Method for identifying camellia group plants by taking T-DNA as molecular marker

Abstract

The invention discloses a method for identifying Camellia plants by using T-DNA as a molecular marker, and relates to the technical field of molecular biology, and the method comprises the following steps: extracting a leaf genome of the Camellia plants, amplifying T-DNA fragments in the leaf genome, purifying an amplified product, sequencing, and comparing with a previously constructed T-DNA sequence difference table to obtain an accurate species identification result. According to the present invention, the family is identified according to the existence of the T-DNA sequence, and the variety is identified according to the SNP difference of the T-DNA sequence, such that the identification result is accurate.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, and in particular to a method for identifying plants of the genus Camellia using T-DNA as a molecular marker. Background Art

[0002] Camellia belongs to the family Theaceae, Theales, and is a perennial evergreen shrub or tree. It is the largest genus in the family Theaceae and also its model genus. Camellia plants have important cultural and economic values, such as the ornamental species with beautiful flowers (Camellia ajaponica., et al.) and the tea tree (Camellia sinensis., et al.). In recent years, the number of species in the genus Camellia has increased from 120 to 280, but there is great controversy over 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 relationship of the Camellia plant is of great strategic significance. In the past, the mainstream classification methods were morphological classification, anatomical classification, cytological classification and molecular systematic classification. Morphological classification is mainly based on the morphological differences of plant vegetative organs (roots, stems, leaves) or reproductive organs (flowers, fruits, seeds) for classification research. Among them, the morphological characteristics of flowers, fruits and leaves are important classification bases for Camellia plants, and three major classification systems have been developed: Sealy system, Zhang Hongda system and Min Tianlu system. Anatomical classification is a classification study based on anatomical characteristics such as pollen morphology (size, shape, outer wall decoration) and leaf morphological structure. This classification method provides some auxiliary evidence for traditional morphological classification to a certain extent. 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 systematic classification provides strong evidence for the classification of Camellia plants. Generally, higher plants have three sets of genomes, namely the nuclear genome, the mitochondrial genome and the chloroplast genome. Research based on these three sets of genomic DNA sequences has brought great progress in the classification of Camellia plants.

[0004] Recently, a T-DNA sequence named CaTA was found in the genomes of all the plants of the Camellia tea group (Thea), and based on the sequence difference information of CaTA, a total of 225 CaTA allele sequences and 2 CaTA structures were typed. The conservation of CaTA sequences can reflect the relationship between biological species and provide clues for phylogenetic reconstruction; the variability of sequences can reveal the characteristic nucleic acid sequences of biological species, which is an important basis for the identification of species of the Camellia tea group. Therefore, technicians in this field are committed to developing an accurate identification method for the species of the Camellia tea group based on T-DNA sequence information. Summary of the invention

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

[0006] To achieve the above object, the present invention provides a method for identifying plants of the genus Camellia using T-DNA as a molecular marker, characterized in that the method comprises the following steps:

[0007] Step 1, extracting the genome of tea plant leaves;

[0008] Step 2, amplifying the T-DNA fragment in the leaf genome;

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

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

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

[0012] Step 1.1, chop 1g of frozen leaves or fresh leaves into pieces and put them into a pre-cooled mortar, add liquid nitrogen to grind, transfer the ground leaves into a 10ml centrifuge tube containing 5ml extraction buffer, and vortex thoroughly to mix;

[0013] Step 1.2: Precool the centrifuge at 4°C and centrifuge at 9000 rpm for 20 min, discard the supernatant; add 4 ml of lysis buffer preheated at 65°C to the remaining precipitate, vortex and mix, and place in a 65°C water bath for 40 min, inverting and mixing during the process to ensure full lysis;

[0014] Step 1.3, after the water bath, add 4 ml of a mixture of chloroform and isoamyl alcohol in a volume ratio of 24:1, invert more than 50 times, let stand for 5 minutes, centrifuge at 10000rpm for 20 minutes, transfer the supernatant to a new 10 ml centrifuge tube to avoid aspirating the precipitate, add 0.6 times the volume of pre-cooled isopropanol of the supernatant, slowly invert and mix, let stand for 10 minutes, centrifuge at 10000rpm at room temperature for 10 minutes, discard the supernatant, add 2 ml of 70% ethanol to the precipitate for washing, centrifuge at 10000rpm for 2 minutes, pour off the supernatant, and dry under ventilation until the precipitate becomes transparent;

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

[0016] 8.0Tris / HCl, 1mM pH 8.0EDTA, incubate at 65℃ for 10-30min, add RNaseA, incubate at 37℃ for 30min, add equal volumes of chloroform and isoamyl alcohol in a volume ratio of 24:1, slowly turn over and mix, let stand at room temperature for 5min, and centrifuge at 10000rpm for 10min;

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

[0018] Step 1.6: Dissolve the DNA in 200 μl TE buffer at 4°C for 1 to 2 days and store at -20°C.

[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 a PCR product, wherein the nucleotide sequence of the primer pair is 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 of step 2 is: 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.

[0024] In another preferred embodiment of the present invention, the amplification PCR reaction procedure of step 2 is: 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 constructed specifically based on the statistical unique variant base information in all CaTA allele sequences of plants from different tea groups.

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

[0027] In another preferred embodiment of the present invention, step 3 specifically includes, after purifying the amplified product, using amplification primers to perform sequencing reaction, comparing the sequencing result with the previously constructed T-DNA sequence difference table of tea plants to obtain accurate species identification results.

[0028] Technical Effects

[0029] The present invention identifies the family or genus by the presence or absence of the T-DNA sequence. The T-DNA sequence is a clear, highly specific and easily identifiable DNA fragment, and the T-DNA insertion fragment will not be amplified and spread in the genome after insertion. There is a large amount of hybridization in tea plants. The T-DNA sequence is long enough and the insertion time is old enough, so it is enough to produce a series of variants. The variety is identified by the SNP difference of the T-DNA sequence, making the identification result more accurate.

[0030] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a flow chart of a method for identifying plants of the genus Camellia using T-DNA as a molecular marker in a preferred embodiment of the present invention;

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

[0033] Figure 3 a-3f are schematic diagrams of T-DNA sequence comparisons of different varieties of tea plants in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0034] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0035] The present invention provides an application for identifying the species of tea plants. The method uses amplification primers to perform PCR to amplify the more conservative CaTA gene fragment in the genome of tea plants, and then continues to use amplification primers to determine the sequences of CaTA and CaTG gene fragments, thereby quickly identifying the species of tea plants. The principle flow chart is as follows Figure 1 shown.

[0036] Specifically, the method for identifying the species of tea plants comprises the following steps:

[0037] (1) Extracting tea plant leaf genome using the modified CTAB method:

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

[0039] 2) Precool the centrifuge at 4°C and centrifuge at 9000 rpm for 20 min, discard the supernatant; add 4 ml of lysis buffer (Table 2) preheated at 65°C to the remaining precipitate, vortex to mix, and place in a 65°C water bath for 40 min, inverting to mix and fully lyse;

[0040] Table 1 DNA extraction buffer preparation

[0041]

[0042] Table 2 DNA extraction lysis buffer preparation

[0043]

[0044]

[0045] 3) After the water bath is over, add a mixture of chloroform and isoamyl alcohol (24:1) (4 ml) in the same volume as the lysate, invert more than 50 times, let stand for 5 minutes, centrifuge at 10,000 rpm for 20 minutes, transfer the supernatant to a new 10 ml centrifuge tube to avoid aspirating the precipitate, add 0.6 times the volume of pre-cooled isopropanol of the supernatant, slowly invert and mix, let stand for 10 minutes, centrifuge at 10,000 rpm for 10 minutes (room temperature), discard the supernatant, add 2 ml of 70% ethanol to the precipitate for washing, and centrifuge at 10,000 rpm for 2 minutes. Pour off the supernatant and dry under ventilation until the precipitate becomes transparent;

[0046] 4) Add 3 ml of TE [10 mM Tris / HCl (pH 8.0), 1 mM EDTA (pH 8.0)] buffer to a 10 ml centrifuge tube to dissolve, incubate at 65°C for 10-30 min, add RNase A, incubate at 37°C for 30 min, add an equal volume of chloroform: isoamyl alcohol (24:1) mixture, slowly invert to mix, let stand at room temperature for 5 min, and centrifuge at 10,000 rpm for 10 min;

[0047] 5) Carefully pipette the supernatant into a 10ml centrifuge tube, add 0.2 times the volume of 5M NaCl solution, add 2 times the volume of pre-cooled anhydrous ethanol, invert and mix, let stand for 10 minutes, centrifuge at 10000rpm for 10 minutes. Discard the supernatant, add 2ml 70% ethanol to wash the DNA precipitate, centrifuge at 10000rpm for 2 minutes. Pour off the supernatant and dry under ventilation until transparent;

[0048] 6) Add 200 μl TE buffer to dissolve the DNA (4°C, 1 to 2 days) and store at -20°C.

[0049] (2) Amplification of CaTA gene fragments

[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 a PCR product with a fragment size of approximately 3000 bp. The nucleotide sequence of the primer pair and the PCR reaction system are shown in Table 3, and the PCR reaction program 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 program

[0058]

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

[0060] (3) Agarose gel electrophoresis

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

[0062] (4) Sequence determination and alignment

[0063] After the amplified product was purified, sequencing reaction 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 CaTA nucleic acid sequence difference table of tea plants we constructed previously to obtain accurate species identification results.

[0064] According to recent literature reports, a T-DNA sequence named CaTA was found in the genome of plants in the Thea group of the genus Camellia, and it was found that the CaTA sequences of different varieties of the genus Camellia were significantly different. Therefore, the unique variant base information in all CaTA allele sequences of Shucha Zao, Baicha No. 1, and Longjing 43 was counted to construct a T-DNA sequence difference table.

[0065] The present invention provides a table of T-DNA specific nucleic acid sequences of three tea group plants, taking the full-length 5687 of the Shucha Zao CaTA sequence as a reference sequence, and the specific sequence differences are as follows:

[0066]

[0067] Related sequencing results and comparisons Figure 3 a-3f, where Figure 3 aThe top is the Shuchazao CaTA reference sequence, the middle is the amplified band of Baicha No. 1, and the bottom is the amplified band of Longjing 43. Baicha No. 1 and Longjing 43 both lack a 10bp sequence (TTCGGAAGCT) at the 3964-3973 position of the Shuchazao CaTA reference sequence. Figure 3 In b, both Baicha No. 1 and Longjing 43 lack base C (cytosine) at position 3806 of the Shuchazao CaTA reference sequence. Figure 3 In c, the base sequence of Longjing 43 at position 3980 of the Shuchazao CaTA reference sequence is G, while the base sequences of Baicha No. 1 and Shuchazao at this position are both A. Figure 3d The base sequence of Longjing 43 at position 3487 of the Shuchazao CaTA reference sequence is C, while the base sequences of Baicha No. 1 and Shuchazao at this position are both A. Figure 3 The base sequence of Baicha No. 1 in the Shucha Zao CaTA reference sequence is T, while the base sequences of Longjing 43 and Shucha Zao are both G at this site. Figure 3 In f, Baicha 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 specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for identifying plants of the genus Camellia using T-DNA as a molecular marker, characterized in that: The method comprises the following steps: Step 1, extracting the genome of tea plant leaves; Step 2, amplifying the T-DNA fragment in the leaf genome; Step 3: Purify the amplified product and sequence it, and compare it with the previously constructed T-DNA sequence difference table of tea plants to obtain accurate species identification results.

2. The method according to claim 1, characterized in that The step 1 uses a modified CTAB method to extract the genome of tea plant leaves.

3. The method according to claim 2, characterized in that The improved CTAB method specifically comprises the following steps: Step 1.1, chop 1g frozen leaves or fresh leaves into pieces and put them into a pre-cooled mortar, add liquid nitrogen to grind, transfer the ground leaves into a 10ml centrifuge tube containing 5ml extraction buffer, and vortex thoroughly to mix; Step 1.2: Precool the centrifuge at 4°C and centrifuge at 9000 rpm for 20 min, discard the supernatant; add 4 ml of lysis buffer preheated at 65°C to the remaining precipitate, vortex and mix, and place in a 65°C water bath for 40 min, inverting and mixing during the process to ensure full lysis; Step 1.3, after the water bath, add 4 ml of a mixture of chloroform and isoamyl alcohol in a volume ratio of 24:1, invert more than 50 times, let stand for 5 minutes, centrifuge at 10000rpm for 20 minutes, transfer the supernatant to a new 10 ml centrifuge tube to avoid aspirating the precipitate, add 0.6 times the volume of pre-cooled isopropanol of the supernatant, slowly invert and mix, let stand for 10 minutes, centrifuge at 10000rpm at room temperature for 10 minutes, discard the supernatant, add 2 ml of 70% ethanol to the precipitate for washing, centrifuge at 10000rpm for 2 minutes, pour off the supernatant, and dry under ventilation until the precipitate becomes transparent; Step 1.4: Add 3 ml of TE buffer to a 10 ml centrifuge tube to dissolve. TE buffer is 10 mM pH 8.0Tris / HCl, 1mM pH 8.0EDTA, incubate at 65℃ for 10-30min, add RNaseA, incubate at 37℃ for 30min, add equal volumes of chloroform and isoamyl alcohol in a volume ratio of 24:1, slowly turn over and mix, let stand at room temperature for 5min, and centrifuge at 10000rpm for 10min; Step 1.5, carefully pipette the supernatant into a 10 ml centrifuge tube, add 0.2 times the volume of the supernatant 5M NaCl solution, add 2 times the volume of the supernatant pre-cooled anhydrous ethanol, invert and mix, let stand for 10 minutes, centrifuge at 10000 rpm for 10 minutes, discard the supernatant, add 2 ml of 70% ethanol to wash the DNA precipitate, centrifuge at 10000 rpm for 2 minutes, pour off the supernatant, and air dry until transparent; Step 1.6: Dissolve the DNA in 200 μl TE buffer at 4°C for 1 to 2 days and store at -20°C.

4. The method according to claim 1, characterized in that The T-DNA fragment in step 2 is a CaTA gene fragment.

5. The method according to claim 4, characterized in that The CaTA region in the sample DNA was amplified using amplification primers CaTA-F2-1 and CaTA-R2-1 to obtain a PCR product, wherein the nucleotide sequence of the primer pair is as follows: SEQ ID No.1CaTA-F1-1:5'-ACGTCGCATTTTACGACCT-3' SEQ ID No. 2CaTA-R1-1: 5'-TCCTCAAGTCTATCGCAGTG-3'.

6. The method according to claim 1, characterized in that The amplification PCR reaction system of step 2 is: 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.

7. The method according to claim 1, characterized in that The amplification PCR reaction program of step 2 is: 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.

8. The method according to claim 1, characterized in that The T-DNA sequence difference table in step 3 is constructed specifically based on the unique variant base information in all CaTA allele sequences of plants from different tea groups.

9. The method according to claim 8, characterized in that The different tea group plants include Shucha Zao, Baicha No. 1 and Longjing 43. The full-length CaTA sequence of Shucha Zao is used as a reference sequence and the differential base sites are listed, wherein the full-length sequence of Shucha Zao CaTA sequence is shown in SEQ ID NO.

3.

10. The method according to claim 1, characterized in that The step 3 specifically includes, after purifying the amplified product, using amplification primers to perform sequencing reaction, comparing the sequencing result with the previously constructed T-DNA sequence difference table of tea plants, and obtaining accurate species identification results.

Images