Application of marker gene PtAP2R1 in identifying vegetative growth stage and reproductive growth stage of Pinus tabulaeformis

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a marker gene PtAP2R1 in identifying vegetative growth stages and reproductive growth stages of Pinus tabulaeformis. The marker gene PtAP2R1 is expressed in large amounts in the vegetative growth stage of Pinus tabulaeformis, and is expressed in small amounts in the reproductive growth stage of Pinus tabulaeformis, and then is continuously expressed at a low level, and the expression amount of the marker gene PtAP2R1 shows a very significant difference between the vegetative growth stage and the reproductive growth stage of Pinus tabulaeformis, and the growth and development stage of Pinus tabulaeformis can be identified according to the expression amount of the marker gene PtAP2R1. Further, the application can efficiently and accurately identify the growth and development stage (vegetative growth and reproductive growth) of Pinus tabulaeformis plants or even selected branches by comparing the expression amount of the marker gene PtAP2R1 in a to-be-tested sample of Pinus tabulaeformis with the expression amount of the marker gene PtAP2R1 in a sample of Pinus tabulaeformis in a known growth stage, thereby providing guidance for forestry production and forest management.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant genetic engineering, and particularly relates to a marker gene PtAP2R1 In the application of identifying the vegetative stage and the reproductive stage of Pinus tabuliformis. BACKGROUND

[0002] Pinus tabuliformis Carriere belongs to Pinaceae and is a needle evergreen arbor with the characteristics of cold tolerance, drought tolerance and poor soil tolerance, and is an important afforestation tree species in the vast northern region of China. After seed germination, Pinus tabuliformis needs to go through a vegetative stage and a reproductive stage, wherein the vegetative stage refers to the growth process from seed germination, seedling growth to the growth before reproduction. In the forestry production practice, the branch regeneration ability of the vegetative stage is strong, and the vegetative stage is more suitable for being selected as asexual propagation material for cutting, grafting, tissue culture and the like. After the tree enters the reproductive stage, the advantages and disadvantages of the growth, dryness and wood properties of the tree gradually appear, and therefore, the selection of excellent Pinus tabuliformis stands and germplasm resources, the determination of offspring trees and the like are mostly selected from the trees in the reproductive stage. However, for a long time, there are many difficulties in determining the vegetative stage and the reproductive stage of Pinus tabuliformis and the time of transition from the vegetative stage to the reproductive stage, and the in-vivo identification has not been achieved. SUMMARY

[0003] The application aims to provide a marker gene PtAP2R1 In the application of identifying the vegetative stage and the reproductive stage of Pinus tabuliformis, the marker gene PtAP2R1 provided by the application can be used for identifying the growth and development stage of Pinus tabuliformis plants or branches, and provides guidance for forestry production and tree management.

[0004] The application provides a marker gene PtAP2R1 In the application of identifying the vegetative stage and the reproductive stage of Pinus tabuliformis, the marker gene PtAP2R1 The encoded amino acid sequence is shown as SEQ ID NO. 1.

[0005] Preferably, the identification method comprises the following steps:

[0006] (1) selecting Pinus tabuliformis tissue materials of different ages grown in the same geographical environment as the reference sample set as the reference sample set; the Pinus tabuliformis tissue materials in the reference sample set comprise Pinus tabuliformis tissue materials in the vegetative stage and Pinus tabuliformis tissue materials in the reproductive stage;

[0007] detecting the expression amount of the marker gene PtAP2R1 in the reference sample set and the sample to be detected;

[0008] (2) sorting by tree age, selecting the Pinus tabulaeformis tissue material in which the expression of the marker gene PtAP2R1 is first significantly reduced as the control sample; the significant reduction is that the expression of the marker gene PtAP2R1 is less than 1 / 2 of the expression of the marker gene PtAP2R1 in the previous tree age reference sample in the reference sample set;

[0009] (3) comparing the expression of the marker gene PtAP2R1 in the to-be-tested sample and the control sample, identifying the growth and development stage of the to-be-tested sample;

[0010] The standard for identification is: if the expression of the marker gene PtAP2R1 in the to-be-tested sample is less than 1 / 2 of the expression of the marker gene PtAP2R1 in the control sample, it is determined that the to-be-tested sample is in the reproductive growth stage; if the expression of the marker gene PtAP2R1 in the to-be-tested sample is equal to or greater than 1 / 2 of the expression of the marker gene PtAP2R1 in the control sample, it is determined that the to-be-tested sample is in the vegetative growth stage.

[0011] Preferably, the Pinus tabulaeformis tissue material includes one or more of the cambium, sprout, branch, and leaf.

[0012] Preferably, the Pinus tabulaeformis tissue material includes the vascular cambium.

[0013] Preferably, the method for detecting the expression of the marker gene PtAP2R1 in the reference sample set and the to-be-tested sample includes: amplifying the marker gene PtAP2R1 using an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 4.

[0014] Preferably, the control sample includes 4-year-old Pinus tabulaeformis tissue material.

[0015] Preferably, the reference sample set includes 2-year-old to 150-year-old Pinus tabulaeformis tissue material.

[0016] Preferably, the reference sample set includes 2-year-old to 123-year-old Pinus tabulaeformis tissue material.

[0017] Preferably, the Pinus tabulaeformis tissue material in the vegetative growth stage includes 2-year-old to 6-year-old Pinus tabulaeformis tissue material; and the Pinus tabulaeformis tissue material in the reproductive growth stage includes 7-year-old to 123-year-old Pinus tabulaeformis tissue material.

[0018] Preferably, the vegetative growth stage pine tissue material includes 2- to 4-year-old pine tissue material; the reproductive growth stage pine tissue material includes 7- to 55-year-old pine tissue material.

[0019] This invention provides marker genes PtAP2R1 Application of the marker gene in differentiating between the vegetative and reproductive growth stages of Pinus tabuliformis PtAP2R1 The encoded amino acid sequence is shown in SEQ ID NO.1. The marker gene described in this invention... PtAP2R1 It is highly expressed during the vegetative growth stage of *Pinus tabuliformis*, tracely expressed during the reproductive growth stage, and then continues to be expressed at low levels. Its expression level shows a highly significant difference between the vegetative and reproductive growth stages. Based on the marker gene... PtAP2R1 The expression levels of marker genes can identify the growth and development stage of *Pinus tabuliformis*. Furthermore, this invention compares the expression levels of marker genes in *Pinus tabuliformis* test samples. PtAP2R1 Expression levels and marker genes in Pinus tabuliformis samples at known growth stages PtAP2R1 The expression level can efficiently and accurately identify the growth and development stage (vegetative growth and reproductive growth) of Pinus tabuliformis plants or even selected branches, providing guidance for forestry production and forest management. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Genetic set of reference samples of Pinus tabuliformis aged 5 to 123 years PtAP2R1 The amount of expression;

[0022] Figure 2 Genes in the test sample and control sample PtAP2R1 The amount of expression. Detailed Implementation

[0023] This invention provides marker genes PtAP2R1 Application of the marker gene in differentiating between the vegetative and reproductive growth stages of Pinus tabuliformis PtAP2R1 The encoded amino acid sequence is shown in SEQ ID NO.1.

[0024] The marker gene described in this invention PtAP2R1The encoded amino acid sequence is shown as SEQ ID NO. 1, specifically as follows: MRTTDTSSIEPVGEITPRSSEETESACVEAATVTHIQSNNEKRRRAPFRSEMAESGTSVSSVVNSITCNENINSLEHEASSKAMELDADVISSNSTQVPPSNEGNHKIFGFSLKIETRNLISRGNLLNAIANDEQQKDPACVTRHFFPLHNGEASQVLMPTECTEGNPFIESHWTPVTSRQPENAETRKKQPENNKPVKKSRRGPRSRSSQFRGVTFYRRTGRWESHIWDCGKQVYLGGFDTAHAAARAYDRAAIKFRGVEADINFTLTDYQEDLDQTSKLSKEEFVHILRRQSTGFSRGSSKYRGVTLHKCGRWEARMGQFLGKKYIYLGLFDSEEEAARAYDKAAIKCNGKEAVTNFDPSLYEKEILEERRESQTSTGCDQNLDLSLGIGHVPTQLKKSPSYKSRAEEDSQLKRIGGSTYSNHPQDSKVLSCLTSKWGENAVPEFQVLPRGHAVYWNSQVLQVPEAMKYKESEAHRSEDNAYEQTATSGSQKYPDHLGNCKKLNMMEDLKEQSFGVPASTVSFWPMKQQQSGWTWQLHARGPTSPVFTTNKSSSPGFSPQIHTAFVPSKHGSHNMGLQWAAQSTDPCMVQTPSPHLSTRLTNGTDSSDHMVGSNLDFPGKKWKRLSVQWAARSTDPCMVQTPSPHLSTRLTNGTDSSDHMVGSNLDFPGKKIKSLSVHRDK;

[0025] the marker gene PtAP2R1PtAP2R1 expressed in large amount in the vegetative growth stage of Pinus tabulaeformis and in trace amount in the reproductive growth stage of Pinus tabulaeformis, and then continuously expressed in low level, and the expression amount of the marker gene in the vegetative growth stage of Pinus tabulaeformis is extremely significantly different from that in the reproductive growth stage of Pinus tabulaeformis, and the growth development stage of Pinus tabulaeformis can be identified according to the expression amount of the marker gene. PtAP2R1

[0026] The method for identification in the present application preferably comprises: (1) selecting tissue materials of Pinus tabulaeformis of different ages grown in the same geographical environment as the sample to be tested as a reference sample set; the tissue materials of Pinus tabulaeformis in the reference sample set comprise tissue materials of Pinus tabulaeformis in the vegetative growth stage and tissue materials of Pinus tabulaeformis in the reproductive growth stage; selecting tissue materials of Pinus tabulaeformis grown in the same geographical environment as the tissue materials of Pinus tabulaeformis in the reference sample set as the sample to be tested; and detecting the expression amount of the marker gene in the reference sample set and the sample to be tested. PtAP2R1

[0027] The tissue materials of Pinus tabulaeformis in the present application preferably comprise cambium, sprout, branch or leaf, and further preferably comprise vascular cambium. The reference sample set in the present application preferably comprises tissue materials of Pinus tabulaeformis of 2 to 150 years old, and further preferably comprises tissue materials of Pinus tabulaeformis of 2 to 123 years old. The tissue materials of Pinus tabulaeformis in the vegetative growth stage in the present application preferably comprise tissue materials of Pinus tabulaeformis of 2 to 6 years old, and further preferably comprise tissue materials of Pinus tabulaeformis of 2 to 4 years old, and more preferably comprise tissue materials of Pinus tabulaeformis of 2, 3 and 4 years old. The tissue materials of Pinus tabulaeformis in the reproductive growth stage preferably comprise tissue materials of Pinus tabulaeformis of 7 to 123 years old, and further preferably comprise tissue materials of Pinus tabulaeformis of 7 to 55 years old, and more preferably comprise tissue materials of Pinus tabulaeformis of 7, 13, 20, 46 and 55 years old. Pinus tabulaeformis usually spends 5 to 7 years in its infancy, and forms embryos and seeds for the first time after 7 years. In actual production, Pinus tabulaeformis after 7 years is referred to as the reproductive growth stage, and Pinus tabulaeformis before 7 years is referred to as the vegetative growth stage. The marker gene PtAP2R1 is expressed in large amount in Pinus tabulaeformis samples of 2 to 4 years old in the vegetative growth stage, and is expressed in extremely significantly reduced amount in Pinus tabulaeformis samples of 7 to 123 years old in the reproductive growth stage, and can effectively reflect and identify the growth development stage of Pinus tabulaeformis, and can be used as a molecular marker for identifying the growth development stage of Pinus tabulaeformis.

[0028] In the present application, the method for detecting the expression amount of the marker gene in the reference sample set and the sample to be tested preferably comprises: amplifying the marker gene by using an upstream primer and a downstream primer. PtAP2R1 PtAP2R1 ​​​; the nucleotide sequence of the upstream primer is preferably as shown in SEQ ID NO. 3, and the nucleotide sequence of the downstream primer is preferably as shown in SEQ ID NO. 4. The present application preferably utilizes Pinus tabulaeformis 18sRNA as the internal reference gene, and the nucleotide sequence of the upstream primer for amplifying the Pinus tabulaeformis internal reference gene 18sRNA is preferably as shown in SEQ ID NO. 5, and the nucleotide sequence of the downstream primer is preferably as shown in SEQ ID NO. 6.

[0029] (2) The marker genes in the reference sample set are sorted according to the tree age, and the Pinus tabulaeformis tissue material in which the expression amount of the marker gene PtAP2R1 is first significantly reduced is selected as the control sample, wherein the significant reduction refers to the expression amount of the marker gene PtAP2R1 being less than 1 / 2 of the expression amount of the marker gene PtAP2R1 in the previous tree age reference sample in the reference sample set. The transition of the vegetative growth stage to the reproductive growth stage of the Pinus tabulaeformis generally occurs at 4-7 years, and in the present application, the control sample preferably comprises 4-year-old Pinus tabulaeformis tissue material.

[0030] (3) The expression amount of the marker gene PtAP2R1 in the test sample and the control sample is compared, and the growth and development stage of the test sample is identified; the identification standard is preferably that if the expression amount of the marker gene PtAP2R1 in the test sample is less than 1 / 2 of the expression amount of the marker gene PtAP2R1 in the control sample, it is determined that the test sample is in the reproductive growth stage; if the expression amount of the marker gene PtAP2R1 in the test sample is equal to or greater than 1 / 2 of the expression amount of the marker gene PtAP2R1 in the control sample, it is determined that the test sample is in the vegetative growth stage.

[0031] In order to further illustrate the present application, the application of the marker genes PtAP2R1 provided by the present application in identifying the vegetative growth stage and the reproductive growth stage of Pinus tabulaeformis is described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0032] Example 1

[0033] Determination of the expression amount of the marker gene PtAP2R1

[0034] (1) Collection of the reference sample set

[0035] ​The cambium of Pinus tabulaeformis at a distance of 15-20 cm from the base of the trunk was collected at 2 years, 3 years, 4 years, 7 years, 13 years, 20 years, 46 years, 55 years and 123 years old in Shangzhuang Forest Farm (111°10'E, 36°48'N, altitude 1570-1930 m) in Xixian County, Linfen City, Shanxi Province, and immediately frozen with liquid nitrogen after collection, and stored in a-80°C ultra-low temperature refrigerator.

[0036] In production practice and related research, Pinus tabulaeformis undergoes the transition from vegetative growth to reproductive growth between 5 years and 7 years, and the 2-year-old, 3-year-old and 4-year-old Pinus tabulaeformis in the present application are in the vegetative growth stage, and the 7-year-old, 13-year-old, 20-year-old, 46-year-old, 55-year-old and 123-year-old Pinus tabulaeformis are in the reproductive growth stage.

[0037] (2) Extraction of total RNA from reference sample set

[0038] The total RNA of the standard sample collected above was extracted by using the RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit (DP441) of Tiangen Biosciences Technology (Beijing) Co., Ltd. The RNA concentration and integrity were determined by Nano microspectrophotometer and 1% agarose gel electrophoresis, respectively.

[0039] (3) Reverse transcription of total RNA from reference sample set

[0040] In an RNase free PCR tube, the reverse transcription reaction of the total RNA obtained in step (2) was completed, and the genomic DNA was removed. The reverse transcription system was prepared according to Table 1:

[0041] Table 1 Reverse transcription system

[0042]

[0043] After gently mixing the reaction system in Table 1, incubate at 42°C for 30 min; heat at 85°C for 5 s to inactivate TransScript RT / RI and gDNA Remover, and store the cDNA at-20°C.

[0044] (4) Primer design

[0045] The primers of Pinus tabulaeformis internal reference genes 18sRNA and gene PtAP2R1 (SEQ ID NO. 2) were designed by using the Primer-BLAST and NCBI Conserved Domain Search of NCBI primer online design website, and the primers of gene 18sRNA and gene PtAP2R1 The primer sequences are as follows:

[0046] PtAP2R1-F (SEQ ID NO. 3): 5'-CCTCTTCATAACGGGGAGGC-3';

[0047] PtAP2R1-R (SEQ ID NO. 4): 5'-GCATTCTCTGGTTGACGGGA-3';

[0048] 18sRNA-F (SEQ ID NO. 5): 5'-CGGCTACCACATCCAAGGAA-3';

[0049] 18sRNA-R (SEQ ID NO. 6): 5'-GCTGGAATTACCGCGGCT-3'.

[0050] (5) Gene PtAP2R1 Real-time fluorescent quantitative PCR (qRT-PCR) detection experiment

[0051] The reverse transcription product cDNA in step (3) was diluted 50 times as a template, and the gene was detected according to the fluorescent quantitative PCR amplification system in Table 2 below. PtAP2R1 The expression amount in the standard sample collected in step (1), the reagent used in the amplification system and the amount used refer to the TransScript One-Step gDAN Removal and cDNASynthesis SuperMix TransScript kit instruction book of the full style gold company, each sample was repeated 3 times in technology and 3 times in biology, and 18sRNA was used as an internal reference for each plate sample.

[0052] Table 2 qRT-PCR reaction system

[0053]

[0054] The reaction was carried out by SYBR Green intercalation fluorescence method, the pre-denaturation reaction program was set to 95℃ for 2min; the PCR reaction program was set to 94℃ for 5s, 60℃ for 30s, 39 cycles; the termination extension reaction program was set to 94℃ for 5s; the melting curve analysis reaction program was set to increase by 0.5℃ every 5s from 65~95℃.

[0055] Gene Figure 1 The expression amount in the reference sample set is shown as follows: the gene PtAP2R1 was highly expressed in 2-year-old and 3-year-old Pinus tabulaeformis, the expression amount of the gene PtAP2R1 in 2-year-old Pinus tabulaeformis was recorded as 1, and the expression amount of the gene PtAP2R1 in 3-year-old Pinus tabulaeformis was 1.35; the gene PtAP2R1 was highly expressed in 2-year-old and 3-year-old Pinus tabulaeformis, the expression amount of the gene PtAP2R1The expression amount in 4-year-old Pinus tabulaeformis was significantly decreased, and the expression amount was 0.34; the expression amount in 7-123-year-old Pinus tabulaeformis was trace, and the expression amount was not more than 0.1. It can be seen that the expression amount of the gene PtAP2R1 The expression amount difference in the process of the transition from the vegetative growth to the reproductive growth is extremely significant, and can effectively reflect and identify the growth and development stage of Pinus tabulaeformis.

[0056] Example 2

[0057] (1) Control sample selection and identification standard setting

[0058] The result of step (5) of Example 1 reflects that the expression amount difference of the marker gene PtAP2R1 in the 4-year-old Pinus tabulaeformis before and after 4 years is extremely significant, so the 4-year-old Pinus tabulaeformis in the vegetative growth stage is selected as the control sample for judging the growth and development stage of Pinus tabulaeformis. If the expression amount of the marker gene PtAP2R1 in the to-be-tested sample is less than 1 / 2 of the expression amount of the marker gene PtAP2R1 in the control sample, it is determined that the to-be-tested sample is in the reproductive growth stage; if the expression amount of the marker gene PtAP2R1 in the to-be-tested sample is equal to or greater than 1 / 2 of the expression amount of the marker gene PtAP2R1 in the control sample, it is determined that the to-be-tested sample is in the vegetative growth stage.

[0059] (2) Collection of the to-be-tested sample and the control sample

[0060] Branches on Pinus tabulaeformis afforestation elite trees are randomly collected, and the vascular cambium thereof is scraped to obtain a to-be-tested sample; at the same time, 4-year-old Pinus tabulaeformis with known tree age in the same geographical location and growth environment is selected, and the cambium at the base of the trunk 15-20 cm from the ground is collected as a control sample. The above materials are immediately frozen with liquid nitrogen after collection, and are stored in a-80℃ ultra-low temperature refrigerator.

[0061] (3) Extraction of total RNA of the to-be-tested sample and the control sample

[0062] The extraction of total RNA of the to-be-tested sample and the control sample is the same as the method in step (2) of Example 1.

[0063] (4) Reverse transcription of total RNA of the to-be-tested sample and the control sample

[0064] The reverse transcription of total RNA of the to-be-tested sample and the control sample is the same as the method in step (3) of Example 1.

[0065] (5) PtAP2R1 Real-time fluorescent quantitative PCR (qRT-PCR) detection of the gene

[0066] Figure 2The real-time quantitative PCR (qRT-PCR) detection of the gene was performed using the same method as in step (5) of Example 1. The results are as follows: PtAP2R1 As shown, the genes in the control sample PtAP2R1 The expression level is recorded as 1, and the gene expression level in the sample to be tested is 1. PtAP2R1 The expression level was 0.31. The gene in the test sample... ​ The expression level of the sample was less than half that of the control sample. Therefore, the developmental stage of the sample was determined to be the reproductive growth stage.

[0067] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Marker gene PtAP2R1 In the application of identifying the age of Pinus tabulaeformis, the marker gene PtAP2R1 The encoded amino acid sequence is shown as SEQ ID NO. 1; The method comprises: (1) selecting Pinus tabulaeformis tissue materials of different ages grown in the same geographical environment as the reference sample set; The Pinus tabulaeformis tissue materials in the reference sample set comprise Pinus tabulaeformis tissue materials in the vegetative growth stage and Pinus tabulaeformis tissue materials in the reproductive growth stage; detecting the expression level of the marker genes in the reference sample set and the sample to be tested PtAP2R1 ; (2) sorting by tree age, selecting the tissue material of Pinus tabulaeformis of the reference sample set, in which the expression of the marker gene PtAP2R1 is first significantly reduced as a control sample; the significant reduction is that the expression of the marker gene PtAP2R1 is less than 1 / 2 of the expression of the marker gene PtAP2R1 in the previous tree age reference sample in the reference sample set; the control sample is the tissue material of Pinus tabulaeformis of 4 years old; (3) Compare the marker genes in the test sample and the control sample. PtAP2R1 The expression level of the tree can be used to identify the tree age of the test sample. The criterion for the identification is: if the expression amount of the marker gene PtAP2R1 in the sample to be tested is less than 1 / 2 of the expression amount of the marker gene PtAP2R1 in the control sample, it is determined that the sample to be tested is 7-123 years old. If the marker gene is present in the sample to be tested PtAP2R1 The expression level is equal to or greater than that of the marker gene in the control sample. PtAP2R1 If the expression level is half, the tree age of the sample to be tested is determined to be 2 or 3 years.

2. Use according to claim 1, characterized in that, The Pinus tabulaeformis tissue materials comprise one or more of the cambium, sprouts, branches and leaves.

3. Use according to claim 2, characterized in that, The Pinus tabulaeformis tissue materials comprise the vascular cambium.

4. Use according to claim 1, characterized in that, Detecting the marker genes in the reference sample set and the test sample PtAP2R1 Methods for measuring expression levels include: amplifying the marker gene using upstream and downstream primers. PtAP2R1 The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

4.

5. The use according to claim 1, characterized in that, The reference sample set comprises Pinus tabulaeformis tissue materials of 2 to 150 years old.

6. Use according to claim 5, characterized in that, The reference sample set comprises Pinus tabulaeformis tissue materials of 2 to 123 years old.

7. Use according to claim 6, characterized in that, The Pinus tabulaeformis tissue materials in the vegetative growth stage comprise Pinus tabulaeformis tissue materials of 2 to 6 years old; and the Pinus tabulaeformis tissue materials in the reproductive growth stage comprise Pinus tabulaeformis tissue materials of 7 to 123 years old.

8. Use according to claim 7, characterized in that, The Pinus tabulaeformis tissue materials in the vegetative growth stage comprise Pinus tabulaeformis tissue materials of 2 to 4 years old; and the Pinus tabulaeformis tissue materials in the reproductive growth stage comprise Pinus tabulaeformis tissue materials of 7 to 55 years old.

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