Penta SSR molecular marker for tea tree parent identification and application of Penta SSR molecular marker

Abstract

The invention discloses a Penta SSR molecular marker for tea tree parent identification and application thereof, and relates to the technical field of molecular markers, and the molecular marker comprises at least one of 25 Penta SSR molecular markers in a Tieguanyin genome. The invention further discloses a primer and a kit for tea tree parent identification and application of the primer and the kit in tea tree variety identification and parent identification. The Penta SSR molecular marker has relatively high polymorphism and good primer specificity, can realize accurate typing of different varieties of tea trees, has relatively strong identification capability, and solves the problems that the SSR marker in the existing tea tree parent identification is insufficient in polymorphism and specificity, not clear enough in typing, and cannot be applied to genetic relationship analysis of the tea trees in a relatively large range.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers, and in particular to a Penta SSR molecular marker for tea tree parent identification and an application thereof. Background Art

[0002] Tea tree is a typical cross-pollinating plant. The paternal parent of tea tree breeding materials obtained without strict isolation measures is unknown. Under traditional hybrid breeding technology, it is difficult to accurately determine the paternal parent based on external morphological indicators. Molecular marker technology can distinguish subtle differences in tea tree genetic information and provide more accurate identification results. By analyzing the molecular marker data of tea tree samples, parentage testing software can be used for calculation and inference. In theory, the parental relationship between tea tree samples can be inferred.

[0003] Since RFLP technology requires high purity and large amount of genomic DNA of the analyzed materials, it requires high technology and is time-consuming to apply, so it is rarely used in the parentage identification of tea trees. The RAPD molecular marker technology created by Williams et al. overcomes the inherent shortcomings of RFLP. It has the characteristics of rapidity, simplicity, high efficiency, and large capacity, and is highly feasible in the parentage identification of tea trees. However, when RAPD technology is used to analyze a large number of varieties and samples, it is time-consuming and expensive, and it is easy to cause misjudgment in analysis. Tea trees also have the characteristics of heterogeneous genetic material, so there are still certain limitations in its application. Xu Qi et al. used SNPs technology to analyze the relationship between different varieties. Li Hongjian developed a core SNP marker for tea variety identification based on KASP technology, which was applied to genotyping of tea varieties, identification or auxiliary identification of tea varieties, and detection of the similarity of any two tea trees. However, SNP and whole genome resequencing analysis are costly, and the experimental process is long and the data analysis is difficult.

[0004] The technology of SSR markers on tea trees is very mature and many markers have been developed. However, the number of repeating units in the existing markers is mostly dinucleotides and trinucleotides, which makes it difficult to distinguish the genotype of tea trees. To a certain extent, there are problems such as the low comparability of genotype data, unclear typing, and low database utilization. In addition, the polymorphism and specificity of some primers are not enough, and they cannot be applied to the kinship analysis of tea trees on a large scale. Although the SSR marker technology on tea trees is quite mature and a variety of markers have been developed, there are still some problems. For example, the genotype data of some markers are not comparable, the typing results are not clear enough, the utilization of the database is not high, and the polymorphism and specificity of the primers are insufficient, which limits its wide application in the kinship analysis of tea trees. Among the existing markers, the repeating units of dinucleotides and trinucleotides occupy a major position. In the research of marker development, John M. Butler mentioned in "Forensic DNA Typing Monograph: Methodology" that the shorter the product motif, the more times it is repeated, and the more serious the Stutter pseudo-peak phenomenon, which will lead to the inability to read the mononucleotide (Mononucleotide, referred to as Mono) SSR normally, and even the dinucleotide (Dinucleotide, referred to as Di) motif is difficult to accurately judge the heterozygosity and homozygosity of the genotype. Therefore, short-segment SSR markers do face some difficulties in identification and analysis in the context of the complexity of the tea plant genome and large-scale gene duplication. However, as the motif length reaches four (Tetranucleotide, Tetra), five (Pentanucleotide, Penta) or six nucleotide (Hexanucleotide, Hexa) repeat sequences, the impact of Stutter on typing determination will be significantly reduced. Among them, the number of Hexa SSRs is small and it is easy to produce mutations that do not conform to the law of repeat motif changes during amplification. In research, Tetra and Penta SSRs with low mutation rates, good stability and easy standardization are often used as high-quality genetic markers. Therefore, this study selected Penta SSR of tea plant for marker development and explored the application of Penta SSR markers in parent identification in tea plant. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a PentaSSR molecular marker for tea tree parent identification and its application. The Penta SSR molecular marker has high polymorphism and good primer specificity, can realize accurate typing of different varieties of tea trees, and has strong identification ability. The marker is used to analyze tea tree samples with known parents and unknown parent relationships. The Penta SSR marker developed and screened based on this research can be used to infer whether there is a parent relationship between tea tree samples with the standard of "MM2 and MM3 ≤ 1" and CKI index greater than 10,000. The content of the present invention helps to solve the problem of missing parent information of tea tree breeding materials from natural hybridization. It solves the problems of SSR marker polymorphism and insufficient specificity in the existing tea tree parent identification, unclear typing, and inability to be applied to tea tree kinship analysis on a large scale.

[0006] The technical solution of the present invention to solve the above technical problem is as follows: Provide a PentaSSR molecular marker for tea tree parent identification, including at least one of the Penta SSR molecular markers at the following positions of the Tieguanyin genome: (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.

[0007] The above molecular markers are named as CsPenta01 - CsPenta25 .

[0008] The invention also provides primers for tea tree parent identification, and amplifies the PentaSSR molecular markers for tea tree parent identification.

[0009] The invention also provides a kit for tea tree parent identification, comprising primers for tea tree parent identification.

[0010] The present invention also provides the application of Penta SSR molecular markers for tea tree parent identification, primers for tea tree parent identification or a kit for tea tree parent identification in tea tree variety identification.

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

[0012] The present invention also provides a method for identifying tea tree parents, comprising the following steps: (1) Extract genomic DNA from the sample to be identified as a template; (2) Design primers based on Penta SSR molecular markers used for tea tree parent identification for PCR amplification, followed by capillary electrophoresis detection and allele and genotype determination; (3) Conduct parental relationship analysis based on the determination results.

[0013] Furthermore, in step (3), CERVUS software was used to perform parental relationship analysis.

[0014] The present invention has the following beneficial effects: 1. Tandem repeat sequences play an important role in genetic research. Since the genome of tea trees is large, heterozygosity is high, and the proportion of repeat sequences is high, the development of SSR markers needs to be screened through systematic experiments to ensure their reliability; in addition, among various SSR sequences, the inter-allele interval of Penta SSR is large, easy to distinguish, and has a low degree of variation, which reduces the probability of false positives during identification. Therefore, the Penta SSR markers obtained by the present invention through a large number of experimental screening and development are of great significance in the research and application of tea trees.

[0015] 2. The Penta SSR molecular marker of the present invention has high polymorphism, and the primer of the present invention has good specificity, can accurately type different varieties of tea trees, and has strong identification ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The capillary electrophoresis peak diagram of SSR molecular markers (1)-(25). DETAILED DESCRIPTION

[0017] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0018] Example 1 (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 measured by previous researchers.

[0019] (2) DNA sample collection, extraction and quality testing: The CTAB method was used to extract the genomic DNA of the samples. 95 tea varieties including Emei Wenchun, Chuancha No. 2, Ziyan, Zijuan, Fuding Dabaicha and the F1 progeny of some varieties were selected for genotyping analysis by polyacrylamide gel electrophoresis for the verification of SSR primers and identification of polymorphism and specificity.

[0020] (3) SSR marker capillary electrophoresis genotyping: The markers screened by polyacrylamide gel were integrated into the selected primers with better quality. Different colors (FAM, TAMRA or HEX) were selected for fluorescent labeling in the upstream region of the primers and then used for PCR amplification. The PCR products were subjected to capillary electrophoresis genotyping using an ABI 3730XL DNA sequencer to obtain the target DNA fragment and peak area. After comparison with the internal reference, 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 screened. The results of primer capillary electrophoresis typing are shown in the figure. Figure 1 The screened primer information and Penta SSR locus information are shown in Tables 1 and 2, respectively.

[0021] Table 1 Information of the selected primers

[0022] Table 2 Information of the selected Penta SSR loci

[0023] Example 2 A method for identifying tea tree parents comprises the following steps: (1) Extract genomic DNA from the sample to be identified as a template; (2) Design primers based on Penta SSR molecular markers used for tea tree parent identification for PCR amplification, followed by capillary electrophoresis detection and allele and genotype determination; (3) Use CERVUS software to analyze the parental relationship. Calculate the number of doublet mismatch sites (MM2), triplet mismatch sites (MM3), kinship index (KI), and cumulative kinship index (CKI) between the offspring and the true candidate parents and the wrong candidate fathers. The KI calculation formula is shown in Table 3. In addition, CKI is the product of the KI of each marker site, that is, CKI=KI1×KI2×KI3×KI4×……×KI n The three indicators MM2, MM3 and CKI are used as the basis for judging whether a parent-child relationship exists.

[0024] Table 3 Calculation formula of complex relationship

[0025] Note: P, Q, R and S are the genotypes of the tested individuals, and p and q are the allele frequencies of the allele types.

[0026] Test Example 1 In order to better reveal the polymorphism of these markers and obtain the number, frequency and genotype composition of alleles, genomic DNA of 95 different tea varieties was extracted, and genotype detection of 25 Penta SSR markers was completed. On this basis, the resolution of these markers in tea variety identification and parent-offspring relationship analysis was evaluated.

[0027] (1) Evaluation of marker polymorphism Results The allele number (N) was calculated using PowerMaker software. A ), Heterozygosity of Observed, H O ), expected heterozygosity (Heterozygosity of Expect, He), diversity index (Polymorphism Information Content, PIC); multi-locus matching analysis and index calculation were performed in GenAlEx 6.51, and the results of polymorphism analysis of different molecular markers are shown in Table 4.

[0028] Table 4 Polymorphism analysis of different molecular markers

[0029] Note: N is the number of individuals with successful tag typing. A is the number of alleles, Ne is the effective number of alleles, Ho is the observed heterozygosity, He is the expected heterozygosity, uHe is the unbiased expected heterozygosity, PIC is the polymorphic information content, and PID is the probability identity value of each primer.

[0030] As shown in Table 4, a total of 176 alleles were detected in the genotype detection of 95 samples using 25 SSR molecular markers. The average number of alleles detected per marker was 7, with a range of 3-14. Among them, CsPenta10 detected the most genotypes, which was 14, indicating that its genotype polymorphism was high. CsPenta11 detected the least genotypes, and the genotype polymorphism of this marker was low. The average polymorphic information content (PIC) of the 25 markers was 0.532, ranging from 0.234 to 0.786. Among them, CsPenta07 had the highest PIC value, with high polymorphism, and CsPenta16 had the lowest PIC value, with low polymorphism. There were 15 markers with PIC values ​​greater than 0.5, and the PIC values ​​of the 24 markers except CsPenta16 were all greater than 0.25, indicating that the 24 markers had medium to high polymorphism. The observed heterozygosity Ho varied from 0.232 to 0.750, with an average of 0.524. The expected heterozygosity He varied from 0.247 to 0.812, with an average of 0.574.

[0031] (2) Marking identification capability According to the gene frequency, the probability of identity (PID) of each primer is estimated. This value represents the probability that two random organisms present the same genotype. The higher the probability of identity, the weaker the discrimination ability of the primer; conversely, the lower the probability of identity, the stronger the discrimination ability of the primer. The calculation results of the PID value of the primer are shown in Table 4.

[0032] Among the 25 primers, the PID value of primer CsPenta07 was the smallest, 0.06, and it had a higher identification ability. The PID value of CsPenta16 was the largest, 0.58, and it had a lower identification ability, which was consistent with the previous PIC value results. The average PID value of the 25 primers was 0.24, and the PID values ​​of all primers were less than 1. It can be concluded that the 25 primers have strong identification abilities.

[0033] (3) Parental analysis The genotype data of the above 95 tea varieties were selected as candidate parents. The progeny materials to be analyzed were divided into those with known parents and those with known maternal parents but unknown paternal parents. Among them, the materials with known parents as controls came from three hybrid combinations, namely: 'Emei Wenchun' and 'Chuanmu 217' (EW×CM), 'Emei Wenchun' and 'Zi Yan' (EW×ZY), 'Emei Wenchun' and 'Chuancha 2' (EW×CC), where EW, CM, ZY and CC refer to 'Emei Wenchun', 'Chuanmu 217', 'Zi Yan' and 'Chuancha 2', respectively, with 8 plants in each hybrid combination, for a total of 24 plants. The materials with known maternal parents and unknown paternal parents were: 33 open-pollinated excellent seedlings of 'Emei Wenchun'.

[0034] The value range and average value (data in brackets) of the calculation results of MM2, MM3 and CKI of the known offspring of the parents are shown in Table 5. The true male parent is the male parent of each of the three hybrid combinations, and the wrong male parent is 94 varieties of the 95 tea tree varieties analyzed before, excluding the true male parent. It can be seen that in the true parent relationship, MM2 and MM3 are between 0 and 1, and CKI is above 10,000; while in the wrong male parent relationship, MM2 and MM3 are both above 2, with average values ​​of 4.97 and 7.91 respectively, and CKI is 0. It shows that the markers provided by the present invention can clearly distinguish the true male parent from the wrong male parent.

[0035] Table 5 Calculation results of MM2, MM3 and CKI of offspring with known parents

[0036] Using the Penta SSR markers of the present invention, paternal analysis was performed on 33 offspring whose maternal parents were EW and whose paternal parents were unknown. The 95 tea varieties analyzed previously were used as candidate paternal parents, and the inferred paternal information of 11 offspring was obtained. The results are shown in Table 6. Among them, ZN, MSBH and CM54 are "Zhe Nong 117", "Ming Shan Bai Hao 131" and "Chuan Mu 54", respectively, and the numbers in brackets are the average values ​​of multiple offspring.

[0037] Table 6 Analysis results of 11 offspring with unknown sires

[0038] It can be seen from Tables 5 and 6 that the SSR marker combination of the present invention can accurately identify the true father of the progeny material to be analyzed, and the SSR marker combination has a strong parent identification ability.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Penta SSR molecular marker for tea tree parent identification, characterized in that: Include at least one of the Penta SSR molecular markers at the following positions in the Tieguanyin genome: (1) Chr14: 147517898-7516946; (2) Chr11: 4551391-4552713; (3) Chr4: 231810508-231811495; (4) Chr 5: 64924311-64925507; (5) Chr8: 206387566-206384771; (6) Chr7: 200511093-200510506; (7) Chr1: 15 9941788-159942551; (8) Chr2: 119126442-119126466; (9) Chr4: 151401844-151402468; (10) Chr5: 135 404988-135405617; (11) Chr7: 134349289-134349913; (12) Chr8: 209551692-209552316; (13) Chr9: 13 2905641-132906270; (14) Chr10: 186584679-186585303; (15) Chr12: 161573160-161573784; (16) Chr1 3: 157922384-157923008; (17) Chr14: 2930025-29300649; (18) Chr5: 34755747-34753554; (19) Chr9: 1 4487778-14487156; (20) Chr1: 36439108-36441070; (21) Chr8: 996992-999688; (22) Chr5: 11005559-1 1008526; (23) Chr12: 120319311-120317190; (24) Chr9: 32296-31112; (25) Chr3: 17974510-17976438.

2. A primer for tea tree parent identification, characterized in that: Amplify the Penta SSR molecular marker for tea tree parent identification as described in claim 1.

3. A method for identifying tea tree parents, characterized in that: The following steps are involved: (1) Extract genomic DNA from the sample to be identified as a template; (2) Designing primers according to the Penta SSR molecular marker for tea tree parent identification according to claim 1 for PCR amplification, and then performing capillary electrophoresis detection and using it for allele and genotype determination; (3) Conduct parental relationship analysis based on the determination results.

4. The method for identifying tea plant parents as claimed in claim 3, characterized in that: In step (3), CERVUS software was used to perform parental relationship analysis.

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