Application of PtoERD3 gene structure variation in evaluation of poplar lignin content

Abstract

The application discloses application of a PtoERD3 gene structure variation in evaluation of poplar lignin content, wherein the structure variation is located in a promoter region of a poplar PtoERD3 gene, is a 54bp chromosome structure variation SV fragment, and has a nucleotide sequence as shown in SEQ ID NO. 1. The structure variation can be used to accurately determine the lignin content of the poplar, can be used to accurately and efficiently screen excellent plants with high-quality wood quality traits in the early growth stage of the poplar, effectively shortens a breeding cycle, and provides theoretical support for molecular design breeding of poplar wood quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, in particular to a molecular marker related to poplar lignin content, and especially to application of PtoERD3 gene structural variation in evaluating poplar lignin content. BACKGROUND

[0002] Populus tomentosa is an important fast-growing timber tree species in northern China, and the lignin content and composition in its wood directly affect the pulping efficiency and biomass energy conversion potential. In recent years, structural variation (SV) has become an important entry point for analyzing the mechanism of poplar wood quality due to its significant impact on gene expression regulation and phenotypic diversity.

[0003] Structural variation involves deletion, duplication, inversion, translocation, etc. of large fragments (≥50bp) of chromosomes, which can affect multiple genes or regulatory elements, leading to changes in gene expression, protein structure changes or chromosome rearrangements, and often more significant and diverse effects on complex traits. Lignin is a key component of plant secondary cell walls, and its biosynthesis is regulated by enzymes such as cinnamyl alcohol dehydrogenase (CAD) and coffee acyl-CoA methyltransferase (CCoAOMT) in the phenylpropanoid metabolic pathway. The significant differences in lignin content and composition in natural populations of P. tomentosa indicate that there is a complex molecular mechanism in the genetic regulatory network.

[0004] Currently, related research lacks functional analysis of lignin-related SV. Therefore, with the continuous development of modern sequencing and molecular breeding technologies, it is necessary to develop key SV molecular markers that can affect lignin content and determine the genetic effects of key SV molecular markers on wood properties of trees, in order to quickly and accurately screen new forest germplasm with excellent wood quality and provide theoretical support for molecular design breeding of poplar wood quality. SUMMARY

[0005] In order to overcome the above problems, the present inventors, based on the strategy of genome-wide association analysis, relying on the P. tomentosa germplasm resource population, used GEMMA (v0.98.5) software to detect SV sites significantly related to lignin content at the whole genome level, and detected a 54bp insertion variation SV fragment located in the promoter region of PtoERD3 gene, which has a nucleotide sequence as shown in SEQ ID NO. 1, or a nucleotide sequence with 90% or more homology to the nucleotide sequence. Using the insertion variation SV fragment as a molecular marker for detecting the lignin content of poplar, the lignin content of poplar can be accurately judged, and high-quality wood quality traits of superior trees can be accurately and efficiently screened in the early growth stage of poplar, significantly shortening the breeding cycle, providing theoretical support for molecular design breeding of poplar wood quality, thereby completing the present application.

[0006] Specifically, the present application aims to provide the following aspects:

[0007] In a first aspect, a molecular marker related to the content of poplar lignin is provided, which is a 54bp chromosome structure variation SV fragment,

[0008] The chromosome structure variation SV fragment has a nucleotide sequence as shown in SEQ ID NO. 1, or a nucleotide sequence with 90% or more homology to the nucleotide sequence.

[0009] In a second aspect, a primer pair for detecting the molecular marker of the first aspect is provided, which comprises primer P1 and primer P2, the primer P1 has a sequence as shown in SEQ ID NO. 3, and the primer P2 has a sequence as shown in SEQ ID NO. 4.

[0010] In a third aspect, the application of the molecular marker of the first aspect in detecting the content of poplar lignin is provided, which comprises the following steps:

[0011] First, the genomic DNA of the poplar to be tested is used as a template for PCR amplification;

[0012] Then, according to the amplification product, it is determined whether the molecular marker is contained, and then the content of poplar lignin is determined.

[0013] In a fourth aspect, the application of the molecular marker of the first aspect in regulating the content of poplar lignin is provided, and the regulation of the content of poplar lignin by the molecular marker is achieved by overexpressing PtoERD3 gene or silencing PtoERD3 gene,

[0014] The content of lignin is reduced by overexpressing PtoERD3 gene in poplar plants, and the content of lignin is increased by silencing PtoERD3 gene in poplar plants.

[0015] In a fifth aspect, a method for genetic improvement of poplar is provided, which is to increase the content of poplar lignin, and the method comprises the steps of subculturing and breeding poplar individuals without the molecular marker of the first aspect, and eliminating poplar individuals with the molecular marker.

[0016] The present application has the following beneficial effects:

[0017] (1) The molecular marker related to the content of poplar lignin provided by the present application is located in the upstream promoter region of the PtoERD3 gene of poplar, which negatively regulates the content of poplar lignin, and by determining the structure variation, the content of poplar lignin can be accurately determined, and high-quality wood quality traits of superior plants can be accurately and efficiently screened in the early growth stage of poplar, and the breeding cycle is significantly shortened.

[0018] (2) The primer pair for detecting the molecular marker provided by the application can be effectively used in molecular marker assisted breeding, thereby realizing short time, low cost and high accuracy screening of excellent tree species with high lignin content;

[0019] (3) The application of the molecular marker in regulating the lignin content of poplar is realized by overexpressing PtoERD3 gene or silencing PtoERD3 gene, and the PtoERD3 gene silencing mutant can significantly increase the lignin content of poplar, which has important significance for breeding poplar plants with excellent wood quality traits and innovation of poplar germplasm resources. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The position of the most significant SV in the PtoERD3 gene promoter region in Example 1 is shown; Figure 2 The verification electrophoresis map of the candidate individual SV in Example 2 is shown; Figure 3 The fluorescence signal result map of the transient transcription SV in Example 3 is shown; Figure 4 The quantitative analysis result of the relative fluorescence activity of the transient transcription SV in Example 3 is shown; Figure 5 The RT-qPCR detection of the transcription level of PtoERD3 in the overexpression plants (OE-2, OE-3, OE-5) in Example 6 is shown, wherein the error line represents the standard deviation; Figure 6 The RT-qPCR detection of the transcription level of PtoERD3 in the interference silencing plants (RNAi1, RNAi6, RNAi8) in Example 6 is shown, wherein the error line represents the standard deviation; Figure 7 The phenotype of the interference silencing plant (denoted as RNAi-PtoERD3), the phenotype of the wild type poplar (denoted as WT) and the phenotype of the PtoERD3 overexpression plant (denoted as OE-PtoERD3) are shown; Figure 8 The plant height of RNAi-PtoERD3, WT and OE-PtoERD3 is shown; Figure 9 The ground diameter of RNAi-PtoERD3, WT and OE-PtoERD3 is shown; Figure 10 The lignin content of RNAi-PtoERD3, WT and OE-PtoERD3 is shown. DETAILED DESCRIPTION

[0021] The application will be further described in detail through preferred embodiments and examples. Through these descriptions, the features and advantages of the application will become more apparent.

[0022] The word "exemplary" is used herein in the sense of being an example, not necessarily a preferred example. Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

[0023] The present inventors have found that, as an important member of a plant stress response gene family, the ERD3 gene (Early Responsive to Dehydration 3 gene) exhibits unique functions in the regulation of lignin metabolism in poplar, and the gene can negatively regulate the lignin content and improve the wood quality traits of poplar.

[0024] Therefore, in order to quickly and accurately screen new forest germplasm with excellent wood varieties, the present application provides a molecular marker related to the lignin content of poplar, which is a 54bp chromosomal structural variation SV fragment,

[0025] The chromosomal structural variation SV fragment has a nucleotide sequence as shown in SEQ ID NO. 1, or a nucleotide sequence with 90% or more homology to the nucleotide sequence.

[0026] Preferably, the nucleotide sequence of the chromosomal structural variation SV fragment is as shown in SEQ ID NO. 1.

[0027] According to a preferred embodiment of the present application, the molecular marker is located in the upstream promoter region of the PtoERD3 gene of poplar, preferably the P. tomentosa, and the sequence of the upstream promoter of the PtoERD3 gene is as shown in SEQ ID NO. 2.

[0028] Among them, the P. tomentosa in the P. alba group is a native tree species unique to China, widely distributed, with the central distribution area being the middle and lower reaches of the Yellow River, and plays an important role in forestry production and ecological environment construction in northern China, and is a pioneer tree species for forest cultivation in northern China, so the P. tomentosa is preferably selected as the research object in the present application.

[0029] In a further preferred embodiment, the molecular marker is located at the 1725bp of the nucleotide sequence shown in SEQ ID NO. 2.

[0030] Based on the strategy of whole genome association analysis, the present inventors identified a structural variation (SV) related to the lignin content of poplar, which negatively regulates the lignin content of poplar.

[0031] According to a preferred embodiment of the present application, when the structural variation exists in the upstream promoter region of the PtoERD3 gene of poplar, the lignin content is low; when the structural variation does not exist in the upstream promoter region of the PtoERD3 gene of poplar, the lignin content is high.

[0032] Therefore, by determining the structural variation, the lignin content of the poplar can be accurately judged, and the superior plants with high-quality wood quality traits can be accurately and efficiently screened in the early growth stage of the poplar, and the breeding cycle is significantly shortened.

[0033] According to a preferred embodiment of the present application, the molecular marker is obtained by a method comprising the following steps:

[0034] Firstly, the SV site and the lignin content of the poplar are subjected to association analysis, and a significantly associated SV site is obtained;

[0035] Then, the obtained significantly associated SV site is annotated, and the position thereof is obtained.

[0036] In a further preferred embodiment, the GEMMA software is used to perform SVGWAS analysis on 49380 high-confidence SVs and the lignin content of 302 poplar materials, and a PCA matrix is added to correct the population structure, 38 significant SV sites are screened, the most significantly associated site is annotated to the promoter region of PtoERD3 (p=1.02E-8), and specifically, an insertion type structural variation with a size of 54 bp located at the 1725th bp of the nucleotide sequence shown in SEQ ID NO. 2.

[0037] According to a preferred embodiment of the present application, the method further comprises the step of verifying the obtained SV.

[0038] Preferably, the verification is that genomic DNA of a plurality of individuals randomly selected from a poplar germplasm resource library is extracted to verify the SV, and then the lignin content of the selected individuals is evaluated.

[0039] In the present application, the poplar germplasm resource library is preferably a poplar hybrid population germplasm resource library planted in Guan County, Shandong, and the number of selected individuals is 24.

[0040] It is found through verification that the lignin content of a non-variant individual (i.e., an individual without the SV fragment) is 22.3±0.6%, when the candidate individual is a homozygous mutant, the lignin content of the candidate poplar individual is the lowest, which is reflected in that the lignin content of the candidate individual (18.2±1.2%) is 18.39% lower than that of the non-variant (22.3±0.6%); when the candidate individual is a heterozygous mutant, the lignin content (18.89±0.6%) is 15.29% lower than that of the non-variant. Among them, non-variant refers to an individual carrying a homozygous genotype without the SV, i.e., both alleles do not contain the SV (wild type homozygous); homozygous variation refers to an individual carrying a homozygous genotype of structural variation (SV), i.e., both alleles contain the SV; heterozygous variation refers to an individual carrying alleles with and without SV, forming a heterozygous genotype.

[0041] That is, the homozygous mutant plant with SV variation has lignin content reduced by 18.39% compared with the homozygous plant without SV variation; the heterozygous mutant plant has lignin content reduced by 15.29% compared with the homozygous plant without SV variation.

[0042] Further, since the molecular marker in the application is located in the promoter region of PtoERD3 gene, in order to determine whether the insertion variation of the promoter affects the promoter activity, the double luciferase experiment is preferably used for verification.

[0043] The verification shows that the SV can significantly enhance the transcription of PtoERD3 and positively regulate the expression of PtoERD3.

[0044] In the second aspect of the application, a primer pair for detecting the molecular marker in the first aspect is provided, and the primer pair comprises primer P1 and primer P2, the primer P1 has the sequence shown in SEQ ID NO. 3, and the primer P2 has the sequence shown in SEQ ID NO. 4.

[0045] In the application, the primer pair can be used to effectively perform PCR amplification on the molecular marker related to the lignin content of poplar, and then the detection of the molecular marker is realized through electrophoresis.

[0046] In the application, in order to exclude the quality problem of the DNA template of the poplar to be detected and improve the accuracy of detection, the primer P1 and the primer P2 are preferably designed according to the upstream and downstream sequences of the SV position to obtain an amplified product with a specific length.

[0047] Preferably, the primer pair in which 1-20 bases are added to the 5' end and the 3' end of the nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 respectively and the same DNA fragment (the DNA sequence between the upstream primer and the downstream primer is the same) can be obtained is included in the primer pair of the application.

[0048] According to a preferred embodiment of the application, the genomic DNA of the poplar to be detected is used as a template, and the primer pair P1 and P2 are used for PCR amplification, and then the amplified product is identified,

[0049] Preferably, when the amplified product contains the molecular marker in the first aspect, the lignin content of the poplar is low; when the amplified product does not contain the molecular marker in the first aspect, the lignin content of the poplar is high.

[0050] More preferably, electrophoretic identification is performed on the amplification product, when a single band of 308bp is amplified, it indicates that the plant has the structural variation, and the poplar lignin content is the lowest; when a single band of 254bp is amplified, it indicates that the plant does not have the structural variation, and the poplar lignin content is the highest; when double bands of 308bp and 254bp are amplified, it indicates that the plant is a hybrid mutation, and the poplar lignin content is between the above two.

[0051] Therefore, the above primer pair for detecting the molecular marker can be effectively used for molecular marker assisted breeding, and thus high-quality tree species with high lignin content can be screened in a short time, at low cost and with high accuracy.

[0052] In a third aspect, the application provides application of the molecular marker in the first aspect in detection of the poplar lignin content.

[0053] Preferably, the application comprises the following steps:

[0054] Firstly, the genomic DNA of the poplar to be detected is used as a template to perform PCR amplification.

[0055] Preferably, the primer pair used in the PCR amplification is primer P1 and primer P2.

[0056] Then, according to the amplification product, it is determined whether the molecular marker is contained, and thus the lignin content of the poplar is determined.

[0057] Preferably, it is detected whether the amplification product has the SV structural variation in the first aspect, if not, the poplar lignin content is high; if yes, the poplar lignin content is low.

[0058] According to a preferred embodiment of the application, electrophoretic analysis is performed on the amplification product, and three electrophoretic results are obtained: a single band of 308bp, a single band of 254bp and double bands of 308bp / 254bp.

[0059] When a single band of 308bp is amplified, it is determined that the poplar lignin content is the lowest; when a single band of 254bp is amplified, it is determined that the poplar lignin content is the highest; when double bands of 308bp and 254bp are amplified, it is determined that the poplar lignin content is between the above two.

[0060] In a fourth aspect, the application provides application of the molecular marker in the first aspect in regulation of the poplar lignin content.

[0061] As mentioned above, the molecular marker of the present application can significantly enhance the transcription of PtoERD3, positively regulate the expression of PtoERD3, and thus, the regulation of the content of lignin in poplar by the molecular marker is achieved by overexpressing PtoERD3 gene or silencing PtoERD3 gene, i.e., reducing the content of lignin by overexpressing PtoERD3 gene in poplar plants, and increasing the content of lignin by silencing PtoERD3 gene in poplar plants.

[0062] According to a preferred embodiment of the present application, the PtoERD3 gene comprises the nucleotide sequence as shown in SEQ ID NO. 5, preferably the nucleotide sequence of the PtoERD3 gene is as shown in SEQ ID NO. 5.

[0063] According to a preferred embodiment of the present application, the method for reducing the content of lignin in poplar by the molecular marker comprises the following steps:

[0064] Step 1, constructing an overexpression vector of PtoERD3 gene, transforming into Agrobacterium to obtain positive overexpression engineering bacteria.

[0065] According to a preferred embodiment of the present application, the overexpression vector is obtained by constructing the CDS sequence of PtoERD3 gene on pBI121 vector, and the CDS sequence of PtoERD3 gene is as shown in SEQ ID NO. 6.

[0066] In a further preferred embodiment, the CDS sequence of PtoERD3 gene is obtained by overexpression primers P3 and P4, and the sequence of the overexpression primer P3 is as shown in SEQ ID NO. 7, and the sequence of the overexpression primer P4 is as shown in SEQ ID NO. 8.

[0067] Step 2, using the positive overexpression engineering bacteria to infect poplar materials, and after cultivation and identification, obtaining overexpression positive seedlings.

[0068] Preferably, the callus method is used to infect the poplar materials.

[0069] According to a preferred embodiment of the present application, the identification of the overexpression positive seedlings comprises DNA level identification and transcription level identification.

[0070] In a further preferred embodiment, the DNA level identification is PCR identification, and the reaction procedure is as follows: 95℃ for 2 min; (94℃ for 30 s; 58℃ for 30 s; 72℃ for 60 s) for 35 cycles; 72℃ for 5 min; 4℃ for ∞.

[0071] The plant identified by PCR is a PtoERD3 gene overexpression plant.

[0072] According to a preferred embodiment of the present application, the identification of the transcription level is performed by an RT-qPCR experiment, and a primer pair of PtoERD3-qPCR-F and PtoERD3-qPCR-R is used, wherein the sequence of the primer PtoERD3-qPCR-F is shown as SEQ ID NO. 9, and the sequence of the primer PtoERD3-qPCR-R is shown as SEQ ID NO. 10.

[0073] In a further preferred embodiment, the reaction procedure for the identification of the transcription level is as follows: 95℃ for 3 min; (95℃ for 5 sec; 60℃ for 30 sec; 72℃ for 15 sec) for 40 cycles; 72℃ for 5 min; 4℃ for ∞.

[0074] In the present application, through the detection of the transcription level, it is found that the overexpression plants numbered as OE-2, OE-3 and OE-5 have a higher expression, which is 99.5 times, 151.3 times and 130 times of the wild type plant (WT), respectively.

[0075] Step 3: transplanting the overexpression positive seedlings to obtain poplar plants with reduced lignin content.

[0076] According to a preferred embodiment of the present application, the method for improving the lignin content of poplar by molecular markers comprises the following steps:

[0077] Step i: constructing an interference silencing vector of the PtoERD3 gene, and transferring into Agrobacterium to obtain positive gene silencing engineering bacteria;

[0078] Step ii: infecting poplar materials with the positive gene silencing engineering bacteria, and culturing the transgenic seedlings to identify the gene silencing positive seedlings;

[0079] Step iii: transplanting the gene silencing positive seedlings to obtain poplar plants with improved lignin content.

[0080] In step i, the interference silencing vector of the PtoERD3 gene is obtained by constructing the interference fragment of the PtoERD3 gene on the pBI121 vector, and preferably, the nucleotide sequence of the interference fragment of the PtoERD3 gene is shown as SEQ ID NO. 11.

[0081] Preferably, the interference fragment of the PtoERD3 gene is obtained by interference silencing primers P5 and P6, wherein the sequence of the interference silencing primer P5 is shown as SEQ ID NO. 12, and the sequence of the interference silencing primer P6 is shown as SEQ ID NO. 13.

[0082] In step ii, the identification of the gene silencing positive seedlings comprises the identification at the DNA level and the identification at the transcription level.

[0083] Preferably, the identification of the transcription level is carried out by RT-qPCR experiment, and the primer pair used is PtoERD3-qPCR-F and PtoERD3-qPCR-R, wherein the nucleotide sequence of the primer PtoERD3-qPCR-F is shown as SEQ ID NO. 9, and the nucleotide sequence of the primer PtoERD3-qPCR-R is shown as SEQ ID NO. 10.

[0084] In a further preferred embodiment, the reaction procedure for the identification of the transcription level is as follows: 95℃ for 3 min; (95℃ for 5 sec; 60℃ for 30 sec; 72℃ for 15 sec) for 40 cycles; 72℃ for 5 min; 4℃ for ∞.

[0085] In the present application, through the detection of the transcription level, it is found that the silencing plants numbered as RNAi-1, RNAi-6 and RNAi-8 have higher expression, which are 0.06 times, 0.12 times and 0.11 times of the wild type plant (WT), respectively.

[0086] In the present application, the PtoERD3 gene silencing mutant can significantly increase the content of poplar lignin, and is of great significance for the selection of poplar plant with excellent wood quality traits and the innovation of poplar germplasm resources.

[0087] In the fifth aspect of the present application, a method for genetic improvement of poplar is provided, wherein the genetic improvement is to increase the content of poplar lignin, and the method comprises the step of subculturing and selecting poplar individuals without the molecular marker of the first aspect, and eliminating poplar individuals with the molecular marker.

[0088] In the present application, a 54bp insertion mutation SV fragment in the upstream promoter region of PtoERD3 gene is used as a molecular marker for detecting the content of poplar lignin, which can quickly and accurately distinguish the content of poplar lignin, and provides a powerful tool for poplar breeding.

[0089] Examples

[0090] The present application is further described below by specific examples, but these examples are merely exemplary and do not constitute any limitation on the scope of protection of the present application.

[0091] Unless otherwise specified, the reagents involved in the following examples are commercially available conventional reagents, the methods used are conventional methods in the art, and the parameters are conventional settings.

[0092] Example 1 Obtaining structure variation SV related to Populus tomentosa lignin content

[0093] The molecular marker related to the content of poplar lignin is obtained according to the following steps:

[0094] The GEMMA (v0.98.5) software was used to perform SVGWAS analysis on 49380 high-confidence SVs and the lignin content of 302 P. tomentosa materials at the whole genome level, and the PCA matrix was added to correct the population structure, and 38 significant SV sites were screened out. One of the significantly associated sites was annotated to the promoter region of PtoERD3 (p = 1.02E-8), the nucleotide sequence of the PtoERD3 gene is shown as SEQ ID NO. 5, the nucleotide sequence of the promoter region is shown as SEQ ID NO. 2, and the significantly associated SV site is located at the 1725bp of the nucleotide sequence shown in SEQ ID NO. 2, as shown in Figure 1

[0095] Among them, 49380 high-confidence SVs were obtained by using Manta software to identify population structure variations.

[0096] The 302 P. tomentosa materials were from 302 germplasms planted in 83 locations across the Yellow River distribution area and planted in Guan County, Shandong Province, China.

[0097] The lignin content was determined based on the Klason standard method established by the U.S. Renewable Energy Laboratory (NREL), and the specific operation was as follows: the ground sample was first placed in a 72% sulfuric acid solution for hydrolysis, and after dilution, it was further hydrolyzed under dilute acid conditions. At this time, most of the lignin remained in solid form, and the lignin content was calculated by weighing the solid mass.

[0098] The GWAS results were obtained by R software package CMplot (https: / / github.com / YinLiLin / CMplot).

[0099] Example 2 Molecular marker of Populus tomentosa lignin content based on SV and its identification method

[0100] In order to exclude the problem of DNA template quality of the detected poplar, upstream and downstream primers for amplifying SV sequences were designed according to the upstream and downstream of the SV position. Considering various principles of primer design, primer blast tool (NCBI, https: / / blast.ncbi.nlm.nih.gov) was used for primer design, and primers SV-F (primer P1) and SV-R (primer P2) were obtained, and their nucleotide sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0101] Then 24 individuals numbered 1-24 were randomly selected from the P. tomentosa hybrid population germplasm resource library planted in Guan County, Shandong Province, to extract genomic DNA for SV verification, and to evaluate the lignin content of the samples.

[0102] ​The specific steps are as follows:

[0103] (1) The crude extraction of Populus tomentosa DNA to be detected:

[0104] (i) Sample processing: Take 1 piece of leaf to be extracted and place it in a 2 mL centrifuge tube, add 1 grinding bead, freeze in liquid nitrogen, and then crush in a tissue crusher for 5 min to a powder.

[0105] (ii) Buffer treatment: Add 500 μL of TBS buffer to the tube, mix well, and then heat in a 65°C water bath for 10 min.

[0106] (iii) Initial centrifugation and precipitation: centrifuge at 12000 rpm for 15 min, transfer the supernatant to a new tube, add an equal volume of isopropanol, and stand at room temperature for 30 min until a white flocculent DNA precipitate appears.

[0107] (iv) Nucleic acid collection: centrifuge at 12000 rpm for 15 min, carefully discard the supernatant, and add 1 mL of 75% ethanol to wash the precipitate.

[0108] (v) Ethanol removal: centrifuge at 7500 rpm for 10 min, and completely aspirate the residual liquid (the residual liquid on the tube wall can be removed with a pipette).

[0109] (vi) Drying and dissolution: dry in a 40°C oven for 2 h, add 50 μL of ddH2O to dissolve the DNA precipitate, and store in a -20°C refrigerator for standby use.

[0110] (2) Identification of SV

[0111] Using the Populus tomentosa DNA obtained in step (1) above as a template, PCR amplification was performed, and the reaction system is shown in Table 1:

[0112] Table 1

[0113]

[0114]

[0115] The reaction conditions are as follows: 95°C for 3 min; (94°C for 30 s; 58°C for 30 s; 72°C for 60 s) for 35 cycles; 72°C for 5 min; 4°C for ∞.

[0116] The amplification results are shown in Figure 2 , which can amplify a 308 bp band to prove the existence of SV, only amplify a 254 bp band to prove the absence of SV, and amplify double bands to prove hybrid variation.

[0117] (3) Comparison of identification results based on SV-based molecular markers and lignin content determination

[0118] The lignin content of 24 randomly selected individual P. tomentosa was compared with the SV-based molecular markers, and the results are shown in Table 2.

[0119] Table 2

[0120] Plant number Variation type Lignin content 1 Homozygous variation 17.5％ 2 Homozygous variation 18.0％ 3 Homozygous variation 19.0％ 4 Homozygous variation 16.8％ 5 Homozygous variation 18.5％ 6 Homozygous variation 17.2％ 7 Homozygous variation 20.4％ 8 No variation 18.2％ 9 No variation 21.5％ 10 No variation 22.0％ 11 No variation 23.0％ 12 No variation 21.8％ 13 No variation 22.5％ 14 No variation 22.2％ 15 No variation 21.9％ 16 Heterozygous variation 22.8％ 17 Heterozygous variation 18.6％ 18 Heterozygous variation 19.2％ 19 Heterozygous variation 18.8％ 20 No variation 19.5％ 21 Heterozygous variation 23.0％ 22 Heterozygous variation 17.8％ 23 Heterozygous variation 18.9％ 24 Example 3 Analysis of regulatory effect of structure variation SV on PtoERD3 gene promoter activity 19.4％

[0121] Among them, homozygous variation refers to an individual carrying a homozygous genotype of a structural variation (SV), i.e., both alleles contain the SV; no variation refers to an individual carrying a homozygous genotype without the SV, i.e., both alleles do not contain the SV (wild type homozygous); heterozygous variation refers to an individual carrying both alleles with and without the SV, forming a heterozygous genotype.

[0122] As can be seen from Table 2, the lignin content of individuals without variation is 22.3±0.6%; when the candidate individual is homozygous mutant, the lignin content of the candidate poplar individual is the lowest, which is reflected in that the lignin content of the candidate individual (18.2±1.2%) is 18.39% lower than that of the individual without variation (22.3±0.6%); when the candidate individual is heterozygous mutant, the lignin content (18.89%±0.6%) is 15.29% lower than that of the individual without variation.

[0123] Figure 3

[0124] Since the SV in the present application is located in the promoter region of the PtoERD3 gene, in order to determine whether the insertion variation of the promoter of the PtoERD3 gene affects its promoter activity, a dual luciferase experiment is used to carry out verification work, and the specific operation is as follows:

[0125] (1) Cloning of two promoters of PtoERD3 gene (with and without SV fragment insertion)

[0126] (i) Extract DNA of poplar samples with and without SV fragment insertion by the method shown in Example 2

[0127] (ii) According to the nucleotide sequence of the promoter of the PtoERD3 gene (as shown in SEQ ID NO. 2), the primer design method is the same as in Example 2, to obtain primers P7 and P8 (the sequence of P7 is shown in SEQ ID NO. 14, and the sequence of P8 is shown in SEQ ID NO. 15).

[0128] Using the extracted DNA as a template, Phanta Max Super-Fidelity DNA Polymerase from Nanjing Vazyme Company was used for PCR amplification, and the PCR experimental reaction system (50 μL) is shown in Table 3:

[0129] Table 3

[0130]

[0131] PCR amplification procedure: 95℃ 3min; (95℃ 25s; 57℃ 30s; 72℃ 60s (amplification efficiency 45s / kb)) 36 cycles; 72℃ 5min; 4℃ ∞.

[0132] (iii) The product of the PCR reaction was subjected to agarose gel electrophoresis, and after detection, it was purified using the DNA Clean-up Kit reagent box of Jiangsu Kangwei Shijis Century Biotechnology Co., Ltd. After purification, the purity and concentration of the DNA purified product were determined using the instrument.

[0133] (2) Construction of pGreenII 0800-Pro-LUC and pGreenII 0800-Pro+SV-LUC vectors

[0134] The pGreenII 0800-LUC plasmid was subjected to single enzyme digestion with Sal I endonuclease, and the enzyme digestion reaction system is shown in Table 4:

[0135] Table 4

[0136]

[0137] The enzyme digestion reaction conditions were: 37℃ water bath for 7-9h, and the plasmid after enzyme digestion was recovered by gel and used as a vector skeleton, which was stored at -20℃.

[0138] (3) Ligation and transformation

[0139] (3.1) Ligation of expression vectors

[0140] The two promoter fragments were constructed into the 0800 vector using the seamless cloning kit Uniclone One Step Seamless Cloning Kit of Beijing Jinsha Biotechnology Co., Ltd. to obtain recombinant vectors. The ligation was performed at 50℃ for 10min, and the ligation product was used to transform E. coli DH5α competent cells.

[0141] (3.2) Transformation of E. coli DH5α competent cells

[0142] Take 50 μl DH5a competent cells, add 5 μl ligation product, mix gently and stand on ice for 30 minutes. Then, place the mixture in a 42°C water bath for 45 seconds, quickly transfer to ice for 2 min, avoid shaking during the period to ensure transformation efficiency. Next, add 700 μl of sterile LB liquid medium without antibiotics, mix and beat evenly, then place in a 37°C, 220 rpm shaker for 15 minutes to recover the bacteria. After recovery, centrifuge at 6000 rpm for 1 minute to collect the bacteria, resuspend the bacteria with 100 μl supernatant, and spread on LB solid plate medium containing kanamycin (100 mg / ml), and incubate at 37°C for 14-16 hours.

[0143] (3.3) Bacterial liquid PCR identification of positive clones

[0144] Use a sterilized gun head to pick single colonies from pGreenII 0800-Pro-LUC and pGreenII 0800-Pro+SV-LUC plates in 250 μl LB liquid medium containing kanamycin, and incubate at 37°C, 200 rpm / min for about 3 hours as amplification template. Another set of positive and negative controls were set up using gene PCR purified product and ddH2O (double distilled water) as template, and Taq Plus Master Mix from Nanjing Vazyme was used for PCR amplification.

[0145] The PCR reaction system is shown in Table 5:

[0146] Table 5

[0147]

[0148]

[0149] The sequence of primer 0800-R is shown in SEQ ID NO. 16.

[0150] The reaction conditions are: 95°C for 3 min; (94°C for 30 s; 58°C for 30 s; 72°C for 60 s) for 35 cycles; 72°C for 5 min; 4°C for ∞.

[0151] The PCR product was detected by 1% agarose gel electrophoresis, and the colonies that could amplify the same size band as the positive control were positive clones.

[0152] (3.4) Extraction of positive clone plasmid

[0153] The PCR positive clones were taken to 6 ml LB liquid medium containing kanamycin, and cultured at 37°C, 200 rpm overnight. Plasmid extraction was performed using the plasmid extraction kit from Jiangsu Kangwei Reagent Co., Ltd., and sequencing was performed by Beijing Ruibo Xingke Biotechnology Co., Ltd. After the sequence determination was compared and no error was found, the vector construction was completed.

[0154] (4) Transformation of Agrobacterium with recombinant plasmid

[0155] (4.1) About 1 μg of pGreenII 0800-Pro-LUC and pGreenII 0800-Pro+SV-LUC recombinant plasmid was added to 100 μL of Agrobacterium GV3101 competent cells, and mixed gently.

[0156] (4.2) In turn, stand on ice for 5 min, freeze in liquid nitrogen for 1 min, immediately put into 37°C water bath for 5 min, and ice bath for 5 min.

[0157] (4.3) Add 700 μL of YEP liquid medium without antibiotics to the bacterial solution, mix thoroughly, and incubate at 28°C, 200 rpm for 2-3 hours. After the recovery culture, centrifuge the bacterial solution at 6000 rpm for 1 minute, discard part of the supernatant, and reserve 100 μL of supernatant mixed with the bacterial solution. Using a disposable sterile coating rod, evenly coat the bacterial solution on the surface of YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, and then place it in a 28°C incubator for inverted culture for 72-90 hours.

[0158] (4.4) After single colonies grow, use a sterile gun head to pick several single colony spots and place them in a 2 mL centrifuge tube containing 250 μL of YEP liquid medium (1:1000 addition of Rif, Kana), and incubate at 30°C, 200 rpm on a shaker for 2 hours. After the culture is completed, the bacterial solution is taken for PCR identification, and the method is the same as described in Example 2. The correct positive bacterial solution is added with 50% glycerol and frozen in liquid nitrogen, and stored in a -80°C refrigerator for subsequent experiments.

[0159] (5) Tobacco transient transformation experiment

[0160] (5.1) Agrobacterium containing pGreenII 0800-Pro-LUC and pGreenII 0800-Pro+SV-LUC plasmid was taken from the -80°C refrigerator, and was streaked onto YEP solid medium (1:1000 addition of Rif, Kana), and was incubated in a 28°C dark incubator for 2-3 days.

[0161] (5.2) Use a sterile gun tip to pick a single colony from the plate and inoculate into 3 mL YEP liquid medium (1:1000 Rif, Kana) and incubate overnight at 30°C, 200 rpm in a shaker. Take 1 mL of the bacterial solution and transfer into a 250 mL sterile conical flask containing 100 mL YEP liquid medium (1:1000 Rif, Kana) and incubate for 4-5 h at 30°C, 200 rpm in a shaker until the OD600 is 0.3-0.5.

[0162] (5.3) Transfer 100 mL of the bacterial solution into two 50 mL sterile centrifuge tubes in a super-clean bench, centrifuge at 2560 g, 4°C for 20 min. Collect the bacterial cells. In the super-clean bench, discard the supernatant and resuspend the bacterial cells in a sterile beaker with 100 mL of resuspension solution. The bacterial solution is used for subsequent tobacco transient transformation experiments. The resuspension solution formula is shown in Table 6:

[0163] Table 6

[0164]

[0165] (6.4) Let the resuspended pGreenII 0800-Pro-LUC and pGreenII 0800-Pro+SV-LUC bacterial solution stand at room temperature for 3-4 h. Select tobacco leaves with good growth and use a needleless syringe to inject the mixed bacterial solution into the lower epidermis of the leaves. Place the infected tobacco plants in a greenhouse and cultivate under light for 16 h / dark for 8 h for 48-72 h. Use a molecular imaging system (IVIS Lumina XRMS Series III, PerkinElmer, Waltham, USA) to image, and the results are shown in Figure 3 Figure 4 It can be seen that the fluorescence signal of pGreenII 0800-Pro+SV-LUC is significantly higher than that of pGreenII 0800-Pro-LUC, proving that the SV can significantly improve the transcription activation activity.

[0166] Take about 100 mg of the infected tobacco leaf tissue, grind in liquid nitrogen, and use a dual luciferase reporter assay kit (Dual-Luciferase Reporter Assay System, Promega, Madison, USA) to detect luciferase activity according to the kit instructions. Use a multifunctional enzyme marker (Varioskan LUX, Thermo Fisher, Waltham, USA) to measure the luminescence intensity (LUC) of firefly luciferase. Then add 100 μL Stop&Glo (Promega, Madison, USA) to stop the reaction, and use the multifunctional enzyme marker to measure the luminescence intensity (Rluc) of Renilla luciferase. The ratio of LUC to Rluc is the relative luciferase activity. ​Reagent, to determine the luminescence intensity (REN) of Renilla luciferase. With Renilla luciferase as an internal reference, the ratio of the luminescence intensity of firefly luciferase to Renilla luciferase (LUC / REN) was calculated to measure the activation activity of SV on PtoERD3. The experiment was repeated at least 3 times, and 3 biological replicates were set each time. The control experiment was carried out by empty vector according to the above method, and the results are shown in Figure 4 It can be seen that: SV can significantly enhance the transcription of PtoERD3 and positively regulate the expression of PtoERD3. Example 4 Construction of PtoERD3 gene overexpression vector and interference silencing vector

[0167] Example 5 Genetic transformation of PtoERD3 gene

[0168] (1) Enzymatic digestion of the vector

[0169] The selected overexpression vector is pBI121 vector, which is 13629 bp long, containing 35S strong promoter (CaMV35S) and resistance to kanamycin (Kana), and can be digested with XbaI. The enzyme digestion reaction system is shown in Table 7:

[0170] Table 7

[0171]

[0172] The enzyme digestion reaction conditions are: 37°C water bath for 7-9h. The digested plasmid is recovered by gel and used as a vector skeleton, which is stored at -20°C.

[0173] The products after enzyme digestion are purified by gel recovery. The GelExtraction Kit reagent box of Kangwei Century is used for recovery, and the DNA Clean-up Kit reagent box of Kangwei Century is used for purification.

[0174] (2) The RNA of wild-type poplar was extracted by the plant RNA extraction kit of Beijing Quanshi Jin Biotechnology Co., Ltd. The RNA was reverse transcribed into cDNA by using HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novozyme Bio-tech Co., Ltd.). The cDNA obtained by reverse transcription was used as a template. According to the CDS sequence of PtoERD3 gene (as shown in SEQ ID NO. 6), the overexpression and interference silencing primer design principles are the same as in Example 2, the overexpression primers P3 and P4 (sequences as shown in SEQ ID NO. 7, SEQ ID NO. 8) are obtained, and the PCR experimental reaction system (50μL) is shown in Table 8. The interference silencing primers are P5 and P6 (sequences as shown in SEQ ID NO. 12, SEQ ID NO. 13), and the PCR experimental reaction system (50μL) is shown in Table 9. ​

[0175] Table 8

[0176]

[0177] Table 9

[0178]

[0179]

[0180] The conditions of the above reaction are: 95℃ 3min; (94℃ 30s; 58℃ 30s; 72℃ 60s) 35 cycles; 72℃ 5min; 4℃ ∞.

[0181] The product of the PCR reaction is subjected to agarose gel electrophoresis, and after detection, the Jiangsu Kangwei Century Biotechnology Co., Ltd. DNA Clean-up Kit reagent box is used for purification. After purification, the purity and concentration of the DNA purified product are determined using the instrument.

[0182] The CDS sequence of the PtoERD3 gene is finally obtained, as shown in SEQ ID NO. 6, and the nucleotide sequence of the interference fragment is as shown in SEQ ID NO. 11.

[0183] (3) The expression vector is connected, the E. coli DH5α competent cells are transformed, and the positive clones are identified by PCR. The PtoERD3 gene overexpression PCR reaction system is shown in Table 10:

[0184] Table 10

[0185]

[0186] The sequence of SKP2a-F is as shown in SEQ ID NO. 17.

[0187] The PtoERD3 gene interference silencing PCR reaction system is shown in Table 11:

[0188] Table 11

[0189]

[0190] The conditions of the PtoERD3 gene overexpression PCR reaction and the interference silencing PCR reaction are: 95℃ 2min; (94℃ 30s; 58℃ 30s; 72℃ 60s) 35 cycles; 72℃ 5min; 4℃ ∞.

[0191] The PCR product is detected by 1% agarose gel electrophoresis, and the colony that can amplify the same band as the positive control is the positive clone. Subsequently, after sequence determination and comparison, the vector construction is completed.

[0192] Example 6 Identification of PtoERD3 gene overexpression and interference silencing plants

[0193] (1) Recombinant plasmid transformation of Agrobacterium

[0194] The method of recombinant plasmid transformation of Agrobacterium is referred to Example 3.

[0195] (2) Activation of Agrobacterium

[0196] (2.1) Take out the Agrobacterium liquid containing overexpression vector plasmid and interference silencing vector plasmid from the -80°C refrigerator, streak inoculate on YEP solid medium (1:1000 Rif, Kana) respectively, seal the plate, and invert culture in a 28°C dark incubator for 2-3 days.

[0197] (2.2) Use a sterile gun head to pick single colonies from the plate and inoculate into 3 mL YEP liquid medium (1:1000 Rif, Kana), and place in a shaker at 30°C, 200 rpm for overnight shaking culture. Take 1 mL of bacterial liquid and transfer into a 250 mL sterile conical flask containing 100 mL YEP liquid medium (1:1000 Rif, Kana), and place in a shaker at 30°C, 200 rpm for shaking culture for 4-5 h until the OD600 is 0.3-0.5.

[0198] (2.3) In the clean bench, transfer 100 mL of bacterial liquid into two 50 mL sterile centrifuge tubes, centrifuge at 2560 g, 4°C for 20 min. Collect the bacterial bodies. In the clean bench, discard the supernatant, resuspend the bacterial bodies in a sterile wide-mouth bottle with 100 mL of resuspension liquid, and the bacterial liquid is used for subsequent infection experiments.

[0199] The resuspension liquid is prepared according to the following ingredients and amounts: WPM (2.37 g) + VB1 (0.9 mg) + MES (0.5 g) + sucrose (20 g) + 2,4-D (0.1 mg) + AS (200 mM) + ddH2O (constant volume to 1 L).

[0200] (3) Callus method infection

[0201] Select the leaves of healthy and sterile seedlings (deep green leaves, thick texture), use a sterile scalpel to remove the petiole, the petiole can be left to grow callus, and 2-3 horizontal wounds are made on the main veins of the leaves, and the leaves are placed on the callus culture medium (CIM) with the front side facing down, and placed in a 25°C environment in the dark. After the leaves grow for 20-30 days, white and loose callus will grow at the wound, the callus is peeled off from the leaves, divided into soybean-sized pieces, and placed into new callus culture medium. The prepared callus is transferred into a sterile wide-mouth bottle containing bacterial liquid, and placed in a shaker at 28°C, 160 rpm for 15-20 min of infection.

[0202] The callus was taken out from the bacteria solution with tweezers and the extra bacteria solution was absorbed with filter paper in a clean bench. The infected callus was laid on the cocultivation medium (WPMC) and cultured in dark at 25°C for 2 days.

[0203] (4) Cocultivation

[0204] The callus was taken out from the bacteria solution with tweezers and the extra bacteria solution was absorbed with filter paper in a clean bench. The infected callus was laid on the cocultivation medium (WPMC) and cultured in dark at 25°C for 2 days.

[0205] The cocultivation medium was prepared according to the following components and amounts: WPM (2.37 g) + VB1 (0.9 mg) + sucrose (20 g) + MES (0.5 g) + AS (200 mM) + agar (7.5 g) + ddH2O (constant volume to 1 L).

[0206] (5) Differentiation culture

[0207] After the dark culture, the appropriate plant resistance was selected according to the carrier, and the differentiation medium containing the corresponding antibiotic was prepared.

[0208] The callus was transferred to the differentiation medium (WPMD). The medium was changed once in about 20 days for the first time, and then once in 10 days. During this period, the callus would turn green, harden, and then partially turn red. This stage lasted about two months, and the whole process was cultured in a 25°C light incubator.

[0209] The cocultivation medium was prepared according to the following components and amounts: WPM (2.37 g) + VB1 (0.9 mg) + sucrose (20 g) + MES (0.5 g) + AS (200 mM) + 6-BA (0.5 mg) + NAA (0.1 mg) + TDZ (0.002 mg) + TMT (250 mg) + Cef (250 mg) + kana (20 mg) + agar (7.5 g) + ddH2O (constant volume to 1 L).

[0210] (6) Shoot induction and rooting

[0211] After the adventitious shoots grew to about half a centimeter, sterile tweezers or a surgical knife were used to cut the adventitious shoots (attention was paid to the growth point of the adventitious shoots to avoid selecting adventitious shoots differentiated from the same cell). The adventitious shoots were placed in the shoot elongation medium (WPML) for growth. After a period of elongation culture, when the adventitious shoots grew to 1-2 centimeters, sterile tweezers or a surgical knife were used to cut the adventitious shoots individually and place them in the rooting medium for rooting culture. After about 10 days, the adventitious shoots would grow roots and the rooting culture would become complete plants.

[0212] The elongation medium is prepared according to the following components and amounts: WPM (2.37 g) + IBA (0.2 mg) + sucrose (20 g) + 6-BA (0.5 mg) + TMT (250 mg) + Cef (250 mg) + agar (7.5 g) + ddH2O (to 1 L).

[0213] Figure 5

[0214] (1) The DNA of wild type P. tomentosa, PtoERD3 gene overexpression plants and PtoERD3 gene interference silenced plants was extracted, and the method was referred to Example 2.

[0215] (2) Identification of PtoERD3 gene overexpression plants and PtoERD3 gene interference silenced plants:

[0216] (2.1) Identification at DNA level

[0217] The overexpression plant gDNA and the interference silenced plant gDNA extracted were used as templates, Taq Plus Master Mix was used for PCR identification, and the PCR reaction system was referred to Table 10 and Table 11 in Example 4. The PCR products were directly run electrophoresis, and whether the band was correct was checked. If correct, it was PtoERD3 gene overexpression plant or PtoERD3 gene interference silenced plant.

[0218] (2.2) Identification at transcription level

[0219] The transcription level of PtoERD3 in overexpression and interference silenced plants was detected by RT-qPCR, and the specific steps were as follows:

[0220] (i) The RNA of the leaf tissue to be detected was extracted, and the template cDNA was obtained by reverse transcription. The method of RNA extraction and reverse transcription to obtain cDNA was the same as that in Example 4, and the reaction system (20 μL) was as shown in Table 12:

[0221] Table 12

[0222]

[0223] The reaction program was: 50 ℃ for 5 min; 85 ℃ for 5 sec.

[0224] (ii) RT-qPCR experiments were performed using 2×ChamQ SYBR Color qPCR Master Mix. In this example, the following fluorescent quantitative PCR (polymerase chain reaction) primers were designed based on the PtoERD3 gene sequence of Populus tomentosa. The primers are named PtoERD3-qPCR-F and PtoERD3-qPCR-R, and their nucleotide sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively. The nucleotide sequences of the internal control primers Actin-F and Actin-R are shown in SEQ ID NO. 18 and SEQ ID NO. 19, respectively.

[0225] The reaction system (20 μL) is shown in Table 13:

[0226] Table 13

[0227]

[0228] The PCR reaction program was as follows: 95℃ for 3 min; (95℃ for 5 sec; 60℃ for 30 sec; 72℃ for 15 sec) for 40 cycles; 72℃ for 5 min; 4℃ for ∞.

[0229] The overexpression detection results of RT-qPCR are as follows: Figure 5 As shown, by Figure 6 It can be seen that the overexpression plants numbered OE-2, OE-3 and OE-5 have higher expression levels, which are 99.5 times, 151.3 times and 130 times higher than those of wild-type plants (WT), respectively.

[0230] The results of RT-qPCR interference silencing detection are as follows: Figure 6 As shown, by Example 7 Phenotype analysis of PtoERD3 gene overexpression plants and interference silencing plants It can be seen that the overexpressing plants numbered RNAi-1, RNAi-6 and RNAi-8 have higher expression levels, which are 0.06 times, 0.12 times and 0.11 times that of wild-type plants (WT), respectively.

[0231] Figure 7

[0232] Plants overexpressing the PtoERD3 gene, plants with interference silence, and wild-type Populus tomentosa were simultaneously planted in a greenhouse, and the phenotypic characteristics of the plants were measured and photographed.

[0233] like Figure 7 As shown, from left to right, the phenotypes of the PtoERD3 overexpressing plant (denoted as OE-PtoERD3), the wild-type Populus tomentosa (denoted as WT), and the interference-silenced plant (denoted as RNAi-PtoERD3) are respectively. Figure 8It can be seen that, compared with wild type P. tomentosa, the PtoERD3 interference silenced plants have higher height and thicker diameter, while the PtoERD3 overexpression plants have significantly inhibited growth, shorter height and thinner diameter. Figure 9 and Figure 10 Figures 2A and 2B respectively show the height and diameter analysis results of the overexpression plants, the interference silenced plants and the wild type P. tomentosa.

[0234] Further, Klason method was used to determine the lignin content of the plants, and the results are shown in Figure 3. ​ It can be seen that the lignin content of the PtoERD3 interference silenced plants is increased by 36% compared with the wild type, while the lignin content of the PtoERD3 overexpression plants is significantly reduced by 17.1% compared with the wild type. The above results show that PtoERD3 gene negatively regulates the lignin content of poplar.

[0235] The present application is described in detail above in combination with the specific embodiments and exemplary examples, but these descriptions cannot be understood as limiting the present application. It is understood by those skilled in the art that various equivalent substitutions, modifications or improvements can be made to the technical solutions and embodiments of the present application without departing from the spirit and scope of the present application, and these all fall within the scope of the present application.

Claims

1. A molecular marker associated with Populus lignin content, characterized in that, The molecular marker is a 54bp chromosome structure variation SV fragment, The nucleotide sequence of the chromosome structure variation SV fragment is shown as SEQ ID NO. 1; The molecular marker is located in the upstream promoter region of the PtoERD3 gene of the poplar, the nucleotide sequence of the upstream promoter of the PtoERD3 gene is shown as SEQ ID NO. 2, the poplar is Populus tomentosa, and the nucleotide sequence of the PtoERD3 gene is shown as SEQ ID NO. 5; The molecular marker is located at the 1725bp of the nucleotide sequence shown in SEQ ID NO.

2.

2. A primer pair for detecting the molecular marker of claim 1, characterized in that, The primer pair comprises primer P1 and primer P2, the sequence of primer P1 is shown as SEQ ID NO. 3, and the sequence of primer P2 is shown as SEQ ID NO.

4.

3. The primer pair according to claim 2, characterized in that, The genomic DNA of the poplar to be tested is used as a template, and the primer pair P1 and P2 is used for PCR amplification of the molecular marker related to the lignin content of the poplar, and then identification is performed, If the amplification product contains the molecular marker in claim 1, the lignin content of the poplar is low; if the amplification product does not contain the molecular marker in claim 1, the lignin content of the poplar is high.

4. Use of the molecular marker of claim 1 for detecting the lignin content in poplar, characterized in that, The application comprises the following steps: First, the genomic DNA of the poplar to be tested is used as a template for PCR amplification; The primer pair used for the PCR amplification is primer P1 and primer P2, the sequence of primer P1 is shown as SEQ ID NO. 3, and the sequence of primer P2 is shown as SEQ ID NO. 4; Then, according to the amplification product, it is determined whether the molecular marker is contained, and then the lignin content of the poplar is determined; The amplification product is subjected to electrophoresis analysis, when a single band of 308bp is amplified, it is determined that the lignin content of the poplar is the lowest; when a single band of 254bp is amplified, it is determined that the lignin content of the poplar is the highest; when double bands of 308bp and 254bp are amplified, it is determined that the lignin content of the poplar is between the above two.

5. Use of the molecular marker of claim 1 for modulating the lignin content of a poplar tree, characterized in that, The regulation of the molecular marker on the lignin content of the poplar is realized by overexpressing the PtoERD3 gene or silencing the PtoERD3 gene, The lignin content of the poplar is reduced by overexpressing the PtoERD3 gene in the poplar plant, and the lignin content of the poplar is increased by silencing the PtoERD3 gene in the poplar plant.

6. Use according to claim 5, characterized in that, The method for reducing the lignin content of the poplar by the molecular marker comprises the following steps: Step 1, constructing an overexpression vector of the PtoERD3 gene, transferring into Agrobacterium, and obtaining positive overexpression engineering bacteria; Step 2, using the positive overexpression engineering bacteria to infect poplar materials, and obtaining overexpression positive seedlings after culture and identification; Step 3, transplanting the overexpression positive seedlings to obtain poplar plants with reduced lignin content.

7. Use according to claim 5, characterized in that, The method for increasing the lignin content of the poplar by the molecular marker comprises the following steps: Step i, constructing an interference silencing vector of the PtoERD3 gene, and transferring into Agrobacterium to obtain positive gene silencing engineering bacteria; Step ii, using the positive gene silencing engineering bacteria to infect poplar materials, culturing the transgenic seedlings, and obtaining gene silencing positive seedlings after identification; Step iii, transplanting the gene silencing positive seedlings to obtain poplar plants with increased lignin content.

8. Use according to claim 7, characterized in that, In step i, the interference fragment of PtoERD3 gene is constructed into pBI121 vector to obtain the interference silencing vector of PtoERD3 gene, The nucleotide sequence of the interference fragment of PtoERD3 gene is shown as SEQ ID NO.

11.

9. A method for genetic improvement of a poplar tree, characterized in that, The genetic improvement is to increase the lignin content of poplar, The method comprises the steps of subculture breeding of poplar individuals without the molecular marker of claim 1, and eliminating poplar individuals with the molecular marker.

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