plants are strong frost resistance and high photosynthetic efficiency, and the primer set includes: InDel2 primer pair: SEQ ID NO: 10-12; and InDel3 primer pairs: SEQ ID NO: 16-18.

[0009] Furthermore, the two allele-specific forward primers in the KASP molecular marker primer set are respectively linked to FAM fluorescent markers or HEX fluorescent markers.

[0010] A second aspect of the present invention provides a kit containing the above-described KASP molecular marker primer set for detecting genetic loci related to key traits of environmental adaptation in tea plants.

[0011] The third aspect of this invention provides the application of the above-mentioned KASP molecular marker primer set or kit in screening tea germplasm with key traits of environmental adaptation, specifically: a) Screening for tea plant varieties with strong frost resistance; or / and b) Screening for tea plant germplasm with high photosynthetic efficiency.

[0012] A fourth aspect of this invention provides a method for detecting gene typing of key traits of environmental adaptation in tea plants using the above-mentioned KASP molecular marker primer set or kit, comprising the following steps: S.1 Extract genomic DNA from the tea germplasm resources to be tested; S.2 Using the above-mentioned KASP molecular marker primer set or the kit described in claim 3, the genomic DNA of the tea germplasm resource to be tested is amplified by PCR in a KSAP genotyping instrument; S.3 Identification and analysis were performed based on the FAM and HEX fluorescence signal results after PCR amplification to determine the gene typing of key environmental adaptation traits of the tea trees to be tested. The key environmental adaptation traits of the tea trees are strong frost resistance and high photosynthetic efficiency.

[0013] Compared with the prior art, the present invention has the following advantages: (1) High throughput: It can detect multiple sites simultaneously, making it suitable for large-scale germplasm resource screening.

[0014] (2) High accuracy: Based on multi-environment GWAS verification, the association between markers and traits is stable.

[0015] (3) Wide applicability: Applicable to tea tree populations with different genetic backgrounds.

[0016] (4) Low cost and easy operation: The amount of reagents used is small, the reaction system is small, it supports automated operation, and it is easy to promote. Attached Figure Description

[0017] Figure 1 Manhattan plot based on GLM model for genome-wide association analysis results; Figure 2 Genotyping results for 8 SNP / Indel molecular markers from 176 samples; Figure 3 Box plots showing haplotype analysis of different genotypes at 8 SNP / Indel loci in 176 samples, corresponding to phenotypic traits; Figure 4 Diagrams showing the different stages of tea bud sprouting; Figure 5 Detailed phenotypic statistics of tea tree branch angles; Figure 6 This is a phenotypic statistical chart of the frost resistance index of tea trees in the field during the wintering period. Detailed Implementation

[0018] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0019] Example 1: Genome-wide association analysis and genotyping analysis (1) Field experiment design This invention integrates 210 representative tea tree resources from around the world. After screening and analysis, 176 tea tree resources were used for genotyping. These resources have rich genetic backgrounds and diverse origins. In 2020, the cuttings were transplanted to the same experimental field at the Shengzhou Tea Comprehensive Experimental Base of the Tea Research Institute of the Chinese Academy of Agricultural Sciences, and conventional tea garden field management methods were adopted.

[0020] (2) Phenotypic data Six traits related to growth and development, environmental adaptation, and secondary metabolism—budding stage, branching angle, photosynthetic rate, frost resistance, theanine glucoside, and methylated catechins—were selected for multi-time-point phenotypic analysis. Specifically, the budding stage was divided into several developmental stages and assigned values: the initial budding stage (dormant bud swelling stage), the fish leaf stage, the one bud and one leaf stage, the one bud and two leaves stage, and the one bud and three leaves stage. For detailed information, see [link to relevant documentation]. Figure 4 The angle between the main trunk and lateral branches of a tea tree in the field is recorded as the branching angle. For detailed information, see [link to relevant documentation]. Figure 5 The photosynthetic rate-related parameters were calculated using a handheld chlorophyll fluorometer; the degree of frost damage to overwintering tea leaves in the field was assessed, and the leaves were classified into five grades (Grade 0-4) for frost resistance phenotype identification and statistical analysis. For details, see [link to relevant documentation]. Figure 6 Secondary metabolites were detected by collecting tender shoots of tea trees at the one-bud-two-leaf stage in the field. All phenotypic data described in this invention are based on the BLUE (Best Linear Unbiased Estimator) values ​​of the traits over two consecutive years.

[0021] (3) GWAS analysis and SNP locus determination A GLM model was employed, corrected for kinship and population structure (Q matrix), and GWAS analysis was conducted using phenotypic data to reduce the interference of environmental variation on association results. Furthermore, to reduce the false positive rate, Bonferroni correction was introduced into the GWAS analysis, where the significance level of the SNP was set at [value missing]. P ≤ 0.05 / n (where n is the number of valid SNPs), the corresponding threshold is 7.92 × 10 -8 The significance level of InDel is also set to 1. P ≤ 0.05 / n, the corresponding threshold is 4.97 × 10 -7 For detailed information on SNP / InDel, please refer to Table 1.

[0022] Table 1. Locus-associated genes and detailed information .

[0023] Further genome-wide association analysis identified eight loci significantly associated with the target trait. These were, in order of importance, loci on chromosome 4 located at 230647183 (G / A), 230609847 (G / T), and 230500498-23050059 (+ / -), corresponding to the tea plant genes IMK2, DREB2, and PYL4, respectively, and loci at chromosome 6 located at 18701125 (T / C), corresponding to the tea plant gene MHP1, at the cladistic level. Loci at the end of chromosome 1 at 24645490 were associated with the environmental adaptation trait. The locus associated with photosynthetic rate at position 9 (+ / -) corresponds to the tea gene F3H; the locus significantly associated with frost resistance is located at position 208523002 (+ / -) on chromosome 3, corresponding to the tea gene BEN1; at the secondary metabolite level, the locus significantly associated with theanine glucoside is located at position 141336330 (G / T) on chromosome 2, corresponding to the tea gene HAC12; and the locus associated with methylated catechins is located at position 41281643 (A / G) on chromosome 6, corresponding to the tea gene CCOAMT. GWAS analysis based on the GLM model showed that the chromosomal location information of SNPs significantly associated with the phenotype and their corresponding genes was summarized in […]. Figure 1 The whole genome Manhattan diagram.

[0024] (4) Genotype analysis Further haplotype analysis was performed on SNPs and Indels significantly associated with the trait using the HaplotypeCaller tool in GATK. Three genotypes with significant phenotypic differences were identified at each locus. Specifically: The locus associated with frost resistance is located at chromosome 3, 208523002. The genotypes include homozygous insertion (+ / +), heterozygous (+ / -), and homozygous deletion (- / -), among which the homozygous deletion (- / -) plants have the strongest frost resistance. At chromosome 4, at loci 230647183 and 230609847, which are associated with the budding period, two homozygous types and one heterozygous type were detected. Among them, tea plants with genotypes AA (230647183) and TT (230609847) budded earlier, while GG type plants budded later. The budding-related loci located in the 230500498–23050059 region of chromosome 4 showed that homozygous deletion (- / -) tea plants exhibited earlier budding time. Among the loci on chromosome 6 at position 18701125 that are associated with branching angle, the branching angle of CC-type tea plants is significantly larger. Among the photosynthetic rate-related loci at 246454909 on the terminal 246454909 of chromosome 1, homozygous insert (+ / +) plants had higher photosynthetic rates. Among the loci associated with the accumulation of theanine glucoside at 141336330 on chromosome 2, the content was higher in TT homozygous varieties; Among the sites on chromosome 6 at position 41281643 associated with methylated catechin content, the GG homozygous tea plant accumulates the most of this component.

[0025] For detailed information on the genotype-phenotype associations of all the above loci, please refer to [link to relevant documentation]. Figure 3 .

[0026] Example 2: Development and Application of KASP Tags (1) Design of KASP molecular marker primer set KASP molecular marker primer sets were designed based on SNP / lnDel locus information, and the primer set sequence information for the corresponding loci is shown in Table 2. Three primers were designed for each locus: two specific forward primers (F1, F2) carrying different fluorescent labels, and one universal reverse primer (R). For each relevant locus, the allele-specific forward primers F1 and F2 were linked to different fluorescent labels, either FAM or HEX. HEX fluorescence was used to label the dominant allele. Primers were synthesized by Beijing Novogene Technology Co., Ltd. Primer lengths were 18-30 bp, and amplified fragment lengths were 50-250 bp (including primers). The 3' end of the specific primers should fall on the variant site (Forward or Reverse). Primer sequences should avoid five or more consecutive bases. The TM temperature difference between the three primers for each locus should ideally be within 2 degrees Celsius. GC content is preferably 20%-70%; excessively low or high GC content will affect amplification and will not be used.

[0027] Table 2 Primer sequence information for the corresponding sites .

[0028] (2) Application of KASP tags The KASP marker provided by this invention can be applied to tea tree type identification in three scenarios. Firstly, regarding growth and development, it identifies new tea tree varieties that sprout early to avoid late spring frosts and / or have suitable branching angles. Specifically, it uses SNP1 primer pairs (SEQ ID NO: 1-3); SNP2 primer pairs (SEQ ID NO: 4-6); and InDel1 primer pairs (SEQ ID NO: 7-9) to identify tea tree varieties with early and late budding periods; and SNP3 primer pairs (SEQ ID NO: 13-15) to identify suitable tea tree varieties with a certain branching angle. Secondly, it identifies new tea tree varieties with strong frost resistance and / or high photosynthetic efficiency based on environmental adaptability. Specifically, it uses InDel2 primer pairs (SEQ ID NO: 10-12) to identify frost-resistant tea tree varieties; and InDel3 primer pairs (SEQ ID NO: 16-18) to identify tea tree varieties with high photosynthetic efficiency. Thirdly, regarding the identification of new tea source materials with high content of internal substances that enhance the flavor of tea at the secondary metabolism level, the SNP4 primer pair (SEQ ID NO: 19-21) was used to detect the level of methylated catechins in the new tea varieties; and the SNP5 primer pair (SEQ ID NO: 22-24) was used to detect the level of theanine in the new tea varieties.

[0029] For practical application, DNA was extracted from tea plants for testing. DNA extracted using the conventional CTAB method was sufficient, with 100 ng or more yielding good results. The reaction system was 5 μL, containing: 2.5 μL 2× KASP Master Mix, 0.5 μL primer mixture (containing F1, F2, and R; the specific amount can be adjusted as needed), 1-2 μL template DNA (adjusted according to DNA concentration), and water to make up the difference if less than 5 μL. The PCR program was: 94℃ for 15 min; 94℃ for 20 s, 61-55℃ for 60 s (decreasing by 0.6℃ per cycle), for a total of 10 cycles; 94℃ for 20 s, 55℃ for 60 s, for a total of 26 cycles. Fluorescence signals were detected using a quantitative real-time PCR instrument, and genotypes were automatically determined using genotyping software. Figure 2Taking the genotyping results of 176 samples based on 8 SNP / Indel molecular markers as an example: In this detection, the horizontal axis represents the relative intensity of the FAM fluorescence signal, and the vertical axis represents the relative intensity of the HEX fluorescence signal. The final output is the FAMHEX value (i.e., the normalized ratio of the two fluorescence signals). The FAM and HEX fluorescence signals correspond to the two types of allele-specific primers described in Table 2, respectively. In the scatter plot, the stronger the FAM fluorescence signal and the weaker the HEX fluorescence signal, the larger the FAMHEX value, and the closer the sample point is to the X-axis, indicating that the sample is homozygous for the allele corresponding to the FAM channel; conversely, if the sample point is closer to the Y-axis, it is homozygous for the allele corresponding to the HEX channel. If the sample point is located in the middle region and the FAMHEX value is in the middle range, it indicates that the locus is a heterozygous genotype. The results show that there is a highly consistent correspondence between genotyping and phenotype, and the genotyping results are reliable.

[0030] Therefore, the KASP marker of this application can be used to effectively identify new tea varieties with strong frost resistance and / or high photosynthetic efficiency in terms of environmental adaptability, to identify new tea varieties with early budding and staggered spring frost and / or suitable branching angles at the growth and development level, and to identify new tea varieties with high internal content and improved tea flavor at the secondary metabolism level.

Claims

1. A KASP molecular marker primer set for detecting genetic loci related to key traits of environmental adaptation in tea plants, characterized in that, The key environmental adaptation traits of the tea tree are its strong frost resistance and high photosynthetic efficiency. The primer set includes: InDel2 primer pair: SEQ ID NO: 10-12; and InDel3 primer pairs: SEQ ID NO: 16-18.

2. The KASP molecular marker primer set for detecting genetic loci related to key traits of environmental adaptation in tea trees as described in claim 1, characterized in that, The two allele-specific forward primers in the KASP molecular marker primer set are respectively linked to FAM fluorescent markers or HEX fluorescent markers.

3. A kit containing the KASP molecular marker primer set as described in claim 1 for detecting genetic loci related to key traits of environmental adaptation in tea plants.

4. The application of the KASP molecular marker primer set as described in claim 1 or the kit as described in claim 3 in screening tea germplasm with key traits of environmental adaptation, specifically as follows: a) Screening for tea plant germplasm with strong frost resistance; or / and b) Screening for tea plant germplasm with high photosynthetic efficiency.

5. A method for detecting gene typing of key traits of environmental adaptation in tea plants using the KASP molecular marker primer set of claim 1 or the kit of claim 3, characterized in that, Includes the following steps: S.1 Extract genomic DNA from the tea germplasm resources to be tested; S.2 Using the KASP molecular marker primer set as described in claim 1 or the kit as described in claim 3, the genomic DNA of the tea germplasm resource to be tested is amplified by PCR in a KSAP genotyping instrument; S.3 Identification and analysis were performed based on the FAM and HEX fluorescence signal results after PCR amplification to determine the gene typing of key environmental adaptation traits of the tea trees to be tested. The key environmental adaptation traits of the tea trees are strong frost resistance and high photosynthetic efficiency.

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