Molecular marker for identifying and screening camellia sinensis var. assamica and application thereof

Abstract

The application provides a molecular marker for identifying and screening a few-flowered tea tree and application thereof, and belongs to the technical field of tea tree molecular breeding. CsAPL1 InDel, wherein the pr CsAPL1 InDel has an amplification fragment length of 382 bp, and a nucleotide sequence as shown in SEQ ID NO. 1; in a many-flowered tea tree, the pr CsAPL1 InDel has an amplification fragment length of 283 bp, and a nucleotide sequence as shown in SEQ ID NO. 2. CsAPL1 The application identifies the tea tree flowering amount phenotype by using the molecular marker pr InDel, and then screens the few-flowered tea tree for subsequent breeding, constructs an effective tea tree molecular marker assisted breeding system, realizes accurate and efficient prediction of genotypes to phenotypes, and lays a technical foundation for high-throughput screening of early breeding materials.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology for tea trees, and particularly relates to a molecular marker for identifying and screening tea trees with few flowers and its application. Background Technology

[0002] tea tree( Camellia sinensis Tea is a specialty leaf-based economic crop, characterized by alternating vegetative and reproductive growth throughout its growth cycle. Once it reaches maturity and begins production, significant nutrient competition arises between these two growth processes. Typically, tea trees exhibit a large number of flower buds and a long flowering period, with the time from flower bud differentiation to seed maturity taking approximately 18 months. Furthermore, flower buds and leaf buds develop simultaneously in the leaf axils. This extensive flower bud differentiation and continuous reproductive development consume substantial amounts of the tree's nutrients, inhibiting new shoot growth, hindering normal bud and leaf growth, and ultimately reducing tea yield and quality, thus limiting the improvement of the tea industry's efficiency and effectiveness. Therefore, cultivating new tea varieties with fewer or no flowers is of significant practical importance for promoting vegetative growth and improving tea yield and quality.

[0003] Currently, the identification of flowering phenotypes in tea trees largely relies on field planting and traditional methods such as plant morphology observation and manual counting of flowers during the flowering period. However, tea trees are perennial woody crops with a long juvenile stage, typically requiring 3-5 years or even longer under natural cultivation conditions to enter the reproductive and flowering stage. Furthermore, flowering phenotypes are highly sensitive to environmental factors such as light, temperature, and water. These identification methods have inherent drawbacks, including lengthy experimental cycles and low screening efficiency. They are also easily affected by external factors such as tree age, climate, and field management, making it impossible to accurately evaluate and efficiently pre-select flowering potential in the early stages of seedling development.

[0004] In recent years, with the successful deciphering of multiple tea plant genomes and the widespread application of multi-omics technologies, a large number of key genes regulating important traits in tea plants and their allelic variations have been identified. Based on this, molecular markers closely linked to target traits have been developed, including single nucleotide polymorphisms (SNPs), insertions / deletions (InDels), and simple sequence repeats (SSRs). Among these, InDel (Insertion-Deletion) molecular markers refer to length polymorphism markers formed between different individuals of the same species due to the insertion or deletion of nucleotide fragments in the genome. These markers have significant advantages such as wide distribution, abundant quantity, high genetic stability, simple genotyping detection, and low cost. Currently, there are no effective molecular markers for screening flowering phenotypes. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a molecular marker for identifying and screening tea plants with few flowers and its application. This invention constructs an effective molecular marker-assisted breeding system for tea plants to achieve accurate and efficient prediction of genotype on phenotype, laying a technical foundation for high-throughput screening of early breeding materials.

[0006] This invention provides a molecular marker for identifying and screening tea plants with few flowers, the molecular marker being pr CsAPL1 -InDel, in tea trees with few flowers, the pr CsAPL1 The amplified fragment of -InDel is 382 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1; in the Camellia japonica plant, the pr CsAPL1 The amplified fragment of -InDel is 283 bp in length, and its nucleotide sequence is shown in SEQ ID NO.2.

[0007] Specifically as follows: SEQ ID NO.1 GGAAGGAGGCTTGAGACTTGATATTAGAAAATAGTCTGCTAAATTATTTTTTATTTATTTTTATTTTTTACATATTGGGCATTTTTTTTGAACATTAATATTTAATTTTTTGTAATTTTTTTTATGTAAAAATCATAATTTTATTTTTTTTATTTATTTTATTTTTTACATATTGGGCATTTTTTTTGAA CATTAATATTTAATTTTTTGTAATTTTTTTTATGTAAAATCATAATTTTATTTTTTATTTTTTTTATTTTTTACATGTTGGACATTTTTTTTTAACATTACTATTTAATGTTTTTTTATTTTTTATTTATTTTGTCATTTTATATAAAAATAATTTAACAATCCCTACACTAACAACTATCATCAAAAC SEQ ID NO.2 GGAAGGAGGCTTGAGACTTGATATTAGAAAATAGTCTGTTAAATTATTTATTTATTTATTTTTATTTTTTACATATTGGGCATTTTTTTTGAACATTAATATTTAATTTTTTGTAATTTTTTTATGTAAAAATCATAATTT TATTTTTTTTATTTTTTTTATTTTTTACATGTTGGACATTTTTTTTTAACATTACTATTTAATGTTTTTTTATTTTTTATTTATTTTGTCATTTTATATAAAAATAATTTAACAATCCCTACACTAACAACTATCATCAAAAC This invention provides the application of reagents for amplifying the aforementioned molecular markers in the identification and screening of tea trees with few flowers.

[0008] Preferably, the reagent includes specific amplification primers pr CsAPL1 -InDel.F and pr CsAPL1 -InDel.R; the pr CsAPL1 The sequence of -InDel.F is shown in SEQ ID NO.3, and the pr CsAPL1 The sequence of -InDel.R is shown in SEQ ID NO.4.

[0009] This invention provides a method for identifying the flowering phenotype of tea plants using the aforementioned molecular markers, characterized by comprising the following steps: 1) Using the genomic DNA of the tea plant material to be tested as a template, specific amplification primers pr were used. CsAPL1 -InDel.F and pr CsAPL1 - Perform PCR amplification using InDel.R to obtain the amplification product; 2) Determine the flowering phenotype of the tea tree to be tested based on the amplification product. When the amplification product is 382 bp, the tea tree to be tested is a tea tree with few flowers; when the amplification product is 283 bp, the tea tree to be tested is a tea tree with many flowers.

[0010] Preferably, the tea tree with few flowers mentioned in step 2) refers to tea tree germplasm materials with a flower grade of 1, that is, 0 to 1 flower buds in a single leaf axil and a flower-bearing branch rate of ≤30%.

[0011] Preferably, the PCR amplification reaction system in step 1) comprises, in 50 μL units, the following components: 10×PCR Buffer 5 μL, 2 mM dNTPs 5 μL, 25 mM MgSO4 3 µL, 10 µM pr CsAPL1 -InDel.F 2 µL, 10 µMpr CsAPL1 -InDel.R 2 µL, template DNA 1 µL, KOD-Plus-Neo enzyme 1 µL, balance ddH2O; The concentration of the template DNA is 90~110 ng / µL; The enzyme activity of the KOD-Plus-Neo enzyme is 0.5~1.5 U / μL; The PCR amplification procedure is as follows: 94℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 60℃ annealing for 30 sec, 68℃ extension for 15 sec, 35 cycles; final extension at 68℃ for 5 min.

[0012] Preferably, the length of the amplification product in step 2) is detected by agarose gel electrophoresis.

[0013] This invention provides the application of the aforementioned molecular markers in the assisted breeding of tea trees with few flowers, using the method to identify the flowering phenotype of tea trees, and then screening tea trees with few flowers for subsequent breeding.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a molecular marker for identifying and screening tea plants with few flowers, the molecular marker being pr CsAPL1 -InDel, located in a key gene regulating flowering in tea plants. CsAPL1 The upstream promoter region contains a 99 bp insertion / deletion sequence; in the tea tree with few flowers, the pr CsAPL1 The amplified fragment of -InDel is 382 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1; in the Camellia japonica plant, the pr CsAPL1 The amplified fragment of -InDel is 283 bp in length, and its nucleotide sequence is shown in SEQ ID NO.2.

[0015] This invention applies the developed molecular markers to marker-assisted breeding of Camellia oleifera, which can replace the complicated field phenotypic identification process and complete the screening by indoor genotyping alone. It effectively overcomes the problems of long breeding cycle, low efficiency and large environmental interference in traditional breeding, and realizes efficient, accurate and rapid early prediction and screening of Camellia oleifera germplasm. Attached Figure Description

[0016] Figure 1 For tea trees CsAPL1 Cloning and sequence analysis of gene promoters, where A is the PCR amplification product of the upstream promoter region; B and C are the sequencing alignment results; and D is the predicted result of differentially acting elements caused by sequence variations. Figure 2 To utilize pr CsAPL1 - PCR amplification results of InDel molecular markers on 20 tea varieties, where A consists of 10 varieties with few flowers, namely 'Longjing 43', 'Jiaming No. 1', 'Tieguanyin', 'Juhuachun', 'Pingyang Tezao', 'Lvya Foshou', 'Zhongcha 606', 'Zhongcha 601', 'Xingrencha', and 'Hongya Foshou'; B consists of 10 varieties with many flowers, namely 'Zhongcha 108', 'Huangjinya', 'Zhonghuang No. 2', 'Baiye No. 1', 'Zijuan', 'Zhenong 12', 'Maoxie', 'Qingfeng', 'Benshan', and 'Fengyuanchun'. Figure 3 This is a single pr plant from the F1 generation of a hybrid of 'Longjing 43' and 'Golden Bud'. CsAPL1 - PCR amplification results of the InDel molecular marker, where M is the DNA molecular weight marker. Detailed Implementation

[0017] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0018] The main reagents used in the following examples are as follows: Polysaccharide and Polyphenol Plant Genomic DNA Extraction Kit (Tiangen); High-Fidelity PCR Enzyme KOD-Plus-Neo (TOYOBO); Agarose Gel Recovery Kit (Axygen); pEASY-Blunt Zero Cloning Kit (Transgen); BioWest Agarose (Baygene); 10×TBE Electrophoresis Buffer and DNA Marker (Coolaber).

[0019] The main instruments used in the examples are as follows: PCR amplification instrument, purchased from Eppendorf; horizontal electrophoresis instrument, purchased from Bio-Rad.

[0020] The synthesis of primers and sequencing of cloning vectors used in the examples were all outsourced to Hangzhou Youkang Biotechnology Co., Ltd.

[0021] Example 1

[0022] tea tree CsAPL1 Gene promoter sequence cloning, discovery of differentially expressed sequences, and development of molecular markers.

[0023] 1. Test materials

[0024] The following tea varieties with different flowering characteristics were selected as research materials: 'Longjing 43', 'Jiaming No. 1', 'Tieguanyin', 'Juhuachun', 'Pingyang Tezao', 'Lvya Foshou', 'Zhongcha 606', 'Zhongcha 601', 'Xingrencha', 'Hongya Foshou', 'Zhongcha 108', 'Huangjinya', 'Zhonghuang No. 2', 'Baiye No. 1', 'Zijuan', 'Zhenong 12', 'Maoxie', 'Qingfeng', 'Benshan', and 'Fengyuanchun'.

[0025] 2. Field statistics and grading of flowering traits

[0026] During the peak flowering season of tea trees in November, a field survey of flowering characteristics was conducted on 20 mature tea varieties planted in the experimental field of the Tea Research Institute of the Chinese Academy of Agricultural Sciences. The survey indicators included: the number of axillary flower buds per leaf axil (number per axil) and the percentage of flowering branches (percentage of flowering branches per 100 branches, %). Based on the tea tree flowering grading criteria (Table 1), 'Longjing 43', 'Jiaming No. 1', 'Tieguanyin', 'Juhuachun', 'Pingyang Tezao', 'Lvya Foshou', 'Zhongcha 606', 'Zhongcha 601', 'Xingrencha', and 'Hongya Foshou' were classified as Grade 1, belonging to varieties with few flowers; 'Zhongcha 108', 'Huangjinya', 'Zhonghuang No. 2', 'Baiye No. 1', 'Zijuan', 'Zhenong 12', 'Maoxie', 'Qingfeng', 'Benshan', and 'Fengyuanchun' were classified as Grade 3, belonging to varieties with many flowers (Table 2).

[0027] Table 1. Criteria for Grading Tea Tree Flowering Stage

[0028] Table 2. Statistics and grading of flowering traits of different tea varieties

[0029] 3. Extraction of genomic DNA

[0030] The extraction of genomic DNA from tea plants was performed using a polysaccharide and polyphenol plant genomic DNA extraction kit (catalog number DP360, Tiangen). The specific operating procedures were strictly followed in accordance with the kit's instruction manual.

[0031] 4. CsAPL1 Cloning of gene promoters

[0032] The inventor's preliminary research showed that CsAPL1 This gene is a key regulator of flowering in tea plants, and it is highly expressed in flower buds. Overexpression can promote flowering in Arabidopsis thaliana, and its expression level in the axillary buds of multi-flowered varieties is significantly higher than that in sparsely-flowered varieties. To further investigate whether there are sequence variations in the upstream promoter region of this gene, this study designed specific upstream primer F (SEQ ID NO.5: 5'-TACGGTGTGGACCCCGATTAGGTG-3') and downstream primer R (SEQ ID NO.6: 5'-TCCTCTCCCCATTTTCTCTCTTGT-3') based on pan-genome information of tea plants, and performed PCR amplification using genomic DNA from multiple tea plant varieties as templates.

[0033] PCR amplification was performed using KOD-Plus-Neo high-fidelity enzyme. The total reaction volume was 50 µL, and the system composition was as follows: 5 µL 10× PCR Buffer, 5 µL dNTPs mixture (2 mM each), 3 µL MgSO4 (25 mM), 2 µL each of forward and reverse primers (10 µM), 31 µL ddH2O, 1 µL template DNA (500 ng / µL), and 1 µL KOD-Plus-Neo enzyme (1 U / µL). The amplification program was as follows: 94℃ pre-denaturation for 3 min; then 36 cycles, each cycle including 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 80 s; and a final extension at 68℃ for 10 min. The amplified products were separated by 1.0% agarose gel electrophoresis to obtain a target band of approximately 2000 bp. Figure 1 (A) Subsequently, the target fragment was excised from the gel, ligated into the pEASY-Blunt Zero vector, transformed into E. coli DH5α competent cells, and multiple positive single clones were obtained by screening by bacterial culture PCR and sent for sequencing analysis.

[0034] Using the same PCR amplification primers, sequencing yielded two different lengths. CsAPL1 promoter, i.e., pr CsAPL1 -L type (2042 bp) and pr CsAPL1 -S type (1941 bp), both have multiple SNP mutation sites and one 99 bp InDel structural variant ( Figure 1 (B and C in the text). Cis-acting element predictive analysis shows: pr CsAPL1 -L contains a unique STRE component, pr CsAPL1 -S contains the unique ARE element; the 99 bp InDel sequence contains Box 4, CAAT-box, and TATA-box elements ( Figure 1 (D in the middle).

[0035] 5. pr CsAPL1 Development of InDel molecular markers

[0036] According to pr CsAPL1 -L and pr CsAPL1 To determine the 99 bp insertion / deletion sequence location between -S, specific primers pr were designed. CsAPL1 -InDel.F (SEQ ID NO.3: 5'-GGAAGGAGGCTTGAGACTTGATATT-3') and pr CsAPL1 -InDel.R (SEQ ID NO. 4: 5'-GTTTTGATGATAGTTGTTAGTGTAGGG-3').

[0037] Using the primer sequences and the tested DNA, PCR amplification and agarose gel electrophoresis analysis were performed. The PCR reaction system consisted of: 5 μL 10×PCR Buffer, 5 μL 2 mM dNTPs, 3 µL 25 mM MgSO4, 2 µL each of 10 µM forward and reverse primers, 31 μL ddH2O, 1 µL DNA (100 ng / µL), and 1 µL KOD-Plus-Neo enzyme (1 U / µL). The PCR amplification program was: 94℃ pre-denaturation for 3 min, followed by the following cycles: 98℃ denaturation for 10 sec, 60℃ annealing for 30 sec, 68℃ extension for 15 sec, for a total of 35 cycles, and a final extension at 68℃ for 5 min. The amplified products were separated by electrophoresis on a 2.8% agarose gel.

[0038] Electrophoresis results showed that PCR amplification products of the two types of materials could be clearly distinguished by 2.8% agarose gel electrophoresis, and all 10 varieties with few flowers amplified a 382 bp band. Figure 2 (A in the text), while only 283 bp bands appeared in 10 multiflora varieties ( Figure 2 (B in the text). The difference in the PCR amplification products indicates that pr CsAPL1 InDel molecular markers can effectively reflect the characteristics of tea plants. CsAPL1 Sequence variations in gene promoter regions can serve as a reliable molecular tool for screening germplasm with few flowers.

[0039] Example 2

[0040] pr CsAPL1 -Application of InDel molecular markers

[0041] 1. Test materials

[0042] The test material was the 'Longjing 43' × 'Golden Bud' F1 population obtained through sexual hybridization, and 40 individual plants from this population were randomly selected for the experiment.

[0043] 2. pr CsAPL1 -InDel molecular markers for different individual plants CsAPL1 Genotyping of genes

[0044] Using molecular markers pr CsAPL1InDel performed genotyping on 40 tested individual plants. DNA extraction, PCR amplification, and agarose gel electrophoresis were performed under the same conditions as in Example 1. The results showed that the maternal parent 'Longjing 43' and its offspring (numbered 2, 4, 6, 8, 10, 11, 12, 13, 16, 21, 22, 23, 25, 26, 27, 28, 31, 33, 34, 36, 37) all amplified a 382 bp fragment; while the paternal parent 'Huangjinya' and its offspring (numbered 1, 3, 5, 7, 9, 14, 15, 17, 18, 19, 20, 24, 29, 30, 32, 35, 38, 39, 40) only amplified a 283 bp fragment, with no 382 bp band observed. Figure 3 ).

[0045] 3. Field statistics and grading of flowering traits

[0046] During the peak flowering period in autumn, field surveys and grading of the flowering characteristics of the aforementioned individual plants were conducted. The survey indicators and grading standards were the same as in Example 1. Statistical results showed that the flowering grade of individual plants numbered 2, 4, 6, 8, 10, 11, 12, 13, 16, 21, 22, 23, 25, 26, 27, 28, 31, 33, 34, 36, and 37 was all grade 1, belonging to the category of tea trees with few flowers (Table 3). This result is consistent with genotypic identification.

[0047] Table 3. Statistics and grading of flowering traits of 'Longjing 43' × 'Golden Bud' F1 individual plants

[0048] As can be seen from the above embodiments, pr CsAPL1 -InDel molecular markers can effectively distinguish between tea trees with few flowers and tea trees with many flowers, and can be used as functional markers for screening germplasm of tea trees with few flowers.

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A molecular marker for identifying and screening tea plants with few flowers, characterized in that, The molecular marker is pr CsAPL1 -InDel, in tea trees with few flowers, the pr CsAPL1 The amplified fragment of -InDel is 382 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1; in the Camellia japonica plant, the pr CsAPL1 The amplified fragment of -InDel is 283 bp in length, and its nucleotide sequence is shown in SEQ ID NO.

2.

2. The application of the molecular marker reagent described in claim 1 in the identification and screening of tea trees with few flowers.

3. The application according to claim 2, characterized in that, The reagents include specific amplification primers pr CsAPL1 -InDel.F and pr CsAPL1 -InDel.R; the pr CsAPL1 The sequence of -InDel.F is shown in SEQ ID NO.3, and the pr CsAPL1 The sequence of -InDel.R is shown in SEQ ID NO.

4.

4. A method for identifying the flowering phenotype of tea plants using the molecular markers described in claim 1, characterized in that, Includes the following steps: 1) Using the genomic DNA of the tea plant material to be tested as a template, the specific amplification primers pr in claim 3 are used... CsAPL1 -InDel.F and pr CsAPL1 - Perform PCR amplification using InDel.R to obtain the amplification product; 2) Determine the flowering phenotype of the tea tree to be tested based on the amplification product. When the amplification product is 382 bp, the tea tree to be tested is a tea tree with few flowers; when the amplification product is 283 bp, the tea tree to be tested is a tea tree with many flowers.

5. The method according to claim 4, characterized in that, Step 2) The tea tree with few flowers refers to tea tree germplasm materials that are graded as Grade 1, that is, the number of flower buds in a single leaf axil is 0 to 1, and the rate of branches with flowers is ≤30%.

6. The method according to claim 4, characterized in that, Step 1) The PCR amplification reaction system, in 50 μL increments, comprises the following components: 5 μL of 10×PCR Buffer, 5 μL of 2 mM dNTPs, 3 µL of 25 mM MgSO4, and 10 µM pr CsAPL1 -InDel.F 2 µL, 10 µM pr CsAPL1 -InDel.R 2 µL, template DNA 1 µL, KOD-Plus-Neo enzyme 1 µL, balance ddH2O; The concentration of the template DNA is 90~110 ng / µL; The enzyme activity of the KOD-Plus-Neo enzyme is 0.5~1.5 U / μL; The PCR amplification procedure is as follows: 94℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 60℃ annealing for 30 sec, 68℃ extension for 15 sec, 35 cycles; final extension at 68℃ for 5 min.

7. The method according to claim 4, characterized in that, The length of the amplification product described in step 2) was detected by agarose gel electrophoresis.

8. The application of the molecular marker as described in claim 1 in assisted breeding of camellia trees with few flowers, characterized in that, The flowering phenotype of tea trees is identified using the method described in claim 4, and then tea trees with fewer flowers are screened for subsequent breeding.

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