Application of TCP20-like gene in dormancy release of poplar

Abstract

The application provides application of a TCP20-like gene in dormancy release of a poplar. The sequence of the TCP20-like gene is shown in SEQ ID NO:1. By detecting the expression amount of the TCP20-like gene, whether the poplar is in a dormancy period can be determined.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and particularly relates to application of a TCP20-like gene in dormancy release of poplar. BACKGROUND

[0002] Compared with annual model plants such as Arabidopsis, the research on trees has problems such as long research cycle and difficult cultivation. 84K poplar is a new variety of poplar of the latest generation, and has the advantages of easy rooting, fast growth and strong resistance, and is therefore widely used in the research on trees.

[0003] Unlike annual plants, perennial plants have evolved mechanisms to adapt to environmental changes and synchronize their growth with seasonal changes. This process is strictly regulated by a series of physiological changes, including growth cessation, bud formation, dormancy establishment, dormancy release and subsequent bud break. The dormancy of perennial plants is mainly regulated by photoperiod and temperature. For most woody species, short day conditions (SDs) will accelerate the cessation of growth and the initiation of dormancy, while long day conditions (LDs) will delay these processes. After dormancy establishment, trees need to be exposed to low temperatures for a long time to achieve dormancy release and restore the ability to grow. The genetic regulation of poplar dormancy has been intensively studied: the signal transduction pathways of photoperiod and temperature interweave to form a complex regulatory network, and finally the dormancy signal is converged to key integrators such as poplar flowering locus t1 (FT1), FT2 and short vegetative-like (SVL), thereby regulating dormancy. In this process, in addition to buds, the vascular cambium also has the process of dormancy and dormancy release. The vascular cambium is the secondary meristem of trees, and its activity promotes the lateral growth of plants. Bud dormancy and cambium dormancy are two independent events that occur to some extent with the same molecular mechanisms, but are not completely consistent. In recent years, the annual cycle of trees has mainly focused on the top, and some key regulators of dormancy and dormancy release have also been identified. The research on the dormancy and dormancy release of the vascular cambium is relatively less, and the key factors regulating the dormancy and dormancy release are even fewer. SUMMARY

[0004] The present application aims to at least partially solve one of the technical problems in the related art. The present application provides application of a TCP20-like gene in dormancy release of poplar.

[0005] Specifically, the present application provides the following technical solutions:

[0006] In a first aspect of the present application, application of a TCP20-like gene in regulating dormancy release of poplar is provided, and the sequence of the TCP20-like gene is shown in SEQ ID NO: 1.

[0007] In a second aspect of the present application, there is provided a use of a TCP20-like protein in regulating dormancy release of a poplar, wherein the sequence of the TCP20-like protein is shown as SEQ ID NO: 2.

[0008] In a third aspect of the present application, there is provided a method for determining a development stage of a poplar, comprising:

[0009] obtaining a sample, and detecting an expression level of a TCP20-like gene of the poplar;

[0010] determining the development stage of the poplar based on the expression level of the TCP20-like gene.

[0011] The development stage of the poplar is a dormancy stage or a germination stage of the poplar.

[0012] According to an embodiment of the present application, if the expression level of the TCP20-like gene has no significant difference compared with the expression level of the TCP20-like gene of the poplar in the dormancy stage, it is determined that the poplar is in the dormancy stage.

[0013] If the expression level of the TCP20-like gene has a significant difference compared with the expression level of the TCP20-like gene of the poplar in the dormancy stage, it is determined that the poplar is in the germination stage.

[0014] The significant difference refers to a significant increase in the expression level of the gene.

[0015] According to an embodiment of the present application, the significant increase is at least 4 times, for example, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, or at least 30 times of the expression level of the TCP20-like gene of the poplar in the dormancy stage.

[0016] According to an embodiment of the present application, the sample is obtained from a vascular cambium or an apical bud of the poplar.

[0017] In a fourth aspect of the present application, there is provided an isolated nucleic acid having a sequence shown as SEQ ID NO: 1, which is used to determine whether the poplar is in the dormancy stage.

[0018] In a fifth aspect of the present application, there is provided an isolated protein having a sequence shown as SEQ ID NO: 2, which is used to determine whether the poplar is in the dormancy stage.

[0019] In the present disclosure, the isolated, whether it is an isolated nucleic acid or an isolated protein, for example, the isolated nucleic acid refers to the nucleic acid separated from the naturally occurring environment and made not in the natural state by human operation.

[0020] According to an embodiment of the present disclosure, the protein is encoded by the sequence shown in SEQ ID NO: 1.

[0021] In a sixth aspect of the present disclosure, a poplar vascular cambium dormancy release marker gene is provided, and the gene has the sequence shown in SEQ ID NO: 1.

[0022] The beneficial effects achieved by the present disclosure are at least:

[0023] The inventors of the present disclosure found in the research process that the TCP20-like gene has significant expression difference between the dormant period and the germination period, and the expression amount of the gene in the germination period is obviously higher than that in the dormant period. Therefore, by determining the expression amount of the TCP20-like gene, the poplar can be distinguished whether it is in the dormant period or the transformation period.

[0024] The inventors of the present disclosure found that the TCP20-like gene can promote the dormancy release of the poplar vascular cambium, and further promote the re-growth of the poplar stem. Therefore, the TCP20-like gene can be used as a marker gene for the dormancy release of the poplar vascular cambium. By overexpressing the TCP20-like gene in the plant, the dormancy state of the plant can be released. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a phenotype diagram of poplar buds in the dormant period and the germination period.

[0026] Figure 2 is a phenotype diagram of poplar vascular cambium in the dormant period and the germination period.

[0027] Figure 3 is a result diagram of the expression amount of the TCP20-like gene in the vascular cambium dormancy and dormancy release according to an embodiment of the present disclosure.

[0028] Figure 4 is a result diagram of the expression pattern of the TCP20-like gene in the annual cycle of poplar according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0029] The embodiments of the present disclosure are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present disclosure, and cannot be understood as a limitation of the present disclosure.

[0030] As used herein, the dormancy period of a poplar refers to a period of time during which the poplar stops the growth of the aboveground part and enters a survival adaptation stage with reduced physiological metabolism. For example, the dormancy period of a poplar can occur at a specific time period, such as starting after leaf fall in autumn and ending before bud break in the following spring.

[0031] As used herein, the bud break period of a poplar refers to a spring bud break period during which the poplar is in the bud break period.

[0032] The TCP transcription factor family is a class of plant-specific transcription factors that play an important role in plant growth and development, and is divided into Class I and Class II according to structural differences. For example, TCP20 is a Class I TCP transcription factor. Studies have shown that TCP20 is involved in the regulation of cell expansion, division and differentiation, and also has functions such as stimulating the cell cycle and organ growth. Overexpression of PtoTCP20 in Populus tomentosa can promote secondary growth of the plant, proliferate cambium cells, and increase xylem content. TCP-like genes refer to a class of genes that have structural or functional similarity to members of the TCP transcription factor family. As used herein, the TCP20-like gene refers to a class of genes in poplar that have structural or functional similarity to members of the TCP transcription factor family in the growth and development of poplar.

[0033] According to the specific embodiments, the sequence of the TCP20-like gene is shown in SEQ ID NO: 1.

[0034] ATGGAACCTAAGGGCCCAAATCATCATCAACTACAGGTGCCCTCTTTCTTGAATCCTCCACAAAAAGCAAGCATGTCAGAGAACAACATCAACAACCATAACAAGCAGCCTGCTGAGGTCAAAGATTTCCAAATTATGATTGAAAACAGAGATCATAACAAGAAGCAATTAGCACCAAAGAGAAGCTCCAACAAGGACAGGCACAAGAAAGTGGATGGTAGAGGAAGGAGGATAAGGATGCCAGCTCTTTGTGCGGCGAGGATTTTTCAACTGACGAGAGAACTGGGGAATAAATCTGATGGAGAAACAATTCAGTGGTTGTTGCAACAAGCAGAGCCATCGATTATTGCTGCGACTGGTACTGGGACTTTTCCTGCTTCAGCTCTTGCAGTTGCAGGGGCCTCTGTTTCAGAACAGGGGAACTCTGTTTCAACAGGATTGCATACAAAAATGGAAGGGTTGGGACCCGCTGTTGTTGGGTCCAGAGATAGGACTAACTGGACCATGATGAATACTAATTTAGGAAGATCTAATGTGGCAAGTGGGGTCTGGCCCTCTGTTGGCGGGGTTGGGTCAGGGTTTGTTCCAAATTCTGGTCAGTCAACATCGAATTTTGGAAATGAAAACTCCACTACTTTACCAAAGTATGGGTTCCATGGGGCTGAGTTGCCAAATATTAACATGGGATTAATGAGTTTTTACTCCATGTTTAGTGGCACTAACCAGCAATTCCCAGGATTGGAGCTTGGACTTTCACAGGATGGGCAAAGTGGGATGTTCAATCCTCAAGCTTTGAATCCCTTTTGCCAGCAGATGGTGCAGGGCCGTGGTGTCTTAAATTCGTTGAATCAAGAACAGCAGCAAGAGCAACCTCCTGATAAAGATGATTCCCAAGGATCAAGGCAGTAG (SEQ ID NO: 1)

[0035] The protein expressed by the TCP20-like gene is shown in SEQ ID NO: 2.

[0036] MEPKGPNHHQLQVPSFLNPPQKASMSENNINNHNKQPAEVKDFQIMIENRDHNKKQLAPKRSSNKDRHKKVDGRGRRIRMPALCAARIFQLTRELGNKSDGETIQWLLQQAEPSIIAATGTGTFPASALAVAGASVSEQGNSVSTGLHTKMEGLGPAVVGSRDRTNWTMMNTNLGRSNVASGVWPSVGGVGSGFVPNSGQSTSNFGNENSTTLPKYGFHGAELPNINMGLMSFYSMFSGTNQQFPGLELGLSQDGQSGMFNPQALNPFCQQMVQGRGVLNSLNQEQQQEQPPDKDDSQGSRQ (SEQ ID NO: 2)

[0037] The inventors found that the TCP20-like gene of poplar can be used to regulate the dormancy of poplar, for example, the dormancy process of poplar with increased expression of TCP20-like gene can be eliminated, thereby accelerating the growth of poplar.

[0038] The inventors found that the TCP20-like gene has significant expression difference between the dormancy period and the germination period, and the expression amount of the gene in the transformation period is significantly higher than that in the dormancy period, which indicates that the expression amount of the gene can be used to distinguish the dormancy period and the transformation period.

[0039] Herein, the poplar mentioned can be 84K poplar.

[0040] Herein, the significant difference in gene expression, or the significant difference in gene expression, refers to the statistically significant change in the expression amount of TCP20-like gene between the germination period and the dormancy period. Such significant change can be detected by the difference expression of the techniques commonly used in the art, such as mRNA difference display, RNA sequencing or fluorescence quantitative PCR. In addition, various data analysis methods can be combined, such as volcano plot, cluster analysis or time series analysis to reveal the expression dynamic pattern.

[0041] The results of real-time fluorescence quantitative PCR showed that compared with the dormancy period, the expression amount of TCP20-like gene began to increase significantly after low temperature treatment under long day conditions for 2 weeks, which indicated that TCP20-like gene can promote the dormancy breaking of vascular cambium of poplar, and further promote the re-growth of poplar stem.

[0042] The provided TCP20-like gene can be used as a marker gene for dormancy breaking of vascular cambium of poplar.

[0043] In this paper, the dormancy release or dormancy phenomenon can be seen through various aspects of poplar, such as changes in the apical bud and cambium of poplar, which can reflect whether the poplar is in dormancy or dormancy release. The apical bud of poplar can be directly observed to observe the dormancy or dormancy release of poplar, and the cambium can be observed by slicing (for example, the growth of the vascular cambium of poplar, which indicates dormancy release).

[0044] For example, as shown in Figure 1 . Figure 1 The phenotypic changes of the buds of poplar in the dormancy and germination periods are shown. Figure 2 The phenotypic changes of the vascular cambium of poplar in the dormancy and germination periods are shown. The phenotypic diagrams of the dormancy (10 weeks of short-day treatment) and germination (2 weeks of long-day treatment) periods of 84K poplar obtained by artificial induction are shown. In the bud phenotypic diagram, the buds in the dormancy period form dormant buds, and most of the leaves are dry and shed; in the germination period, the buds begin to crack, and new leaves begin to grow. In the cambium phenotypic diagram, the cambium layer of the dormancy period (10 weeks of short-day treatment) becomes less, the cell wall thickens, and the cambium cell layer of the germination period (2 weeks of long-day treatment) begins to gradually increase, and the cell wall begins to thin. The scale length is 20 μm. The technical solutions of the present application are described below through specific examples. It should be noted that these examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as a limitation on the scope of protection of the present application. Unless otherwise specified, the reagents used in the examples can be obtained by commercial purchase.

[0045] Example 1

[0046] Example 1 studies the expression of TCP20-like in the transcriptome of the vascular cambium of poplar.

[0047] 1. Obtaining of transcriptome material

[0048] The transcriptome material used in this study was derived from 4-year-old natural growing Populus tomentosa. The vascular cambium region of Populus tomentosa was sampled on January 21, 2017 and March 30, 2017, corresponding to the dormant period and the germination period of the annual development process of Populus tomentosa. At each sampling time point, three Populus tomentosa plants with consistent growth were selected. After stripping the bark at a height of 1.3 m above ground, a wood block (about 3 cm long and 1 cm wide) containing phloem, cambium and xylem was obtained from the trunk using an art knife. After marking, it was immediately transferred into liquid nitrogen and then into an ultra-low temperature refrigerator for storage. The vascular cambium part was separated using a freezing microtome. The specific steps are as follows: first, pre-cool the freezing microtome to-20°C in advance, and use a double-sided blade to modify the wood block to remove the excess phloem and xylem on the outside; then adjust the section thickness to 20 µm, make tangential sectioning, and observe the cross section with a microscope during the process to determine the position of the cambium; finally, discard the xylem and phloem materials, and collect the cambium region materials with tin foil paper and store them in an ultra-low temperature refrigerator.

[0049] 2. RNA extraction

[0050] The vascular cambium materials of Populus tomentosa at different periods were used to extract total RNA using EASYspin Plus polysaccharide polyphenol / complex plant RNA rapid extraction kit. The steps are as follows:

[0051] (1) The stored cambium materials at different periods were ground into powder using a grinder, then added to the lysis solution CLB (containing 5% β-mercaptoethanol), vortexed for 30 s to mix, shear DNA and reduce viscosity; after mixing, heat at 65°C for 10 min, invert 2-3 times during the water bath process to help lysis; then centrifuge at 13000 rpm for 10 min;

[0052] (2) Transfer the supernatant to a new centrifuge tube, add 0.5 times the volume of anhydrous ethanol, and mix well using a pipette gun;

[0053] (3) Transfer the mixture of the previous step to the genomic cleanup column, centrifuge at 13000 rpm for 2 min, and discard the waste liquid;

[0054] (4) Take out the genomic cleanup column and place it in a 2 mL centrifuge tube. Add 500 µL of lysis solution RLT Plus to the column, centrifuge at 13000 rpm for 30 s, then add 0.5 times the volume of anhydrous ethanol and mix well by blowing;

[0055] (5) The mixture of the previous step was transferred to the adsorption column RA, centrifuged at 13000 rpm for 2 min each time, and the waste liquid was discarded; 700 μL of deproteinized liquid RW1 was added to the column, and after standing for 1 min, it was centrifuged at 13000 rpm for 2 min, and the waste liquid was discarded;

[0056] (6) 500 μL of the rinsing liquid RW added with anhydrous ethanol was added, centrifuged at 13000 rpm for 30 s to discard the waste liquid, and repeated twice; then the adsorption column was placed in an empty collection tube and centrifuged for 2 min;

[0057] (7) The adsorption column RA was placed in an RNase free centrifuge tube, 40 μL of ddH2O was added to the adsorption membrane, and after standing for 1 min, it was centrifuged and eluted; the eluate was re-added to the adsorption column for enrichment by elution again; and after marking, it was stored at -80°C.

[0058] 3. RNA-seq library construction and data analysis

[0059] After the total RNA of different periods was detected and qualified, the library construction was carried out according to the standard process of Illumina sequencing. Specifically: mRNA was separated from the total RNA of the dormancy and germination layers of the cambium using Dynabeads combined with Oligo dT, and then the mRNA was randomly fragmented using a fragmentation buffer; the first and second strands of cDNA were continuously synthesized, and the cDNA was purified using AMPureXP beads; the constructed cDNA library was amplified by PCR, and after end repair, dA addition at the 3' end, ligation of sequencing adapters, and purification, it was qualified after QC inspection, and then sequenced using the Illumina HiSeq X-ten platform.

[0060] After data filtering, high-quality RNA-Seq data was aligned to the reference genome P. trichocarpa v3.0, and the positions were obtained using TopHat2. The gene expression was calculated using Cuffquant and Cuffnorm software, and the standardized FPKM value was used for K-means analysis to analyze the different expression patterns of the two stages.

[0061] 4. Expression amount of TCP20-like in the cambium transcriptome

[0062] By comparing and analyzing the expression amount in the transcriptome after sequencing, we found that the TCP20-like gene had a significant difference in expression between the dormancy and germination periods. Compared with the dormancy period, the expression amount of TCP20-like gene in the germination period increased significantly, and showed a significant difference from the dormancy period, as shown in Figure 3 The expression amount of the gene in the transformation period was significantly higher than that in the dormancy period, which indicated that the expression amount of the gene could be used to distinguish the dormancy period and the transformation period.

[0063] Example 2

[0064] Example 2 studied the expression pattern of TCP20-like in annual cycle activity of poplar stem.

[0065] 1. Artificial simulation of poplar annual cycle activity

[0066] The wild type (WT) 84K poplar in the laboratory was cut into 2 cm long stem segments and cultured on rooting medium for 45 days (16 h light / 8 h dark, 23°C). Then the poplar seedlings were taken out from the rooting medium with tweezers and transplanted into flowerpots, and cultured in the greenhouse (16 h light / 8 h dark, 21°C) for 45 days. Six poplars with consistent growth were selected for annual cycle activity simulation. To induce growth arrest and dormancy, the plants were transferred from the greenhouse to a light incubator and grown under short day (SD) photoperiod (8 h light, 23°C / 16 h dark, 16°C) for 10 weeks. To meet the low temperature requirement, the dormant plants were placed in a cold room at 4°C for 5 weeks. To induce bud break, the plants were transferred to a warm temperature and LD photoperiod (16 h light, 23°C / 8 h dark, 16°C).

[0067] The 10th stem node and the stem segments below of the treated plants were cut into 1 cm small pieces with scissors and placed in 5 ml centrifuge tubes. After labeling, they were stored in an ultra-low temperature freezer for subsequent experiments. A total of 2 time points were taken for sampling, three plants each time, as three parallel experiments. The sampling times were: when the plants were treated with short day for 10 weeks (dormant period); and when the plants were treated with long day for 2 weeks (germination period).

[0068] 2. RNA extraction of artificial annual cycle materials

[0069] The mortar and pestle for grinding were sterilized at 121°C for 20 min and then pre-cooled in liquid nitrogen. 0.5 g of stem segment material was poured into the mortar containing liquid nitrogen, and when the honeycomb appeared, it was quickly ground into powder. Then the EASYspin Plus polysaccharide polyphenol / complex plant RNA rapid extraction kit was used to extract total RNA. The specific steps are as above.

[0070] 3. Reverse transcription of total RNA

[0071] After extracting total RNA from the 7 period samples, the TransScript one-step method gDNA removal and first strand cDNA synthesis kit was used for reverse transcription. The reverse transcription system was prepared according to the following ratio as shown in Table 1:

[0072] Table 1. Ratio parameters

[0073] Reagent 20 μL system Total RNA 50 ng-2 µg Oligo(dT)20 primer 1 μL 2×TS Ⅱ reaction mixture 10 μL RT / RI enzyme mixture 1 μL gDNA Enzyme ]]> 1 μL RNase-free water To 20 μL

[0074] Gently mix the above system and incubate it in a PCR instrument at 42°C for 15 min. Then heat it at 85°C for 5 s to completely inactivate the RT / RIEnzyme and gDNA Remover. Store the product at -20°C.

[0075] 4. Real-time quantitative PCR

[0076] The cDNA generated by reverse transcription was diluted 10-fold and used as a template. qPCR experiments were performed according to the qPCR reaction system and conditions shown in Table 2.

[0077] Table 2 Reaction conditions

[0078] Reagent 20 μL system TransStart Top Green qPCR SuperMix 10 μL Forward primer (10 μM) 0.5 μL Reverse primer (10 μM) 0.5 μL Template cDNA (cDNA) 1 μL RNase-free water 8 μL

[0079] The forward primer sequence is shown in SEQ ID NO:3.

[0080] TGCGGCGAGGATTTTTCAAC (SEQ ID NO: 3)

[0081] The reverse primer sequence is shown in SEQ ID NO:4.

[0082] GGGTCCCAACCCTTCCATTT (SEQ ID NO:4)

[0083] A two-step reaction was performed, with the reaction program set to 94℃ for 30 s pre-denaturation, followed by 40 cycles of 94℃ for 5 s and 60℃ for 30 s. Finally, the melting curve was analyzed using the following program: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s, and 60℃ for 15 s. Each sample underwent three technical replicates and three biological replicates, with an independent internal control (Pag18S) added to each plate. After data export, expression levels were calculated using the 2-ΔΔCT method, with the expression level in the 45 DLD sample set to 1.

[0084] 5. Expression patterns of TCP20-like structures in the annual stem cycle of poplar trees

[0085] like Figure 4 The results, shown, were obtained by quantitatively analyzing the gene using 18S as an internal reference after samples were collected from 84K poplar trees in an artificially simulated natural environment during dormancy and budding stages in an incubator. The expression level of this gene during dormancy was used as a control (represented as 1), and the expression level during budding relative to dormancy (represented as 1) was compared. Real-time quantitative PCR results showed that, compared to dormancy, the expression level of this gene significantly increased after two weeks of long-day treatment following low-temperature treatment, consistent with transcriptome data. This indicates that the TCP20-like gene can promote the breaking of dormancy in the vascular cambium of poplar trees, thereby promoting the regrowth of poplar stems.

[0086] In summary, it is considered that the TCP20-like gene can be used as a marker gene for the release of dormancy of the vascular cambium of poplar.

[0087] In the description of the present specification, the description referring to the terms "one embodiment", "some embodiments", "an example", "the embodiments", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in the present specification and the features of the different embodiments or examples can be combined and integrated by those skilled in the art without contradiction, if necessary.

[0088] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. TCP20-like Application of the gene as a marker gene in the release of poplar vascular cambium dormancy, the TCP20- like The sequence of the gene is shown as SEQ ID NO:

1.

2. Use of a TCP20-like protein, the sequence of which is given in SEQ ID NO: 2, as a marker in the release of dormancy of the vascular cambium of poplar trees.

3. A method for determining the developmental stage of a poplar tree, characterized in that, Comprising: The expression amount of the gene is detected by taking the vascular cambium of a poplar tree, and the poplar tree is TCP20-like ​ based on TCP20-like the expression amount of the gene, determining the development stage of the poplar; wherein the poplar development stage is a dormancy stage of the poplar or a germination stage of the poplar; if the expression amount of the gene is not significantly different from the expression amount of the gene in the dormancy stage of the poplar, it is determined that the poplar is in the dormancy stage. TCP20-like wherein the poplar development stage is a dormancy stage of the poplar or a germination stage of the poplar; if the expression amount of the gene is not significantly different from the expression amount of the gene in the dormancy stage of the poplar, it is determined that the poplar is in the dormancy stage. If the above TCP20-like If the gene expression level is significantly different from that of poplar trees in the dormant period, then the poplar trees are determined to be in the budding stage. The significant difference referred to means that the gene expression is significantly increased.

4. The method of claim 3, wherein, The significant increase is at least 4 times the gene expression in poplar trees in the dormant phase.

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