KASP molecular marker primer group and kit for detecting tea tree key character related genetic loci 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 key traits of tea trees, a kit and application of the KASP molecular marker primer group. The invention provides four SNP (Single Nucleotide Polymorphism) / InDel sites which are identified on the basis of GWAS (Genome Whole Association Analysis) and are obviously related to key agronomic traits and characteristic flavors of tea trees, and the sites relate to BF (Bud flush), FDI (Frost Damage Index) and secondary metabolites of the tea trees. Based on the loci, the invention develops a corresponding KASP primer group which is used for detecting the key character genotyping of the tea tree 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

Technical Field

[0001] 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 in tea plants. Background Technology

[0002] tea tree( Camellia sinensis As an important economic crop, tea production is a highly globalized agricultural industry. 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 greater emphasis on the improvement of tea varieties.

[0003] 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.

[0004] 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.

[0005] 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

[0006] In order to solve the problems of limited number of molecular markers, low detection efficiency and poor universality in the prior art, the present application provides a set of molecular marker system based on GWAS identification and KASP technology development, which can be used for rapid and accurate typing of key traits of tea tree, and can be applied to rapid screening of excellent varieties with early germination, high resistance and high internal substances in tea tree molecular breeding.

[0007] The present application is realized by the following technical solutions: In a first aspect, the present application provides a KASP molecular marker primer set for detecting genetic loci related to key traits of tea tree growth and development, comprising: a SNP1 primer pair: SEQ ID NO: 1-3; a SNP2 primer pair: SEQ ID NO: 4-6; an InDel1 primer pair: SEQ ID NO: 7-9; a SNP3 primer pair: SEQ ID NO: 13-15.

[0008] Further, the key traits of tea tree growth and development include early germination characteristics of tea tree and specific branch angle characteristics of tea tree.

[0009] In a second aspect, the present application provides a KASP molecular marker primer set for detecting genetic loci related to key traits of tea tree growth and development, comprising: SNP1 (G / A): located at chromosome 4:230647183, with A as the dominant allele; SNP2 (G / T): located at chromosome 4:230609847, with T as the dominant allele; InDel1 (- / +): located at chromosome 4:230500498, with the deletion type as the dominant genotype; SNP3 (T / C): located at chromosome 6:18701125, with C as the dominant allele.

[0010] Further, two allele-specific forward primers in the KASP molecular marker primer set are respectively connected with FAM fluorescent label or HEX fluorescent label.

[0011] In a third aspect, the present application provides a kit for a KASP molecular marker primer set for detecting genetic loci related to key traits of tea tree growth and development.

[0012] In a fourth aspect, the KASP molecular marker primer set of the first aspect or the kit of the third aspect is applied to screening tea tree germplasm with key traits of tea tree growth and development.

[0013] Further, the application comprises: early screening of tea germplasm with early sprouting characteristics; or / and screening of tea germplasm with specific branch angle.

[0014] In the fifth aspect, the present application provides a method for detecting the genotyping of key traits of tea growth and development by using the KASP molecular marker primer set of the first aspect or the kit of the third aspect, comprising the following steps: extracting the genomic DNA of the tea germplasm resource to be tested; using the KASP molecular marker primer set of the first aspect or the kit of the third aspect, performing PCR amplification of the genomic DNA of the tea germplasm resource to be tested on a KSAP genotyping instrument; identifying and analyzing the results of FAM and HEX fluorescence signals after PCR amplification to determine the genotyping of key traits of tea growth and development to be tested.

[0015] In the sixth aspect, the present application provides a KASP molecular marker primer set for detecting genetic loci related to key traits of environmental adaptation of tea, comprising: InDel2 primer pair: SEQ ID NO: 10-12; InDel3 primer pair: SEQ ID NO: 16-18.

[0016] Further, the key traits of environmental adaptation of tea include strong frost resistance characteristics and high photosynthetic efficiency characteristics of tea.

[0017] In the seventh aspect, the present application provides a KASP molecular marker primer set for detecting genetic loci related to key traits of environmental adaptation of tea, comprising: InDel2 (- / +) : located at chromosome 3:208523002, and the dominant genotype is insertion type; InDel3 (- / +) : located at chromosome 1:2464549, and the dominant genotype is insertion type.

[0018] Further, the two allele-specific forward primers in the KASP molecular marker primer set are respectively connected with FAM fluorescent label or HEX fluorescent label.

[0019] In the eighth aspect, the present application provides a kit for detecting a KASP molecular marker primer set for detecting genetic loci related to key traits of environmental adaptation of tea.

[0020] In the ninth aspect, the KASP molecular marker primer set of the sixth aspect or the kit of the eighth aspect is applied in screening tea germplasm with key traits of environmental adaptation characteristics.

[0021] Further, the application comprises: (1) screening tea germplasm with strong anti-freezing ability; or / and (2) screening tea germplasm with high photosynthetic efficiency.

[0022] In the tenth aspect, the present application provides a method for detecting the genotyping of the environmental adaptation key traits of tea plants by using the KASP molecular marker primer set of the sixth aspect or the kit of the eighth aspect, which comprises the following steps: (1) extracting the genomic DNA of the tea germplasm resource to be tested; (2) using the KASP molecular marker primer set of the sixth aspect or the kit of the eighth aspect, performing PCR amplification on the genomic DNA of the tea germplasm resource to be tested in the KSAP genotyping instrument; (3) identifying and analyzing according to the FAM and HEX fluorescence signal results after PCR amplification to determine the genotyping of the environmental adaptation key traits of the tea plants to be tested.

[0023] In the eleventh aspect, the present application provides a KASP molecular marker primer set for detecting the genetic loci related to the key traits of secondary metabolites of tea plants, which comprises: a SNP4 primer pair: SEQ ID NO: 19-21; a SNP5 primer pair: SEQ ID NO: 22-24.

[0024] Further, the key traits of secondary metabolites of tea plants include the high-methylated catechin content characteristics of tea plants and the high-theanine glucoside content characteristics of tea plants.

[0025] In the twelfth aspect, the present application provides a KASP molecular marker primer set for detecting the genetic loci related to the key traits of secondary metabolites of tea plants, which comprises: SNP4 (A / G): located at chromosome 6:41281643, and the dominant allele is G; SNP5 (G / T): located at chromosome 2:141336330, and the dominant allele is T.

[0026] Further, the two allele-specific forward primers in the KASP molecular marker primer set are respectively connected with FAM fluorescent labels or HEX fluorescent labels.

[0027] In the thirteenth aspect, the present application provides a kit for the KASP molecular marker primer set for detecting the genetic loci related to the key traits of secondary metabolites of tea plants.

[0028] In the fourteenth aspect, the KASP molecular marker primer set of the eleventh aspect or the kit of the thirteenth aspect is applied in screening tea germplasm with secondary metabolite key trait characteristics.

[0029] Further, the application comprises: (1) Screening of tea germplasm with high methylation catechin content; or / and (2) Screening of tea germplasm with high theanine glucoside content.

[0030] In the fifteenth aspect, the application provides a method for detecting the genotyping of key traits of secondary metabolites of tea plants by using the KASP molecular marker primer set of the eleventh aspect or the kit of the thirteenth aspect, comprising the following steps: (1) Extracting the genomic DNA of the tea germplasm resource to be detected; (2) Using the KASP molecular marker primer set of the eleventh aspect or the kit of the thirteenth aspect, performing PCR amplification on the genomic DNA of the tea germplasm resource to be detected in the KSAP genotyping instrument; (3) Identifying and analyzing according to the FAM and HEX fluorescence signal results after PCR amplification to determine the genotyping of key traits of secondary metabolites of the tea plant to be detected.

[0031] Compared with the prior art, the application has the following beneficial effects: High throughput: multiple sites can be detected simultaneously, suitable for large-scale screening of germplasm resources.

[0032] High accuracy: based on multi-environment GWAS verification, the marker is stably associated with the trait.

[0033] Wide applicability: suitable for tea plant populations with different genetic backgrounds.

[0034] Low cost and easy operation: small amount of reagent, small reaction system, supporting automated operation, and easy to promote. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is a Manhattan plot based on GLM model for whole genome association analysis results; Figure 2 It is a genotyping detection chart of 8 SNP / Indel molecular markers of 176 samples; Figure 3 It is a box plot of haplotype analysis of different genotypes corresponding to traits of 8 SNP / Indel sites of 176 samples; Figure 4 It is a different stage chart of tea bud germination; Figure 5 It is a detailed chart of phenotype statistics of tea branch angle; Figure 6 It is a chart of phenotype statistics of tea plant frost resistance index during overwintering. DETAILED DESCRIPTION

[0036] Following, the embodiments of the present application will be described in detail by specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure of the specification. The present application can also be implemented or applied by other different embodiments, and various modifications or changes can be made to the details in the specification based on different views and applications without departing from the spirit of the present application. It should be noted that the following examples and features in the examples can be combined with each other without conflict. The methods used in the embodiments of the present application are conventional methods unless otherwise specified, and the reagents used can be obtained from commercial channels.

[0037] Example 1: Genome-wide association analysis and genotype analysis Field experiment design The present application integrates a total of 210 representative tea tree resources from around the world, of which 176 tea tree resources are used for genotype detection after screening analysis. These resources have rich genetic backgrounds and are widely sourced. In 2020, the cuttings were transplanted and planted in the same test field of the Shengzhou Tea Comprehensive Experimental Base of the Tea Research Institute of the Chinese Academy of Agricultural Sciences, and the conventional tea garden field management method was adopted.

[0038] Phenotype data Select sprouting period, branch angle, photosynthetic rate, frost resistance, theanine glucoside, methylated catechin, etc. 6 traits at the levels of growth and development, environmental adaptation and secondary metabolism for multi-time point phenotype statistics. Specifically, the sprouting period is divided into multiple development stages and assigned values, respectively, the initial stage of sprouting (Sprouting initial stage), fish leaf stage (Fish leaf stage), one bud and a leaf stage (One bud and a leaf stage), one bud and two leaves stage (One bud and two leaves stage) and one bud and three leaves stage (One bud and three leaves stage), for details see Figure 4 ; The angle between the main stem and the lateral branch of the tea tree in the field is measured as the branch angle, for details see Figure 5 ; The hand-held chlorophyll fluorescence instrument is used to calculate the photosynthetic rate related parameters; the frost damage degree of the overwintering leaves of the tea tree in the field is evaluated, which is divided into five grades of Grade 0-4 for frost resistance phenotype identification and value statistics, for details see Figure 6 ; The one bud and two leaves stage of the tea tree in the field is used for secondary metabolite detection. The trait data described in the present application are all BLUE (Best Linear Unbiased Estimator) values of the traits in two consecutive years as the phenotype data.

[0039] (3) GWAS analysis and SNP site determination The GLM model is adopted, the relationship and the group structure (Q matrix) are corrected, the phenotype data are combined, the GWAS analysis is carried out, so that the interference of environmental variation on the correlation result is reduced. Further, in order to reduce the false positive rate, the Bonferroni correction is introduced in the GWAS analysis, wherein the significance level of the SNP is set to P ≤ 0.05 / n (n is the effective SNP number), and the corresponding threshold is 7.92 × 10 -8 ; the significance level of the InDel is also set to P ≤ 0.05 / n, and the corresponding threshold is 4.97 × 10 -7 . The detailed information of the SNP / InDel is shown in Table 1.

[0040] Table 1: Site associated genes and detailed information .

[0041] Further, through the whole genome correlation analysis, 8 sites significantly associated with the target traits are identified. In sequence, the sites 230647183 (G / A), 230609847 (G / T) and 230500498-23050059 (+ / -) on the 4th chromosome are significantly associated with the germination period on the growth and development level, and correspond to the tea tree genes IMK2, DREB2 and PYL4 respectively; the site 18701125 (T / C) on the 6th chromosome is associated with the branch angle, and corresponds to the tea tree gene MHP1; the site 246454909 (+ / -) at the end of the 1st chromosome is associated with the photosynthetic rate on the environmental adaptation level, and corresponds to the tea tree gene F3H; the site 208523002 (+ / -) on the 3rd chromosome is significantly associated with the frost resistance, and corresponds to the tea tree gene BEN1; the site 141336330 (G / T) on the 2nd chromosome is significantly associated with the theanine glucoside on the secondary metabolite level, and corresponds to the tea tree gene HAC12; and the site 41281643 (A / G) on the 6th chromosome is associated with the methylation catechin, and corresponds to the tea tree gene CCOAMT. The GWAS analysis result based on the GLM model shows that the SNP and the corresponding gene chromosome position information significantly associated with the phenotype are all summarized in the whole genome Manhattan graph of Figure 1 .

[0042] (4) Genotype analysis Further, the HaplotypeCaller tool in GATK is used to analyze the haplotype of the SNP and the Indel site significantly associated with the traits, and three genotypes significantly different in the phenotype are identified for each site. 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.

[0043] For detailed information on the genotype-phenotype associations of all the above loci, please refer to [link to relevant documentation]. Figure 3 .

[0044] Example 2: Development and Application of KASP Tags KASP molecular marker primer design 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.

[0045] Table 2 Sequence information of primer sets of corresponding sites .

[0046] Application of KASP markers The KASP markers provided by the present application can be applied to the identification of three types of tea plants. One is to identify new tea plant varieties that can avoid the late spring cold and / or have suitable branch angles in terms of growth and development. In detail, the SNP1 primer pair: SEQ ID NO: 1-3; the SNP2 primer pair: SEQ ID NO: 4-6; and the InDel1 primer pair: SEQ ID NO: 7-9 are used to identify tea plant varieties with early and late germination periods. The SNP3 primer pair: SEQ ID NO: 13-15 is used to identify tea plant varieties with suitable planting and certain branch angles. The second is to identify new tea plant varieties with strong frost resistance and / or high photosynthetic efficiency in terms of environmental suitability. In detail, the InDel2 primer pair: SEQ ID NO: 10-12 is used to identify tea plant varieties with frost resistance; and the InDel3 primer pair: SEQ ID NO: 16-18 is used to identify tea plant varieties with high photosynthetic efficiency. The third is to identify new tea plant varieties with high content of substances to improve the flavor of tea leaves in terms of secondary metabolism. In detail, the SNP4 primer pair: SEQ ID NO: 19-21 is used to detect the level of methylated catechins in new tea plant varieties; and the SNP5 primer pair: SEQ ID NO: 22-24 is used to detect the level of tea amino acids in new tea plant varieties.

[0047] The DNA of the tea plant to be detected is extracted by the application. The DNA extracted by the conventional CTAB method can meet the requirements, and 100 ng or more can work better. The reaction system is 5 μL, which contains: 2.5 μL 2× KASP Master Mix, 0.5 μL primer mixture (containing F1, F2, and R, and the specific amount can be adjusted as needed), 1-2 μL template DNA (appropriately adjusted according to the DNA concentration), and water is added to make up to 5 μL if less than 5 μL. The PCR program is: 94℃ 15 min; 94℃ 20 s, 61-55℃ 60 s (decrease by 0.6℃ for each cycle), a total of 10 cycles; 94℃ 20 s, 55℃ 60 s, a total of 26 cycles. The fluorescence signal is detected by a fluorescence quantitative PCR instrument, and the genotype is automatically judged by a genotyping software. Figure 2The 176 samples shown are based on the genotyping results of 8 SNP / Indel molecular markers: in this detection, the abscissa represents the relative intensity of FAM fluorescence signal, the ordinate represents the relative intensity of HEX fluorescence signal, and the final output result is 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. 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 to the X-axis, indicating that the sample is a homozygote for the allele corresponding to the FAM channel; on the contrary, if the sample point is close to the Y-axis, it is a homozygote for the allele corresponding to the HEX channel. If the sample point is in the middle area and the FAMHEX value is in the middle range, it indicates that the site is a heterozygous genotype. The results show that there is a high correspondence between genotyping and phenotype, and the genotyping result is reliable.

[0048] As can be seen, the KASP marker of the present application can effectively identify tea tree new varieties with strong frost resistance and / or high photosynthetic efficiency in terms of environmental applicability, identify tea tree new varieties with early germination to avoid spring cold and / or suitable branch angle in terms of growth and development, and identify new tea tree materials with high content of secondary metabolites to improve the flavor of tea leaves in terms of secondary metabolism.

Claims

1. A KASP molecular marker primer set for detecting genetic loci associated with key traits of growth and development of tea plant, characterized in that, The primer set comprises: SNP1 primer pair: SEQ ID NO: 1-3; SNP2 primer pair: SEQ ID NO: 4-6; InDel1 primer pair: SEQ ID NO: 7-9; SNP3 primer pair: SEQ ID NO: 13-15.

2. The KASP molecular marker primer set for detecting the genetic loci associated with the key traits of tea plant growth and development according to claim 1, characterized in that, The key traits of tea tree growth and development include early germination of tea tree and specific branch angle characteristics of tea tree.

3. The KASP molecular marker primer set for detecting the genetic loci related to the key traits of tea plant growth and development according to claim 1, characterized in that, The genetic loci related to the key traits of tea tree growth and development include: SNP1 (G / A): located at chromosome 4:230647183, the dominant allele is A; SNP2 (G / T): located at chromosome 4:230609847, the dominant allele is T; InDel1 (- / +): located at chromosome 4:230500498, the dominant genotype is deletion; SNP3 (T / C): located at chromosome 6:18701125, the dominant allele is C.

4. The KASP molecular marker primer set for detecting the genetic loci associated with the key traits of tea plant growth and development according to claim 1, wherein, The two allele-specific forward primers in the KASP molecular marker primer set are respectively connected with FAM fluorescent label or HEX fluorescent label.

5. A kit comprising the KASP molecular marker primer set for detecting the genetic loci related to the key traits of tea tree growth and development according to claim 1.

6. Use of the KASP molecular marker primer set according to claim 1 or the kit according to claim 5 in screening tea germplasm with key traits of growth and development.

7. Use according to claim 6, wherein Specifically: a) early screening of tea germplasm with early germination characteristics; or / and b) screening of tea germplasm with specific branch angle.

8. A method of genotyping a key trait of growth and development in tea plant using the KASP molecular marker primer set of claim 1 or the kit of claim 5, characterized in that, The method comprises the following steps: S.1 extracting the genomic DNA of the tea germplasm resource to be tested; S.2 using the KASP molecular marker primer set according to claim 1 or the kit according to claim 5, the genomic DNA of the tea germplasm resource to be tested is subjected to PCR amplification on a KSAP genotyping instrument; S.3 according to the identification analysis of FAM and HEX fluorescence signal results after PCR amplification, the genotyping of the key traits of tea tree growth and development to be tested is determined.

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