A Penta SSR molecular marker for tea tree parentage identification and its application

Abstract

This invention discloses a Penta SSR molecular marker for tea tree parentage identification and its application, relating to the field of molecular marker technology. The molecular marker includes at least one of 25 Penta SSR molecular markers found in the Tieguanyin genome. This invention also discloses primers, kits, and their applications in tea tree variety and parentage identification. This Penta SSR molecular marker exhibits high polymorphism and good primer specificity, enabling precise typing of different tea varieties and demonstrating strong discriminative ability. It solves the problems of insufficient SSR marker polymorphism and specificity, unclear typing, and limited applicability to a wide range of tea tree kinship analysis in existing tea tree parentage identification methods.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to a Penta SSR molecular marker for identifying tea tree parents and its application. Background Technology

[0002] Tea is a typical cross-pollinated plant. Without strict isolation measures, the paternal parent of tea breeding materials is unknown. Under traditional hybridization breeding techniques, it is difficult to accurately determine the paternal parent based solely on external morphological indicators. Molecular marker technology, however, can distinguish subtle differences in the genetic information of tea plants, providing more precise identification results. By analyzing the molecular marker data of tea plant samples, parentage testing software can be used to calculate and infer parentage relationships between tea plant samples, theoretically allowing for the deduction of parentage.

[0003] Because RFLP technology requires high purity and large quantity of genomic DNA for analysis, it is technically demanding and time-consuming, thus its application in tea paternity testing is limited. The RAPD molecular marker technology developed by Williams et al. overcomes the inherent shortcomings of RFLP, offering advantages such as speed, simplicity, efficiency, and large capacity, making it highly feasible for tea paternity testing. However, RAPD technology is time-consuming and expensive when analyzing large numbers of varieties and samples, and it is prone to analytical errors. Furthermore, the heterogeneity of genetic material in tea plants limits its application. Xu Qi et al. used SNP technology to analyze the kinship between different varieties, and Li Hongjian developed core SNP markers for tea variety identification based on KASP technology. These markers are applied to genotyping, identification, and detection of similarity between any two tea plants. However, SNP analysis and whole-genome resequencing-based analysis are costly, involve lengthy experimental procedures, and present significant data analysis challenges.

[0004] SSR marker technology for tea plants is quite mature, and many markers have been developed. However, existing markers predominantly use dinucleotide and trinucleotide repeat units, making it difficult to distinguish tea plant genotypes. This leads to problems such as weak comparability of genotype data, unclear genotyping results, and low database utilization. Furthermore, some primers lack sufficient polymorphism and specificity, limiting their widespread application in tea plant phylogenetic analysis. Despite the maturity of SSR marker technology for tea plants and the development of various markers, some problems remain. For example, the genotype data comparability of some markers is weak, the genotyping results are not clear, and the database utilization is low. The insufficient polymorphism and specificity of primers limit their widespread application in tea plant phylogenetic analysis. Among existing markers, dinucleotide and trinucleotide repeat units dominate. In marker development research, John M. Butler, in his book *Methodology of Forensic DNA Typing*, mentions that the shorter the product motif and the more repetitions, the more severe the stuttering phenomenon. This leads to the inability to properly interpret mononucleotide (Mono) SSRs, and even dinucleotide (Di) motifs are difficult to accurately determine genotype heterozygosity and homozygosity. Therefore, short SSR markers do face some difficulties in identification and analysis given the complexity of the tea plant genome and the large-scale gene repetitions. However, as the motif length reaches four (Tetranucleotide, Tetra), five (Pentanucleotide, Penta), or six (Hexanucleotide, Hexa) nucleotide repeat sequences, the impact of stuttering on genotyping significantly decreases. Among these, Hexa SSRs are relatively few in number and are prone to mutations that do not conform to the repetitive motif variation patterns during amplification. Therefore, Tetra and Penta SSRs, which have low mutation rates, good stability, and are easy to standardize, are often used as high-quality genetic markers in research. Therefore, this study selected Penta SSR markers from tea plants for marker development and explored the application of Penta SSR markers in parentage identification in tea plants. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a PentaSSR molecular marker for tea tree parentage identification and its application. This PentaSSR molecular marker exhibits high polymorphism and good primer specificity, enabling precise typing of different tea varieties and demonstrating strong discriminative ability. The marker can be used to analyze tea samples with known and unknown parental relationships. Based on the PentaSSR marker developed and screened in this study, the "MM2 and MM3 ≤ 1" standard and a CKI index greater than 10000 can be used to infer the existence of parental relationships between tea samples. This invention helps solve the problem of missing parental information in naturally hybridized tea breeding materials. It addresses the issues of insufficient polymorphism and specificity of existing SSR markers in tea tree parentage identification, unclear typing, and the inability to be widely applied to tea tree kinship analysis.

[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A PentaSSR molecular marker for identifying tea tree parents is provided, comprising at least one of the PentaSSR molecular markers located at the following positions in the Tieguanyin genome:

[0007] (1) Chr14: 147517898-7516946; (2) Chr11: 4551391-4552713; (3) Chr4: 231810508-231811495; (4) Ch r5: 64924311-64925507; (5) Chr8: 206387566-206384771; (6) Chr7: 200511093-200510506; (7) Chr1: 1 59941788-159942551; (8) Chr2: 119126442-119126466; (9) Chr4: 151401844-151402468; (10) Chr5: 13 5404988-135405617; (11) Chr7: 134349289-134349913; (12) Chr8: 209551692-209552316; (13) Chr9: 1 32905641-132906270; (14) Chr10: 186584679-186585303; (15) Chr12: 161573160-161573784; (16) Chr 13:157922384-157923008; (17) Chr14: 2930025-29300649; (18) Chr5: 34755747-34753554; (19) Chr9: 14487778-14487156; (20) Chr1: 36439108-36441070; (21) Chr8: 996992-999688; (22) Chr5: 11005559- 11008526; (23) Chr6: 203913408-203912569; (24) Chr9: 32296-31112; (25) Chr3: 17974510-17976438.

[0008] The molecular marker names mentioned above are denoted as follows: CsPenta01 - CsPenta25 .

[0009] The present invention also provides primers for identifying tea tree parents, amplifying the PentaSSR molecular markers used for identifying tea tree parents.

[0010] The present invention also provides a kit for identifying parent tea plants, including primers for identifying parent tea plants.

[0011] The present invention also provides the application of Penta SSR molecular markers for tea parent identification, primers for tea parent identification, or kits for tea parent identification in tea variety identification.

[0012] The present invention also provides the application of Penta SSR molecular markers for tea parent identification, primers for tea parent identification, or kits for tea parent identification in tea parent identification.

[0013] This invention also provides a method for identifying parent tea trees, comprising the following steps:

[0014] (1) Extract genomic DNA from the sample to be identified as a template;

[0015] (2) Primers were designed based on the Penta SSR molecular marker used for tea parent identification for PCR amplification, and then capillary electrophoresis was performed for detection and allele and genotype determination.

[0016] (3) Conduct parental relationship analysis based on the determination results.

[0017] Furthermore, in step (3), the CERVUS software is used to perform parental relationship analysis.

[0018] The present invention has the following beneficial effects:

[0019] 1. Tandem repeat sequences play a crucial role in genetic research. Due to the large size, high heterozygosity, and high proportion of repetitive sequences in the tea plant genome, the development of SSR markers requires systematic experimental screening to ensure their reliability. Furthermore, among various SSR sequences, Penta SSRs exhibit larger allele spacing, are easier to distinguish, and have lower variability, reducing the probability of false positives during identification. Therefore, the Penta SSR marker developed through extensive experimental screening in this invention has significant implications for research applications in tea plants.

[0020] 2. The Penta SSR molecular marker of this invention has high polymorphism, and the primers of this invention have good specificity, enabling accurate typing of different tea varieties and strong identification ability. Attached Figure Description

[0021] Figure 1 Capillary electrophoresis peak diagrams of SSR molecular markers (1)-(25). Detailed Implementation

[0022] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0023] Example 1

[0024] (1) Sequence source: The primer sequences used for marker development were derived from the Tieguanyin genome and the full-length transcriptome data of the buds and leaves of the Emei Wenchun tea variety obtained by previous researchers.

[0025] (2) DNA sample collection, extraction and quality testing: Genomic DNA was extracted from the samples using the CTAB method. Genotyping analysis of 95 tea varieties, including Emei Wenchun, Chuancha No. 2, Ziyan, Zijuan, and Fuding Dabaicha, as well as the F1 progeny of some varieties, was performed by polyacrylamide gel electrophoresis for the verification of SSR primers and the identification of polymorphism and specificity.

[0026] (3) SSR-labeled capillary electrophoresis genotyping: Primers with good quality were selected from those screened by polyacrylamide gel electrophoresis. Different colors (FAM, TAMRA, or HEX) were used for fluorescent labeling of the upstream region of the primers before PCR amplification. PCR products were genotyped by capillary electrophoresis using an ABI 3730XL DNA sequencer to obtain the target DNA fragment and peak area. After comparison with the internal control, the allele band size was read using GeneMapperv 4.0 software. A total of 25 primers with clear bands, high polymorphism, and good specificity were selected. The results of primer capillary electrophoresis genotyping are shown below. Figure 1 As shown, the selected primer information and Penta SSR site information are shown in Table 1 and Table 2, respectively.

[0027] Table 1. Information on the selected primers

[0028]

[0029] Table 2 shows the selected Penta SSR loci.

[0030]

[0031] Example 2

[0032] A method for identifying parent tea trees includes the following steps:

[0033] (1) Extract genomic DNA from the sample to be identified as a template;

[0034] (2) Primers were designed based on the Penta SSR molecular marker used for tea parent identification for PCR amplification, and then capillary electrophoresis was performed for detection and allele and genotype determination.

[0035] (3) Parental relationship analysis was performed using CERVUS software. The number of duotrad mismatches (MM2), the number of tritrad mismatches (MM3), the kinship index (KI), and the cumulative kinship index (CKI) were calculated between offspring and true candidate parents, and between offspring and incorrect candidate fathers. The formula for calculating KI is shown in Table 3. CKI is the product of the KI values ​​of each marker locus, i.e., CKI = KI1 × KI2 × KI3 × KI4 × … × KI n The MM2, MM3, and CKI indicators are used as the criteria for determining whether a parent-child relationship exists.

[0036] Table 3. Calculation Formulas for Complex Relationships

[0037]

[0038] Note: P, Q, R, and S represent the types of genotypes detected in an individual, while p and q represent the allele frequencies of alleles.

[0039] Experimental Example 1

[0040] To better reveal the polymorphism of these markers and obtain the number, frequency, and genotypic composition of alleles, genomic DNA from 95 different tea varieties was extracted, and genotypic detection of 25 Penta SSR markers was completed. Based on this, the resolving power of these markers in tea variety identification and parentage analysis was evaluated.

[0041] (1) Evaluation of the polymorphism of the marker

[0042] The results were used to calculate the allelic number (N) using PowerMaker software. A ), Heterozygosity of Observed (H O The polymorphism of different molecular markers was analyzed using GenAlEx 6.51, including Heterozygosity of Expect (He) and Polymorphism Information Content (PIC). Multisite matching analysis and index calculation were performed. The results of the analysis are shown in Table 4.

[0043] Table 4. Polymorphism Analysis of Different Molecular Markers

[0044]

[0045] Note: N represents the number of individuals successfully genotyped. Adenoted as , where is the number of alleles, is the effective number of alleles, is the observed heterozygosity, is the expected heterozygosity, is the unbiased expected heterozygosity of is , is the polymorphism information content, and is the probability identity value for each primer.

[0046] As shown in Table 4, a total of 176 alleles were detected in 95 samples using 25 SSR molecular markers. On average, 7 Na alleles were detected per marker, with a range of 3-14 alleles. CsPenta10 had the most detected alleles (14), indicating high genotype polymorphism. CsPenta11 had the fewest detected alleles, indicating low genotype polymorphism. The average polymorphic information content (PIC) of the 25 markers was 0.532, ranging from 0.234 to 0.786. CsPenta07 had the highest PIC value, indicating high polymorphism, while CsPenta16 had the lowest PIC value, indicating low polymorphism. A total of 15 markers had a PIC value greater than 0.5. Excluding CsPenta16, the PIC values ​​of the other 24 markers were all greater than 0.25, indicating that all 24 markers exhibited medium to high polymorphism. The observed heterozygosity Ho ranged from 0.232 to 0.750, with an average of 0.524. The expected heterozygosity He ranged from 0.247 to 0.812, with an average of 0.574.

[0047] (2) Marker discrimination ability

[0048] Based on gene frequencies, the Probability of Identity (PID) value of each primer was estimated. This value represents the probability that two random organisms will have the same genotype. A higher PID value indicates a weaker resolving power of the primer; conversely, a lower PID value indicates a stronger resolving power. The PID values ​​of the primers are shown in Table 4.

[0049] Among the 25 primers, primer CsPenta07 had the lowest PID value of 0.06, indicating high discrimination ability, while primer CsPenta16 had the highest PID value of 0.58, indicating lower discrimination ability, which is consistent with the previous PIC values. The average PID value of the 25 primers was 0.24, and the PID values ​​of all primers were less than 1, which shows that all 25 primers have strong discrimination ability.

[0050] (3) Parental analysis

[0051] The genotypic data of the aforementioned 95 tea varieties were selected as candidate parents. The offspring materials to be analyzed were divided into those with known parents and those with known maternal parents but unknown paternal parents. The control materials with known parents came from three hybrid combinations: 'Emei Wenchun' and 'Chuanmu 217' (EW×CM), 'Emei Wenchun' and 'Ziyan' (EW×ZY), and 'Emei Wenchun' and 'Chuancha No. 2' (EW×CC). EW, CM, ZY, and CC refer to 'Emei Wenchun', 'Chuanmu 217', 'Ziyan', and 'Chuancha No. 2', respectively. Each hybrid combination had 8 plants, totaling 24 plants. The 'Emei Wenchun' variety with known maternal parents but unknown paternal parents consisted of 33 superior seedlings from open-pollinated 'Emei Wenchun'.

[0052] The range and average values ​​of the calculated results for the offspring MM2, MM3, and CKI (data in parentheses) of the known parents are shown in Table 5. The true paternal parents are the respective paternal parents of the three hybrid combinations, while the incorrect paternal parents are the 94 tea varieties analyzed previously, excluding the true paternal parents. It can be seen that in the true parental relationships, MM2 and MM3 are both between 0 and 1, and their CKI values ​​are both above 10000; while in the incorrect paternal relationships, MM2 and MM3 are both above 2, with average values ​​above 4.97 and 7.91 respectively, and their CKI values ​​are both 0. This demonstrates that the markers provided by this invention can clearly distinguish between true and incorrect paternal parents.

[0053] Table 5. Calculation results of MM2, MM3, and CKI in known offspring of both parents.

[0054]

[0055] Using the Penta SSR marker of this invention, paternal parent analysis was performed on 33 offspring with EW as the maternal parent and unknown paternal parent. Using the previously analyzed 95 tea varieties as candidate paternal parents, inferred paternal parent information was obtained for 11 offspring. The results are shown in Table 6. ZN, MSBH, and CM54 are 'Zhenong 117', 'Mingshan Baihao 131', and 'Chuanmu 54', respectively. The numbers in parentheses are the average values ​​of multiple offspring.

[0056] Table 6. Analysis results of the paternal lineage of 11 offspring with unknown paternal lineage.

[0057]

[0058] As shown in Tables 5 and 6, the SSR marker combination of the present invention accurately identified the true paternal parent for the offspring material to be analyzed, and the SSR marker combination has a strong parent identification capability.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Primers for identifying parent tea trees, characterized in that, The primer pairs consist of the following primer pairs for amplifying Penta SSR molecular markers used for tea parentage identification:

2. A method for identifying parent tea trees, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be identified as a template; (2) PCR amplification was performed using the primers for identifying tea tree parents as described in claim 1, followed by capillary electrophoresis detection and use for allele and genotype determination; (3) Conduct parental relationship analysis based on the determination results.

3. The method for identifying tea tree parents as described in claim 2, characterized in that, In step (3), the CERVUS software is used to perform parental relationship analysis.

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