A method for identifying camellia sect. thea plants using t-dna as a molecular marker

Abstract

The application discloses a method for identifying Camellia sect. Thea plants by taking T-DNA as a molecular marker, relates to the technical field of molecular biology, extracts leaf genome of the Camellia sect. Thea plants, amplifies a T-DNA fragment in the leaf genome, sequences after purifying the amplification product, and compares with previously constructed T-DNA sequence difference tables to obtain accurate species identification results. The application identifies families and genera by the presence or absence of T-DNA sequences, identifies varieties by SNP differences of the T-DNA sequences, and makes the identification results more accurate.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a method for identifying Camellia species in the Camellia section using T-DNA as a molecular marker. Background Technology

[0002] *Camellia* belongs to the order Theales and family Theaceae in the class Dicotyledonous. It comprises perennial evergreen shrubs or trees, and is the largest and type genus in the Theaceae family. Plants in the *Camellia* genus have significant cultural and economic value, for example, being known for their beautiful ornamental flowers (*Camellia ajaponica*, et al.) and the tea plant (*Camellia sinensis*, et al.). In recent years, the number of species in the *Camellia* genus has increased from 120 to 280, but considerable controversy remains regarding the classification of *Camellia* plants.

[0003] As a common group of crops with multiple important economic and cultural values, the study of the classification and systematic relationships of Camellia plants is of significant strategic importance. The mainstream classification methods in the past were morphological classification, anatomical classification, cytological classification, and molecular systematics. Morphological classification is mainly based on the morphological differences of plant vegetative organs (roots, stems, leaves) or reproductive organs (flowers, fruits, seeds). The morphological characteristics of flowers, fruits, and leaves are important classification criteria for Camellia plants, leading to the development of three major classification systems: the Sealy system, the Zhang Hongda system, and the Min Tianlu system. Anatomical classification is based on anatomical characteristics such as pollen morphology (size, shape, exine ornamentation) and leaf morphology and structure, providing some supplementary evidence to traditional morphological classification. Cytological classification mainly classifies Camellia plants based on chromosome number, morphological structure, cell ploidy, and karyotype analysis. With the rapid development of molecular technology, molecular systematics has provided stronger evidence for the classification of Camellia plants. Higher plants typically possess three sets of genomes: the nuclear genome, the mitochondrial genome, and the chloroplast genome. Research based on the DNA sequences of these three sets of genomes has brought significant advancements to the classification of Camellia species.

[0004] Recently, a T-DNA sequence named CaTA has been discovered in the genomes of all plants in the *Thea* section of the *Camellia* genus. Based on the sequence differences of CaTA, a total of 225 CaTA allele sequences and 2 CaTA structures have been identified. The conservation of CaTA sequences can reflect the phylogenetic relationships of species and provide clues for phylogenetic reconstruction; sequence variability can reveal the characteristic nucleic acid sequences of species and is an important basis for the identification of *Thea* species. Therefore, those skilled in the art are dedicated to developing an accurate method for identifying *Thea* species based on T-DNA sequence information. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide an accurate method for identifying species of Camellia species in the Camellia section.

[0006] To achieve the above objectives, the present invention provides a method for identifying Camellia species in the Camellia section using T-DNA as a molecular marker, characterized in that the method includes the following steps:

[0007] Step 1: Extract the genome from the leaves of tea plants;

[0008] Step 2: Amplify the T-DNA fragment in the leaf genome;

[0009] Step 3: After purifying and sequencing the amplified product, compare it with the previously constructed T-DNA sequence difference table of tea group plants to obtain accurate species identification results.

[0010] In a preferred embodiment of the present invention, step 1 uses a modified CTAB method to extract the genome from the leaves of tea plants.

[0011] In another preferred embodiment of the present invention, the improved CTAB method specifically includes the following steps:

[0012] Step 1.1: Cut 1g of frozen or fresh leaves into small pieces and place them in a pre-cooled mortar. Add liquid nitrogen and grind. Transfer the ground leaves into a 10ml centrifuge tube containing 5ml of extraction buffer and vortex thoroughly to mix.

[0013] Step 1.2: After pre-cooling the centrifuge at 4℃, centrifuge at 9000rpm for 20min and discard the supernatant; add 4ml of lysis buffer preheated at 65℃ to the remaining precipitate, vortex and mix well, then incubate in a 65℃ water bath for 40min, inverting the container during the process to ensure complete lysis.

[0014] Step 1.3: After the water bath, add 4 ml of a 24:1 mixture of chloroform and isoamyl alcohol, invert the mixture more than 50 times, let it stand for 5 minutes, centrifuge at 10,000 rpm for 20 minutes, transfer the supernatant to a new 10 ml centrifuge tube, avoiding aspirating the precipitate, add 0.6 times the volume of pre-cooled isopropanol to the supernatant, slowly invert and mix, let it stand for 10 minutes, centrifuge at 10,000 rpm at room temperature for 10 minutes, discard the supernatant, add 2 ml of 70% ethanol to the precipitate and wash, centrifuge at 10,000 rpm for 2 minutes, discard the supernatant, and air dry until the precipitate becomes transparent;

[0015] Step 1.4: Add 3 ml of TE buffer to a 10 ml centrifuge tube to dissolve the contents. The TE buffer is 10 mM pH.

[0016] After incubating at 65°C for 10–30 min with 8.0 Tris / HCl and 1 mM pH 8.0 EDTA, add RNase A and incubate at 37°C for 30 min. Then add equal volumes of chloroform and isoamyl alcohol in a volume ratio of 24:1, mix slowly by inverting, let stand at room temperature for 5 min, and centrifuge at 10,000 rpm for 10 min.

[0017] Step 1.5: Carefully aspirate the supernatant into a 10ml centrifuge tube, add 0.2 times the volume of 5M NaCl solution to the supernatant, add 2 times the volume of pre-cooled anhydrous ethanol to the supernatant, mix by inversion, let stand for 10 minutes, centrifuge at 10000 rpm for 10 minutes, discard the supernatant, add 2ml of 70% ethanol to wash the DNA precipitate, centrifuge at 10000 rpm for 2 minutes, discard the supernatant, and air dry until transparent;

[0018] Step 1.6: Dissolve the DNA in 200 μl of TE buffer at 4℃ for 1-2 days, then store at -20℃.

[0019] In another preferred embodiment of the present invention, the T-DNA fragment in step 2 is a CaTA gene fragment.

[0020] In another preferred embodiment of the present invention, the CaTA region in the sample DNA is amplified using amplification primers CaTA-F2-1 and CaTA-R2-1 to obtain PCR products, wherein the nucleotide sequences of the primer pairs are as follows:

[0021] SEQ ID No.1CaTA-F1-1:5'-ACGTCGCATTTTACGACCT-3'

[0022] SEQ ID No. 2CaTA-R1-1: 5'-TCCTCAAGTCTATCGCAGTG-3'.

[0023] In another preferred embodiment of the present invention, the amplification PCR reaction system in step 2 is as follows: 25 μL KOD high-fidelity DNA polymerase, 2 μL each of 10 μM upstream and downstream amplification primers, 1 μL template, 20 μL enzyme-free water, and a total volume of 50 μL.

[0024] In another preferred embodiment of the present invention, the amplification PCR reaction program in step 2 is as follows: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 s, annealing at 55°C for 10 s, extension at 68°C for 5-10 s / kb, 33 cycles, final extension at 68°C for 5 min, and storage at 4°C.

[0025] In another preferred embodiment of the present invention, the T-DNA sequence difference table in step 3 is specifically constructed based on the unique variant base information in all CaTA allele sequences of different tea groups.

[0026] In another preferred embodiment of the present invention, the different tea group plants include Shuchazao, Baicha No. 1 and Longjing 43, with the full-length CaTA sequence of Shuchazao as a reference sequence and the differential base sites listed, wherein the full-length CaTA sequence of Shuchazao is shown in SEQ ID NO. 3.

[0027] In another preferred embodiment of the present invention, step 3 specifically includes purifying the amplification product, performing a sequencing reaction using amplification primers, comparing the sequencing results with the previously constructed T-DNA sequence difference table of tea group plants, and obtaining accurate species identification results.

[0028] Technical effect

[0029] This invention identifies genera and families based on the presence or absence of T-DNA sequences. T-DNA sequences are well-defined, highly specific, and easily identifiable DNA fragments, and the inserted T-DNA fragments do not amplify or spread throughout the genome after insertion. Tea plants exhibit extensive hybridization, and their T-DNA sequences are long enough and inserted at an ancient time to generate a range of variants. By identifying varietals through SNP differences in T-DNA sequences, the identification results are made more accurate.

[0030] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0031] Figure 1 This is a flowchart of a preferred embodiment of the method for identifying Camellia species in the Thea section using T-DNA as a molecular marker.

[0032] Figure 2 This is a schematic diagram of the amplification bands of T-DNA from different tea varieties in a preferred embodiment of the present invention;

[0033] Figure 3 a-3f is a schematic diagram comparing the T-DNA sequences of different tea varieties in a preferred embodiment of the present invention. Detailed Implementation

[0034] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0035] This invention provides an application for identifying species of Tea section plants. The method uses amplification primers to amplify a relatively conserved CaTA gene fragment in the genome of Tea section plants via PCR. Then, amplification primers are used to determine the sequences of the CaTA and CaTG gene fragments, thereby rapidly identifying the species of Tea section plants. The principle flowchart is shown below. Figure 1 As shown.

[0036] Specifically, the above-mentioned method for identifying plant species in the Tea section includes the following steps:

[0037] (1) The modified CTAB method was used to extract the genome from the leaves of tea plants:

[0038] 1) Cut 1g of frozen leaves (or fresh leaves) into small pieces and place them in a pre-cooled mortar. Add liquid nitrogen and grind. Transfer the ground leaves to a 10ml centrifuge tube containing 5ml of extraction buffer (Table 1) and vortex thoroughly to mix.

[0039] 2) After pre-cooling at 4℃, centrifuge at 9000rpm for 20min and discard the supernatant; add 4ml of lysis buffer (Table 2) preheated at 65℃ to the remaining precipitate, vortex to mix, and incubate in a 65℃ water bath for 40min, inverting to mix during the process to ensure complete lysis;

[0040] Table 1. Preparation of DNA Extraction Buffer

[0041]

[0042] Table 2. Preparation of DNA Extraction and Lysis Buffer

[0043]

[0044]

[0045] 3) After the water bath, add 4 ml of a chloroform:isoamyl alcohol (24:1) mixture equal in volume to the lysis buffer. Invert the solution at least 50 times, let it stand for 5 minutes, and centrifuge at 10,000 rpm for 20 minutes. Transfer the supernatant to a new 10 ml centrifuge tube, avoiding aspirating the precipitate. Add 0.6 times the volume of pre-chilled isopropanol to the supernatant, gently invert to mix, let it stand for 10 minutes, and centrifuge at 10,000 rpm for 10 minutes (room temperature). Discard the supernatant. Add 2 ml of 70% ethanol to the precipitate and wash. Centrifuge at 10,000 rpm for 2 minutes. Discard the supernatant and allow the solution to air dry until the precipitate becomes transparent.

[0046] 4) Dissolve the contents of a 10ml centrifuge tube in 3ml of TE buffer [10mM Tris / HCl (pH 8.0), 1mM EDTA (pH 8.0)], incubate at 65℃ for 10-30min, add RNase A, incubate at 37℃ for 30min, add an equal volume of chloroform:isoamyl alcohol (24:1), slowly invert to mix, let stand at room temperature for 5min, and centrifuge at 10000rpm for 10min.

[0047] 5) Carefully transfer the supernatant to a 10ml centrifuge tube, add 0.2 volumes of 5M NaCl solution, and 2 volumes of pre-cooled anhydrous ethanol. Mix well by inverting, let stand for 10 minutes, and centrifuge at 10,000 rpm for 10 minutes. Discard the supernatant, add 2ml of 70% ethanol to wash the DNA precipitate, and centrifuge at 10,000 rpm for 2 minutes. Discard the supernatant and allow to air dry until clear.

[0048] 6) Dissolve the DNA in 200 μl of TE buffer (4℃, 1-2 days), and store at -20℃.

[0049] (2) Amplification of the CaTA gene fragment

[0050] The CaTA region in the sample DNA was amplified using amplification primers SEQ ID No.1 (CaTA-F2-1) and SEQ ID No.2 (CaTA-R2-1) to obtain PCR products with a fragment size of approximately 3000 bp. The nucleotide sequences of the primer pairs and the PCR reaction system are shown in Table 3, and the PCR reaction procedure is shown in Table 4.

[0051] SEQ ID No.1(CaTA-F1-1):

[0052] 5'-ACGTCGCATTTTTACGACCT-3'

[0053] SEQ ID No.2(CaTA-R1-1):

[0054] 5'-TCCTCAAGTCTATCGCAGTG-3'

[0055] Table 3 PCR Reaction System Table 1 PCR Reaction System

[0056]

[0057] Table 4 PCR reaction procedures

[0058]

[0059] The reaction procedure for fragment amplification is shown in the table above. The annealing temperature varies with the primer melting temperature, and is generally about 5°C lower than the primer melting temperature. The reaction system is shown in the table below. The template concentration should not be too high, and should be controlled at 10–20 ng / μL.

[0060] (3) Agarose gel electrophoresis

[0061] The PCR products were subjected to agarose gel electrophoresis to detect the amplification effect. The results of a preferred embodiment are as follows: Figure 2 As shown.

[0062] (4) Sequence determination and alignment

[0063] After purifying the amplification product, sequencing was performed using amplification primers SEQ ID No.1 (CaTA-F2-1) and SEQ ID No.2 (CaTA-R2-1). The sequencing results were compared with the previously constructed CaTA nucleic acid sequence difference table of tea group plants to obtain accurate species identification results.

[0064] According to recent literature, a T-DNA sequence named CaTA has been found in the genomes of all plants in the *Thea* section of the *Camellia* genus, and significant differences in the CaTA sequences have been observed among different *Camellia* varieties. Therefore, a T-DNA sequence difference table was constructed by statistically analyzing the unique base variations in all CaTA allele sequences of Shuchazaozao, Baicha No. 1, and Longjing 43.

[0065] This invention provides a list of T-DNA-specific nucleic acid sequences characteristic of three tea plants, using the full-length 5687-character Suchazaozao CaTA sequence as a reference sequence. The specific sequence differences are as follows:

[0066]

[0067] Related sequencing results and comparisons, for example Figure 3 As shown in a-3f, where Figure 3 The top of image shows the reference sequence for Shucha Zao CaTA, the middle image shows the amplified band of Baicha No. 1, and the bottom image shows the amplified band of Longjing 43. Both Baicha No. 1 and Longjing 43 have a 10bp deletion (TTCGGAAGCT) at sites 3964-3973 of the Shucha Zao CaTA reference sequence. Figure 3 Both Bai Cha No. 1 and Longjing 43 in the early-stage Sucha CaTA reference sequence have a deletion of the base C (cytosine) at position 3806. Figure 3 In c, Longjing 43 has a G base sequence at position 3980 of the Shucha Zao CaTA reference sequence, while Baicha No. 1 and Shucha Zao both have an A base sequence at this position. Figure 3dLongjing 43 has a C base sequence at position 3487 of the early-maturing CaTA reference sequence, while Baicha No. 1 and early-maturing Baicha both have an A base sequence at this position. Figure 3 In the *e* white tea variety, the base sequence at position 4030 of the *Shucha Zao* CaTA reference sequence is T, while the base sequence at this position is G for both *Longjing 43* and *Shucha Zao*. Figure 3 f Zhongbai Tea No. 1 has an A at position 2046 of the Shucha Zao CaTA reference sequence, while Longjing 43 and Shucha Zao both have a G at this position.

[0068] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for distinguishing three Camellia species (Tea section) – Shuchazaozao, Baicha No. 1, and Longjing 43 – using the CaTA gene fragment as a molecular marker, characterized in that… The method includes the following steps: Step 1: Extract the genome from the leaves of tea plants; Step 2: Amplify the CaTA gene fragment in the leaf genome; amplify the CaTA region in the sample DNA using amplification primers to obtain PCR products. The nucleotide sequences of the primers are as follows: 5'-ACGTCGCATTTTACGACCT-3' and 5'-CACTGCGATAGACTTGAGGA-3'; Step 3: After purifying and sequencing the amplified product, compare it with the CaTA gene sequence differences of tea plants to obtain accurate identification results; using the full-length CaTA sequence of Shuchazao as the reference sequence, where the full-length CaTA sequence of Shuchazao is shown in SEQ ID NO.3, the CaTA gene sequence differences are as follows: Shuchazao: Loci 3964-3973 are not missing, i.e., TTCGGAAGCT is retained; loci 3806 is C; loci 3980 is A; loci 3487 is A; loci 4030 is G; and loci 2046 is G. Longjing 43: Loci 3964-3973 are missing TTCGGAAGCT; loci 3806 is missing C; loci 3980 is G; loci 3487 is C; loci 4030 is G; and loci 2046 is G. Baicha No. 1: Loci 3964-3973 are missing TTCGGAAGCT; loci 3806 is missing C; loci 3980 is A; loci 3487 is A; loci 4030 is T; and loci 2046 is A.

2. The method as described in claim 1, characterized in that, Step 1 involves extracting the genome from tea plant leaves using a modified CTAB method; the modified CTAB method specifically includes the following steps: Step 1.1: Cut 1g of frozen or fresh leaves into small pieces and place them in a pre-cooled mortar. Add liquid nitrogen and grind. Transfer the ground leaves into a 10ml centrifuge tube containing 5ml of extraction buffer and vortex thoroughly to mix. Step 1.2: After pre-cooling the centrifuge at 4℃, centrifuge at 9000rpm for 20min and discard the supernatant; add 4ml of lysis buffer preheated at 65℃ to the remaining precipitate, vortex and mix well, then incubate in a 65℃ water bath for 40min, inverting the container during the incubation to ensure complete lysis; Step 1.3: After the water bath, add 4 ml of a 24:1 mixture of chloroform and isoamyl alcohol, invert the mixture more than 50 times, let it stand for 5 minutes, centrifuge at 10,000 rpm for 20 minutes, transfer the supernatant to a new 10 ml centrifuge tube, avoiding aspirating the precipitate, add 0.6 times the volume of pre-cooled isopropanol to the supernatant, slowly invert to mix, let it stand for 10 minutes, centrifuge at 10,000 rpm at room temperature for 10 minutes, discard the supernatant, add 2 ml of 70% ethanol to the precipitate and wash, centrifuge at 10,000 rpm for 2 minutes, discard the supernatant, and air dry until the precipitate becomes transparent; Step 1.4: Add 3 ml of TE buffer to a 10 ml centrifuge tube to dissolve the TE buffer (10 mM pH 8.0 Tris / HCl, 1 mM pH 8.0 EDTA). Incubate at 65°C for 10-30 min, then add RNase A and incubate at 37°C for 30 min. Add equal volumes of chloroform and isoamyl alcohol in a volume ratio of 24:1, mix slowly by inverting, let stand at room temperature for 5 min, and centrifuge at 10000 rpm for 10 min. Step 1.5: Transfer the supernatant to a 10ml centrifuge tube, add 0.2 times the volume of 5M NaCl solution, add 2 times the volume of pre-cooled anhydrous ethanol, mix by inversion, let stand for 10 minutes, centrifuge at 10000rpm for 10 minutes, discard the supernatant, add 2ml of 70% ethanol to wash the DNA precipitate, centrifuge at 10000rpm for 2 minutes, discard the supernatant, and air dry until transparent; Step 1.6: Dissolve the DNA in 200 μl of TE buffer at 4℃ for 1-2 days, and store at -20℃.

3. The method as described in claim 1, characterized in that, The amplification PCR reaction system in step 2 is as follows: 25 μL KOD high-fidelity DNA polymerase, 2 μL each of 10 uM upstream and downstream amplification primers, 1 μL template, 20 μL enzyme-free water, and a total volume of 50 μL.

4. The method as described in claim 1, characterized in that, The amplification PCR reaction program in step 2 is as follows: pre-denaturation at 98℃ for 3 min, denaturation at 98℃ for 10 s, annealing at 55℃ for 10 s, extension at 68℃ for 5-10 s / kb, 33 cycles, final extension at 68℃ for 5 min, and storage at 4℃.

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