Molecular marker suitable for high-product seedling stage breeding of populus tomentosa and application of molecular marker

Abstract

The invention provides a molecular marker closely linked with a populus tomentosa seedling stage growth vigor character. The method can accurately evaluate the growth vigor of the populus tomentosa at the molecular level in the seedling stage, high-yield new populus germplasm can be bred through an efficient molecular marker-assisted breeding means in the seedling stage, and compared with mature-stage adult breeding in traditional industrial breeding, the method greatly shortens the populus breeding period and improves the breeding efficiency of populus tomentosa. And an efficient means is provided for improved variety breeding of industrial materials of populus tomentosa.

Description

Technical Field

[0001] The present invention belongs to the technical field of poplar breeding, and specifically relates to a molecular marker related to the growth potential such as poplar height, ground diameter, breast diameter and volume, and its application. Background Art

[0002] As the most abundant renewable resource on earth, wood has important industrial and economic value for human production and life. With the rapid development of the economy, the requirements for wood production are getting higher and higher. To meet this demand, sufficient high-quality wood fiber industrial raw materials are needed.

[0003] As an important timber tree species, poplar has the characteristics of fast growth, high quality, strong adaptability, etc. At the same time, it has the advantages of rapid growth, short rotation period, easy interspecific hybridization and asexual reproduction, and convenient genetic manipulation. It is considered to be the most promising fibrous energy tree species in the Northern Hemisphere. However, poplar is a perennial tree species, and it takes a long time to carry out genetic improvement by traditional cross-breeding methods. Screening molecular markers that can improve poplar growth and wood production by using genomics methods can effectively improve the above disadvantages.

[0004] The growth and yield traits of forest trees are complex quantitative traits controlled by numerous minor genes. Traditional single-trait GWAS (Genome-Wide Association Studies) and QTL (quantitative trait locus) mapping have insufficient detection ability for the core regulatory genes of un-domesticated forest trees. The goal of plant breeding is to concentrate multiple ideal traits in one variety. However, multi-trait combined breeding is often accompanied by trait mutual exclusion (multi-trait linkage drag effect) and linkage resistance caused by closely linked genes, resulting in difficulty in improving one trait while improving another trait.

[0005] At present, the poplars obtained by breeding still cannot meet people's needs. How to further improve the breeding efficiency, shorten the breeding time, and increase the wood yields of multiple individuals in the breeding population requires further research to achieve a method for improving the combined breeding ability of multiple expected traits. Summary of the Invention

[0006] In order to overcome the above problems, the inventors of the present invention conducted intensive research, integrated the strategies of genome-wide association analysis and selective sweep, screened out molecular markers significantly related to the growth potential traits such as poplar height, breast diameter, ground diameter and volume, which can be directly applied to molecular marker-assisted breeding of poplar. In addition, primer pairs for amplifying the molecular markers, applications of the molecular markers, and poplar genetic improvement methods are provided, which accelerate the improvement of molecular-assisted breeding technology for forest tree quantitative traits and improve the accuracy and efficiency of forest tree improved variety selection.

[0007] The obtained markers can make an accurate evaluation of the overall growth potential of Populus tomentosa at the molecular level, and accurate and efficient molecular marker-assisted breeding can be carried out in the seedling stage to select high-yield poplar varieties. Compared with the mature stage selection in traditional industrial breeding, the poplar breeding cycle is greatly improved, and an efficient means is provided for molecular marker-assisted selection breeding of Populus tomentosa, thereby completing the present invention.

[0008] The purpose of the first aspect of the present invention is to provide a molecular marker tightly linked to the growth potential traits of Populus tomentosa seedlings, wherein the molecular marker is an InDel molecular marker, and the molecular marker InDel is located at base position 594919 of chromosome 7 of the poplar genome and has GA / G and GA / GA genotype polymorphisms.

[0009] The molecular marker is located in the coding region (CDS region) of the gene PtoHAT22.

[0010] The nucleotide sequence of the gene PtoHAT22 is shown in SEQ ID NO:3.

[0011] The second aspect of the present invention aims to provide a primer pair for amplifying a molecular marker tightly linked to the growth potential traits of Populus tomentosa seedlings, the primer pair comprising primer P1 and primer P2, primer P1 having a nucleotide sequence as shown in SEQ ID NO:1, and primer P2 having a nucleotide sequence as shown in SEQ ID NO:2.

[0012] The third aspect of the present invention aims to provide the use of the molecular marker tightly linked to the growth potential trait of Populus tomentosa seedlings in any of the following aspects (1) to (4):

[0013] (1) Screening or assisting in the screening of new varieties of Populus tomentosa with high growth potential at the seedling stage; (2) Molecular marker-assisted breeding of Populus tomentosa; (3) Preparing products for screening or assisting in the screening of new varieties of Populus tomentosa with high growth potential; (4) Preparing products for molecular marker-assisted breeding of Populus tomentosa.

[0014] The fourth aspect of the present invention aims to provide a method for genetic improvement of poplars. Preferably, the method comprises the steps of successive breeding of individuals with the GA / G genotype marked by the above-mentioned InDel molecular marker and eliminating individuals with all other genotypes except for the marker.

[0015] The poplar genetic improvement method comprises the following steps:

[0016] Step 1, extracting genomic DNA of Populus tomentosa;

[0017] Step 2, using Populus tomentosa genomic DNA as a template, performing PCR amplification;

[0018] Step 3, determining the genotype of the molecular markers of the tested Populus tomentosa;

[0019] Step 4, evaluating the growth potential of the tested poplar according to the genotype detection results;

[0020] Step 5, selecting and breeding individuals with the GA / G genotype marked by the above InDel molecular marker.

[0021] The present invention has the following beneficial effects:

[0022] (1) The gene identification step provided by the present invention uses the PCA principal component analysis method to fit multiple poplar growth-related complex quantitative traits. By reducing the dimension and extracting comprehensive variables, the key regulatory genes that affect the overall growth potential of poplars can be effectively identified. Compared with the previous gene identification method that can only identify a single quantitative trait, the identified genes have greater efficacy.

[0023] By jointly analyzing QTL analysis and single-marker T test for main effect QTL intervals, we can identify several QTL intervals with the highest confidence from the whole genome, and then perform phenotype-genotype association tests on the molecular markers in the candidate intervals to detect the key genes and functional sites that cause phenotypic variation.

[0024] The combined application of the above steps can accelerate the improvement of molecular-assisted breeding technology for quantitative traits of forest trees and improve the accuracy and efficiency of breeding of forest tree varieties.

[0025] (2) The PtoHAT22 gene provided by the present invention is significantly correlated with the overall growth potential of poplars, and is of great significance for increasing the yield of improved forest tree species;

[0026] (3) The PtoHAT22 gene contains an EAR domain and is a transcriptional repressor. Its Arabidopsis homologous gene AT4G37790 can inhibit the growth of Arabidopsis. The molecular marker InDel provided by the present invention, which is significantly correlated with the overall growth potential of Populus tomentosa, can terminate the translation of the PtoHAT22 gene protein prematurely, resulting in the ineffectiveness of the HD-ZIP functional domain of the PtoHAT22 gene and the removal of the inhibitory effect. Compared with the SNP variant site, it has a higher regulatory effect on individual traits.

[0027] This InDel molecular marker can make an accurate evaluation of the growth potential of poplar at the molecular level. It can carry out precise and efficient molecular marker-assisted breeding in the poplar seedling stage to select high-yield poplar varieties. Compared with the mature stage selection in traditional industrial breeding, it has greatly improved the poplar breeding cycle and provided an efficient means for molecular marker-assisted selection breeding of Populus tomentosa.

[0028] (4) The molecular markers and the Populus tomentosa genetic improvement method provided by the present invention have been verified for genetic improvement in a natural population of 500 Populus tomentosa plants, indicating that it has a wide range of applications, broad application prospects and excellent economic value, and can greatly improve the economic value of industrial poplar production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Showing the schematic diagram of the HD-ZIP II subfamily structure;

[0030] Figure 2 Showing the significant difference comparison chart of the PC 1 index between the GA / GA and GA / G genotype populations of the present invention;

[0031] Figure 3 Showing the significant difference comparison chart of the tree height index between the GA / GA and GA / G genotype populations of the present invention;

[0032] Figure 4 Showing the significant difference comparison chart of the DBH index between the GA / GA and GA / G genotype populations of the present invention;

[0033] Figure 5 Showing the significant difference comparison chart of the ground diameter index between the GA / GA and GA / G genotype populations of the present invention;

[0034] Figure 6 Showing the significant difference comparison chart of the volume index between the GA / GA and GA / G genotype populations of the present invention;

[0035] Figure 7 Showing the amino acid sequence alignment chart of the GA / GA genotype and GA / G genotype of the PtoHAT22 gene of the present invention;

[0036] Figure 8 Showing the significant difference comparison chart of the tree height index of the 302-plant population with the GA / GA and GA / G genotypes of the present invention;

[0037] Figure 9 Showing the significant difference comparison chart of the DBH index of the 302-plant population with the GA / GA and GA / G genotypes of the present invention;

[0038] Figure 10 Showing the significant difference comparison chart of the ground diameter index of the 302-plant population with the GA / GA and GA / G genotypes of the present invention;

[0039] Figure 11 Showing the significant difference comparison chart of the volume index of the 302-plant population with the GA / GA and GA / G genotypes of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will be described in detail below through specific embodiments, and the features and advantages of the present invention will become clearer and more definite with these descriptions.

[0041] The first aspect of the present invention provides a molecular marker closely linked to the growth vigor trait of Populus tomentosa seedlings. The molecular marker is an InDel molecular marker, and the InDel is located at the 594,919th base of chromosome 7 of the Populus tomentosa genome, with GA / G and GA / GA genotype polymorphisms.

[0042] The genotypes of the InDel molecular marker are GA / G and GA / GA.

[0043] The molecular marker is located in the coding region (CDS region) of the gene PtoHAT22.

[0044] The gene PtoHAT22 is a transcriptional repressor gene containing an EAR transcriptional repression domain and an HD-ZIP (homeodomain-leucine zipper) functional domain.

[0045] The nucleotide sequence of the gene PtoHAT22 is as shown in SEQ ID NO:0. (National GeneBank: Genomesequencing of Populus tomentosa; Project number: CNP0004290, Sample number: CNS0752799, Assembly number: CNA0069009; Location of PtoHAT22: Chromosome 7, Start position 594380, End position 596001, see specifically http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / )

[0046] The growth vigor traits of Populus tomentosa seedlings closely linked to the molecular marker include the tree height, ground diameter, breast diameter, and volume of Populus tomentosa.

[0047] Individuals with the GA / G genotype of the molecular marker have a higher tree height, larger breast diameter, larger ground diameter, larger volume, and higher overall growth vigor than those with the GA / GA genotype.

[0048] The second aspect of the present invention provides a primer pair for amplifying a molecular marker closely linked to the growth vigor trait of Populus tomentosa seedlings. The primer pair includes primer P1 and primer P2. Primer P1 has the nucleotide sequence shown in SEQ ID NO:1, and primer P2 has the nucleotide sequence shown in SEQ ID NO:2.

[0049] The third aspect of the present invention provides a method for obtaining an InDel molecular marker, and the method includes the following steps:

[0050] Step 1: Measure the phenotypic traits of the Populus tomentosa hybrid population;

[0051] Step 2: Extract genomic DNA from the Populus tomentosa population;

[0052] Step 3: Perform PCA analysis on the phenotypic traits to fit the data;

[0053] Step 4: Use the genetic map for QTL mapping and perform T - test phenotypic - marker association analysis on the significant QTL intervals;

[0054] Step 5: Obtain genes and InDel molecular markers related to the growth potential of Populus tomentosa.

[0055] The fourth aspect of the present invention provides the application of the molecular markers tightly linked to the growth potential traits of Populus tomentosa seedlings in any one of the following aspects (1) to (4):

[0056] (1) Screening or assisting in screening new Populus tomentosa varieties with high tree height, diameter at breast height, ground diameter, and volume at the seedling stage; (2) Molecular marker - assisted breeding of Populus tomentosa; (3) Preparing products for screening or assisting in screening new Populus tomentosa varieties with high growth potential; (4) Preparing products for molecular marker - assisted breeding of Populus tomentosa.

[0057] The growth potential of the Populus tomentosa includes tree height, ground diameter, diameter at breast height, and volume.

[0058] The fifth aspect of the present invention provides a method for genetic improvement of Populus tomentosa. Preferably, the method includes the step of successive selection of individuals with the GA / G genotype of the above - mentioned InDel molecular marker and elimination of all other genotype individuals outside this marker.

[0059] The method for genetic improvement of Populus tomentosa includes the following steps:

[0060] Step 1: Extract genomic DNA of Populus tomentosa;

[0061] Step 2: Use the genomic DNA of Populus tomentosa as a template for PCR amplification;

[0062] Step 3: Determine the genotype of the InDel molecular marker of the Populus tomentosa to be tested;

[0063] Step 4: Evaluate the growth potential of the Populus tomentosa to be tested according to the genotype detection results;

[0064] The growth potential of the Populus tomentosa includes tree height, ground diameter, diameter at breast height, and volume.

[0065] Step 5: Successively select individuals with the GA / G genotype of the InDel molecular marker for breeding.

[0066] Principal component analysis (PCA) is an effective means of extracting key information from complex phenotypic traits. It can convert a set of related variables into a set of smaller uncorrelated variables as principal components (PCs), while retaining the original information to the greatest extent. By constructing a genetic map and performing QTL mapping, major functional intervals highly related to the target trait can be captured across the entire genome. Subsequently, a phenotypic-single marker student test (T-test) is conducted on the candidate intervals to obtain the main functional sites of the candidate QTL intervals. The combined application of the above methods can help us identify major functional genes at the whole genome level and single variant site level, improving the accuracy and efficiency of forest tree breeding.

[0067] HD-ZIP (homeodomain-leucine zipper) proteins are plant-specific transcription factors that play important roles in plant growth, development, and environmental responses. Their gene family consists of two functional domains, namely the homeodomain (HD) and the leucine zipper (LZ). According to their sequence conservation and functional characteristics, HD-ZIP is further divided into four subfamilies (HD-ZIP I, HD-ZIP II, HD-ZIP III, and HD-ZIP IV). Among them, the subfamily II proteins have a conserved LxLxL domain (called the EAR domain, Leu×Leu×Leu, as Figure 1 shown) at the N-terminus, which is mainly involved in the responses of plant growth, development, and stress (Genes (Basel) 17; 12(8):1256(2021)). The EAR domain can interact with the transcriptional repressor TPL / TPR to form a transcriptional repression complex, enabling the transcription factor to exert its transcriptional repression function, while the HD-ZIP domain binds to specific motifs of downstream genes to regulate the functions of downstream genes (Plant Biotechnol J 16(2):495-506(2017)).

[0068] Therefore, developing a molecular-assisted breeding design strategy for identifying major genes and functional sites based on multi-phenotypic combined breeding can have a higher breeding efficiency than traditional single-phenotypic breeding, greatly shortening the breeding time and improving the combined breeding ability of multiple wood yield traits in the breeding population.

[0069] Examples

[0070] Example 1

[0071] (1) In spring, the tree height, diameter at breast height (DBH), ground diameter, and volume of 80 two-year-old hybrid poplar populations [3-85-1×LM50] (male parent LM50, female parent 3-85-1, 3 clonal replicates) of Populus tomentosa were collected. The res.pca in the R package was used to extract key information from the four growth traits of tree height, DBH, ground diameter, and volume. The first three PCs explained 91.4% of the phenotypic variation of the four traits (PC1 - 60.7%, PC2 - 18.5%, PC3 - 12.3%, Table 1). Therefore, the first three PCs were used as comprehensive growth traits and retained for QTL mapping.

[0072] Table 1 Principal component analysis of four phenotypic traits by PCA

[0073] Principal components PCs Standard deviation Variance proportion Cumulative proportion PC1 2.5835 0.6068 0.6068 PC2 1.4269 0.1851 0.7919 PC3 1.1607 0.1225 0.9143 PC4 0.6635 0.04002 0.95437 PC5 0.46449 0.01961 0.97398 PC6 0.33397 0.01014 0.98412 PC7 0.30347 0.00837 0.99249 PC8 0.19141 0.00333 0.99582 PC9 0.14945 0.00203 0.99785 PC10 0.12382 0.00139 0.99925 PC11 0.09104 0.00075 1

[0074] (2) Genomic DNA was extracted using the genotype samples described in Example 1 and used to construct a resequencing library according to the sample preparation instructions of Illumina. Then, Trimmomatic v0.39 and Fastp v0.23.1 were used to identify read segments contaminated with adapters and trim the adapter sequences from the read segments. After checking the quality of the raw sequencing data using FastQC v0.10.1, all high-quality reads were mapped to the Populus tomentosa reference genome using BWA v0.7.11. After alignment, SNPs and InDels were called using SAMtools v1.13 and GATK v4.2. GATK was used to remove variant clusters with 3 or more SNPs and InDels within a 10bp window region. The OneMap package in the R package was used to align the hybrid parents and offspring populations, and markers that did not conform to the genetic laws were deleted. The Lep-MAP3 and Kosambi functions were used to construct a genetic map for the hybrid population, and then the genetic map was imported into the OneMap package for Bin map construction to eliminate redundant genetic information. Using the ICIM function of the GACD v1.2 software, QTL mapping was performed on the three PCs based on the constructed Bin map, and the statistical significance LOD threshold was determined by 1000 iterations of calculation (LOD threshold = 6.40, Type I error < 0.05). Finally, the QTL interval with the highest LOD value was selected as the significant QTL interval (LOD = 12.5511).

[0075] (3) SNPs and InDels within the significant QTL intervals were extracted using VCFtools v0.1.17, and a T-test for growth vigor traits was performed on the markers using Plink v1.90. Through the Benjamini-Hochberg FDR correction test, a significant association was detected for the InDel molecular marker at position 594,919 on chromosome 7 (FDR_BH = 0.009, Table 2). This locus is within the CDS region of the gene Ptom.007G.00087 (PtoHAT22), and the genotype is GA / G. Through single-locus phenotype-genotype variation analysis, it was found that when the genotype is GA / G, the growth vigor of poplar is significantly higher than that of the genotype GA / GA( Figures 2 - 6 , Table 3-8, *(P < 0.05), **(P < 0.01), *** (P < 0.001)).

[0076] Table 2 T-Test Analysis of Growth Vigor Traits at Loci in the Significant Interval

[0077]

[0078]

[0079]

[0080] Table 3 Significance Test of PC 1 Index Differences in Populations Carrying Two Genotypes

[0081]

[0082] Table 4 Significance Test of Tree Height Index Differences in Populations Carrying Two Genotypes

[0083]

[0084]

[0085] Table 5 Significance Test of DBH Index Differences in Populations Carrying Two Genotypes

[0086]

[0087] Table 6 Significance Test of Ground Diameter Index Differences in Populations Carrying Two Genotypes

[0088]

[0089] Table 7 Significance Test of Volume Index Differences in Populations Carrying Two Genotypes

[0090]

[0091]

[0092] Table 8 Genotypes and phenotypic traits of InDel loci of individuals in the Populus tomentosa hybrid population

[0093]

[0094]

[0095]

[0096] (4) RNA of leaves of 80 genotype individuals in the Populus tomentosa hybrid population was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Novoprotein Scientific Co., Ltd.). The RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novoprotein Scientific Co., Ltd.). Referring to the Populus tomentosa reference genome as the template, the primer blast tool (provided by the National Center for Biotechnology Information (NCBI), USA) was used to design primers according to the various principles of primer design as shown in Table 9. Using the primers shown in Table 9, PCR amplification was performed with the obtained genomic cDNA as the template as shown in Table 9.

[0097] Table 9 Primer sequences

[0098] Primer name Primer sequence P1 (SEQ ID NO.1) ATGGGGTGCCTTGATGATGGGT P2 (SEQ ID NO.2) ACATGCTGCCGAAGGATTAGTGAAGG

[0099] The CDS sequence (SEQ ID NO.0) of PtoHAT22 of individuals with the InDel genotype GA / GA is as follows (underlined are primers, and the base marked in red with shadow is the InDel molecular marker):

[0100]

[0101] The amino acid codons are shown in Table 10:

[0102] Table 10

[0103]

[0104] According to the degeneracy of codons, the amino acid sequence (SEQ ID NO.4) of the PtoHAT22 gene (GA / GA genotype) is as follows:

[0105] MGCLDDGCNTGLVLGLGFTTTNLENTSRPADNNKRLIKPQIKPLMTGFEPSLSLGLSAETYSLVDGKKGCEESVGAHDQLYRQASPHSAVSSFSSGRVKRERDLSSEDIEVERVSSRVSDEDEDGTNARKKLRLTKEQSALLEESFKQHSTLNPKQKQALARQLNLRPRQVEVWFQNRRARTKLKQTEMDCEFLKKCCETLTDENRRLQKELQDLKSLKMAQPFYMHMPAATLTMCPSCERIGGVGEGASKSPFSMATKPHFYNSFTNPSAAC* (“*” is the stop codon)

[0106] The CDS sequence (SEQ ID NO.3) of PtoHAT22 in individuals with the InDel genotype GA / G is as follows (the base marked in red and shaded is the InDel molecular marker):

[0107]

[0108]

[0109] According to the codon degeneracy, the amino acid sequence (SEQ ID NO.5) of the PtoHAT22 gene (GA / G genotype) is as follows:

[0110] MGCLDDGCNTGLVLGLGFTTTNLENTSRPADNNKRLIKPQIKPLMTGFEPSLSLGLSAETYSLVDGKKGCEESVGAHDQLYRQASPHSAVSSFSSGRVKRERTLAVKI*

[0111] The amino acid sequence alignment of the GA / GA genotype and the GA / G genotype of the PtoHAT22 gene is as Figure 7 shown.

[0112] The PtoHAT22 gene contains a homeodomain and a leucine zipper domain LZ. These two functional domains enable the PtoHAT22 gene to regulate plant growth and development. In individuals with the GA / G genotype, the PtoHAT22 gene lacks 1 bp of the base sequence, resulting in premature termination of amino acid coding, thus affecting the function of the PtoHAT22 gene and ultimately affecting plant growth and development( Figure 1 , Figure 7 ).

[0113] Extract the RNA of Populus tomentosa individuals and perform reverse transcription to obtain cDNA. Determine the 305th base in the CDS region of the PtoHAT22 gene encoded by the candidate Populus tomentosa individuals, determine the genotype of this site, and judge the overall growth potential phenotype of the poplar individuals based on the genotype of this site. Refer to the Populus tomentosa reference genome as a template, comprehensively consider the primer design principles, extract the genomic cDNA sequence of PtoHAT22 and perform primer design as shown in Table 11. Using the primers shown in Table 11, perform PCR amplification with the obtained genomic cDNA as a template, as shown in Table 11.

[0114] Table 11 Primer sequences for extracting the PtoHAT22 genome

[0115] Primer name Primer sequence P3 (SEQ ID NO.3) TCATGATCAGTTGTATCGTCAAG P4 (SEQ ID NO.4) AGATTGCTCTTTGGTGAGTCTAAGT

[0116] The CDS sequence of the PtoHAT22 gene (SEQ ID NO.0) is as follows (the underlined part is the primer, and the position of the InDel is marked in shaded red), and the InDel molecular marker is located between primers P3 and P4:

[0117]

[0118]

[0119] Determine the genotype of the InDel molecular marker of the poplar individuals to be tested, and evaluate the comprehensive growth potential score of the poplar individuals to be tested according to the genotype detection results. When the 305th base in the CDS region of the PtoHAT22 gene is the GA / G genotype, the growth potential of the Populus tomentosa individuals is significantly higher than that of the individuals with the GA / GA genotype ( Figures 2 - 6 , Table 3-8, *(P<0.05), **(P<0.01), *** (P<0.001)).

[0120] Select and breed the individuals with the InDel molecular marker of the GA / G genotype for breeding.

[0121] Example 2

[0122] In spring, randomly select 302 Populus tomentosa individuals with different genotypes from 1047 natural populations of Populus tomentosa (Beijing Forestry University Base, Guan County, Shandong), and measure their tree height, breast diameter, ground diameter and volume. Collect 3 functional leaves (i.e., the 4th to 6th leaves at the top of the stem), immediately put them into liquid nitrogen (-196 °C) for preservation, and extract the individual genomic cDNA. Use the kit related to the PtoHAT22 gene to determine the genotype of the 305th base in the CDS region of the PtoHAT22 gene. As shown in Tables 12 - 16, Figures 8 - 11 as shown.

[0123] In this example, among the 302 Populus tomentosa individuals, there are 237 individuals with the InDel molecular marker GA / G genotype and 65 individuals with the GA / GA genotype. The average tree height of individuals with the GA / G genotype in the population is 503.4 cm, and the average tree height of individuals with the GA / GA genotype is 458.5 cm; the average diameter at breast height of individuals with the GA / G genotype is 2.94 cm, and the average diameter at breast height of individuals with the GA / GA genotype is 2.66 cm; the average ground diameter of individuals with the GA / G genotype is 3.40 cm, and the average ground diameter of individuals with the GA / GA genotype is 3.13 cm; the average volume of individuals with the GA / G genotype is 1531.7 cm 3 , and the average tree height of individuals with the GA / GA genotype is 1158.7 cm 3 .

[0124] Therefore, PtoHAT22 and the InDel molecular marker can significantly change the growth potential traits of Populus tomentosa. The kit developed using this gene and marker can quickly and accurately identify fast-growing and excellent germplasms at the early stage of poplar growth and development, thus greatly improving the breeding accuracy of target traits and significantly shortening the breeding cycle.

[0125] Table 12 Genotypes and phenotypic traits of InDel loci in 302 individuals

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Table 13 Significance test of differences in tree height indexes of 302 individuals with two genotypes

[0135]

[0136]

[0137] Table 14 Significance test of differences in diameter at breast height indexes of 302 individuals with two genotypes

[0138]

[0139] Table 15 Significance test of differences in ground diameter indices of a population of 302 plants carrying two genotypes

[0140]

[0141] Table 16 Significance test of differences in volume indices of a population of 302 plants carrying two genotypes

[0142]

[0143]

[0144] The present invention has been described in detail above in combination with specific embodiments and / or exemplary examples and the accompanying drawings. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications or improvements can be made to the technical solutions and their implementation manners of the present invention, and these all fall within the scope of the present invention. The protection scope of the present invention is subject to the appended claims.

Claims

1. A molecular marker tightly linked to the growth vigor trait of Populus tomentosa seedlings. The molecular marker is an InDel molecular marker, and the InDel of the molecular marker is located at the 594919th base of chromosome 7 of the Populus tomentosa genome, with GA / G and GA / GA genotype polymorphisms.

2. The molecular marker according to claim 1, characterized in that The molecular marker is located in the coding region of the gene PtoHAT22. The nucleotide sequence of the gene PtoHAT22 is shown in SEQ ID NO:

3.

3. The molecular marker according to claim 1, characterized in that, The growth vigor traits of Populus tomentosa seedlings tightly linked to the molecular marker include the tree height, ground diameter, breast diameter, and volume of Populus tomentosa.

4. The molecular marker according to claim 1, characterized in that, Individuals with the GA / G genotype of the molecular marker have a higher tree height, larger breast diameter, larger ground diameter, larger volume, and overall higher growth vigor.

5. A primer pair for amplifying the molecular marker closely linked to the Populus tomentosa seedling growth vigor trait described in claim 1 or 2, wherein the molecular marker is characterized in that The primer pair includes primer P1 and primer P2. Among them, primer P1 has the nucleotide sequence shown in SEQ ID NO:1, and primer P2 has the nucleotide sequence shown in SEQ ID NO:

2.

6. A method for obtaining a molecular marker tightly linked to the growth vigor trait of Populus tomentosa seedlings as claimed in claim 1 or 2, the method comprising the following steps: Step 1, measuring the phenotypic traits of the Populus tomentosa hybrid population; Step 2, extracting genomic DNA from the Populus tomentosa population; Step 3, performing PCA analysis on the phenotypic traits to fit the data; Step 4, performing QTL mapping using the genetic map and performing T-test phenotypic-marker association analysis on the significant QTL interval; Step 5, obtaining genes and InDel molecular markers related to the growth vigor of Populus tomentosa.

7. An application of the molecular marker tightly linked to the growth vigor trait of Populus tomentosa seedlings as claimed in claim 1 or 2 in any one of the following aspects (1) to (4): (1) Screening or assisting in screening new Populus tomentosa varieties with high growth vigor at the seedling stage; (2) Molecular marker-assisted breeding of Populus tomentosa; (3) Preparing products for screening or assisting in screening new Populus tomentosa varieties with high growth vigor; (4) Preparing products for molecular marker-assisted breeding of Populus tomentosa.

8. A method for genetic improvement of Populus tomentosa, characterized in that, The method includes successive selection of individuals with the GA / G genotype of the InDel molecular marker as claimed in claim 1 or 2.

9. The method according to claim 8, characterized in that The method includes the following steps: Step 1, extracting genomic DNA of Populus tomentosa; Step 2, using the genomic DNA of Populus tomentosa as a template for PCR amplification; Step 3, determining the genotype of the InDel molecular marker of the Populus tomentosa to be tested; Step 4, evaluating the growth vigor of the Populus tomentosa to be tested according to the genotype detection result; Step 5, successively selecting individuals with the GA / G genotype of the InDel molecular marker for breeding.

10. The method according to claim 9, characterized in that, In step 4, the growth vigor of the Populus tomentosa includes tree height, ground diameter, breast diameter, and volume.

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