SNP (Single Nucleotide Polymorphism) marker related to poplar leaf rust and application

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker related to poplar leaf rust and application, and relates to the technical field of genetic breeding of poplar. The marker is located at the 14699534th bp position on a chromosome 13 of a reference sequence of a 4.1 version of a populus trichocarpa genome. Through multi-model whole genome association analysis (GWAS), it is determined that the gene is a key regulation and control site affecting the leaf rust of the poplar, meanwhile, expression of genes related to the leaf rust is affected, a new choice is provided for breeding leaf rust resistant poplar germplasm, and the gene has important application value in accelerating the poplar breeding process.

Description

Technical Field

[0001] The present invention relates to an SNP (single nucleotide polymorphism) marker related to poplar leaf rust and its application, belonging to the technical field of poplar genetic breeding. Background Art

[0002] Populus spp. is an important tree species for economic and ecological forests. It grows rapidly and has a wide range of wood uses. At the same time, it plays an important role in ecological protection and carbon sequestration. However, due to reasons such as improper variety selection, unsuitable planting range, or ineffective technical measures, poplars face many disease threats during cultivation. Among them, leaf rust caused by Melampsora sp. occurs most commonly, seriously affecting the development of poplar timber forests and protection forests.

[0003] At present, the prevention and control strategies for poplar leaf rust mainly include chemical control and disease-resistant breeding. Chemical control has a high cost. Long-term use is likely to cause the pathogen to develop drug resistance and pollute the environment and ecological systems. Traditional disease-resistant breeding mainly relies on field phenotypic screening and hybridization breeding. However, the growth cycle of poplars is relatively long, and traditional breeding has low efficiency, and the disease resistance is greatly affected by environmental factors. Determining the resistance difference to leaf rust at the seedling stage can effectively predict the disease resistance performance in the later growth stage and accelerate the identification and utilization of disease-resistant germplasm resources.

[0004] Therefore, providing a molecular marker related to poplar leaf rust and its application for the early selection of poplars, reducing the breeding cost, shortening the breeding cycle, and accelerating genetic progress; by directly identifying this marker locus and screening poplar germplasms with different leaf rust resistances, excellent varieties resistant to leaf rust can be accurately and efficiently selected, which has become an urgent technical problem to be solved in this technical field. Summary of the Invention

[0005] In order to overcome the above problems, the present invention proposes a molecular marker related to poplar leaf rust resistance and its application for the early selection of poplars, reducing the breeding cost, shortening the breeding cycle, and accelerating genetic progress. By directly identifying this marker locus and screening poplar germplasms with different leaf rust resistances, excellent varieties resistant to leaf rust can be accurately and efficiently selected.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] An SNP molecular marker related to poplar leaf rust resistance, which is located at the 14,699,534th bp on chromosome 13 of the reference sequence of Populus trichocarpa genome version 4.1, with T / C polymorphism.

[0008] Preferably, the alleles of the SNP molecular marker are T and C.

[0009] Preferably, the genotypes corresponding to the alleles of the SNP molecular marker are TT genotype, TC genotype, and CC genotype, respectively.

[0010] The present invention also provides a method for obtaining the above SNP molecular marker related to poplar leaf rust resistance, comprising the following steps:

[0011] Step 1, extracting leaf DNA of poplar cultivated germplasm resources and performing whole-genome resequencing;

[0012] Step 2, measuring the phenotypic traits of poplar leaf rust resistance;

[0013] Step 3, performing multi-model GWAS (Genome-wide association studies) analysis to screen for key loci.

[0014] Preferably, in Step 1, the poplar cultivated germplasm resources are germplasms of the section Populus, section Aigeiros, inter-sectional hybrids, etc.

[0015] Preferably, Step 1 is specifically as follows:

[0016] Using the improved cetyltrimethylammonium bromide (CTAB) method to extract total DNA from poplar germplasm resource leaves, constructing an Illumina sequencing library, sequencing with Illumina HiSeqPE150, reading paired-end reads with a length of 150 bp, with a sequencing depth of approximately 30×, performing quality control on the original reads using Perl scripts, and then using the BWA v.0.7.8 software to align high-quality paired-end sequencing reads to the Populus trichocarpa reference genome v4.1 (https: / / phytozome-next.jgi.doe.gov / info / Ptrichocarpa_v4_1); performing SNP calling using the Genome Analysis Toolkit (GATK) v.4.0.4.0 with default parameters (SNP calling refers to the process of detecting and identifying single nucleotide polymorphisms (SNPs) from genomic sequencing data through bioinformatics methods).

[0017] Preferably, Step 2 is specifically as follows:

[0018] Grading according to the proportion of uredinia on diseased leaves in the total leaf area, and then calculating the disease index of each cultivated germplasm according to the grading and identification results.

[0019] Preferably, Step 3 is specifically as follows:

[0020] Using Vcftools v.0.1.16, quality control was performed on the original file with the filtering criteria: minor allele frequency (MAF) 0.05, Geno 0.02; subsequently, using Plink v2.0, linkage disequilibrium (LD) analysis was performed on the SNPs after the initial screening, and the screening criteria were set as LD 50-1-0.8 to screen out SNPs with high linkage disequilibrium. Finally, 1,348,536 SNPs were obtained for association analysis; among them, there were 4 single-locus genome-wide association study (GWAS) models: the MLM (Mixed linear model) model, CMLM (Compressed mixed linear model) model, SUPER (Settlement of MLM under progressively exclusive relationship) model based on the GAPIT3 software, and the LMM (Linear mixed model) model based on the GEMMA (Genome-wide efficient mixed model association algorithm) software. The significance threshold was set as p < 1 / N, where N is the number of SNPs. Subsequently, candidate genes were detected in the 20 kb regions upstream and downstream of the significant loci.

[0021] Preferably, in step 3, among the 4 single-locus GWAS models, the MLM model and the CMLM model detected the fewest significant loci. The LMM model of the GEMMA software detected 12 significant loci, and the SUPER model detected the most significant loci, which was 24.

[0022] Preferably, in step 3, the MLM model, CMLM model, and SUPER model produced similar association results. Among them, 1 significant locus was detected by all 4 GWAS models, and 3 significant loci were detected by two models (the GEMMA model and the SUPER model) (although the SUPER model has a risk of false positives, its consistency with other models further enhanced the genetic stability of these loci).

[0023] Preferably, in step 3, to further search for stable genetic loci related to leaf rust, subsequent comparison and analysis were carried out. The Chr13_14699534 (T / C) locus was detected in both the SUPER model and the GEMMA model. Although its p-value did not reach the significance level in the MLM model and the CMLM model, it was close to the significant range, indicating that these models produced more statistically significant results for the core loci.

[0024] Preferably, in step 3, through further analysis, it was found that the genotype TT at the Chr13_14699534 (T / C) locus mainly appears in highly resistant and a few disease-resistant germplasms, while the highly susceptible germplasms and only individual susceptible germplasms are genotype CC, and the phenotypic differences corresponding to the two genotypes are significant. Subsequently, through Sanger sequencing, this locus was verified, and the results were consistent with the GWAS analysis results.

[0025] Preferably, in step 3, a gene BBX29 (Potri.013G150500) was annotated in the upstream region of the Chr13_14699534 (T / C) locus. Through qRT-PCR analysis, it was found that the relative expression level of BBX29 in disease-resistant germplasms was significantly lower than that in susceptible germplasms.

[0026] Through the above analysis process, the present invention identified SNP loci related to poplar leaf rust resistance. The alleles of the SNP marker located at position 14,699,534 on chromosome 13 of the Populus trichocarpa genome version 4.1 reference sequence are T and C, corresponding to three genotypes, namely TT genotype, TC genotype, and CC genotype.

[0027] The above research results prove that the poplar germplasm with the genotype TT of the SNP marker located at position 14699534 on chromosome 13 of the Populus trichocarpa genome version 4.1 has significantly higher rust resistance than the poplar germplasm with the genotype CC.

[0028] The object of the present invention is to provide the application of the above SNP molecular markers related to poplar leaf rust resistance.

[0029] The above object of the present invention is achieved by the following technical solutions:

[0030] The application of SNP molecular markers related to poplar leaf rust resistance in regulating poplar leaf rust resistance.

[0031] Compared with the prior art, the beneficial effects of the present invention:

[0032] The present invention uses poplar cultivated germplasm resources such as Sect. Populus, Sect. Aigeiros, and Hybrids between Sections as materials. Through leaf rust resistance identification and combined with multi-model genome-wide association analysis (GWAS), an SNP marker located at locus 14,699,534 on chromosome 13 of poplar is identified to be significantly associated with the leaf rust resistance phenotype. This molecular marker can stably indicate different leaf rust resistance types, shorten the breeding cycle, and accelerate the breeding process of disease-resistant new varieties. By using the molecular marker in the present invention, a reliable and efficient technical means can be provided for the disease resistance screening of poplar germplasm resources and molecular-assisted breeding for leaf rust resistance.

[0033] The following further illustrates the present invention through specific embodiments and drawings, but it does not mean limiting the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a representative image example of the front and back sides of poplar leaves with different degrees of leaf rust infection in Example 1 of the present invention;

[0035] Figure 2 It is the leaf rust resistance distribution of poplar cultivated germplasm in Example 1 of the present invention;

[0036] Figure 3-1 It is the Manhattan plot of single-site GWAS analysis in Example 1 of the present invention;

[0037] Figure 3-2 It is the QQ plot of single-site GWAS analysis in Example 1 of the present invention;

[0038] Figure 4 It is the haplotype analysis of the SNP locus (Chr13_14699534(T / C)) in Example 1 of the present invention;

[0039] Figure 5 It is the Sanger sequencing of the genotypes of the SNP locus (Chr13_14699534(T / C)) in different germplasms in Example 1 of the present invention;

[0040] Figure 6 It is the qRT-PCR (Quantitative Real-Time PCR) analysis of the annotated genes of the SNP locus (Chr13_14699534(T / C)) in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The following further describes the present invention in combination with specific embodiments. It should be understood that the following text is only used to describe one or several specific implementation manners of the present application, and does not strictly limit the scope of protection specifically requested by the present application. Without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0042] Unless otherwise specified, the reagents involved in the following examples are all conventional reagents that can be purchased on the market, and the methods used are all common methods in the technical field of the present invention.

[0043] Example 1

[0044] In Example 1 of the present invention, a systematic evaluation of the resistance to leaf rust of poplar cultivated germplasm resources such as Populus sect. Leuce, Populus sect. Aigeiros, and inter-sectional hybrids was first carried out; multi-model GWAS analysis was performed through whole-genome resequencing to discover SNP loci significantly associated with target traits and genes; by genotyping specific loci of the test population, early selection of poplar resistant to leaf rust was achieved, thereby saving breeding costs, time, and accelerating genetic progress. Specifically as follows:

[0045] 1. Materials and Methods

[0046] 1.1 Poplar Cultivated Germplasm Materials and Experimental Design

[0047] In March 2021, the collected poplar cultivated germplasm resources, including 14 germplasms of Populus sect. Leuce, 80 germplasms of Populus sect. Aigeiros, and 41 inter-sectional hybrids (37 Populus sect. Aigeiros × Populus sect. Tacamahaca, 1 Populus sect. Turanga × Populus sect. Tacamahaca, 3 Populus sect. Aigeiros × Populus sect. Populus), a total of 135, were cuttaged and propagated in the greenhouse. After growing for two months, they were transplanted to the nursery of the Chinese Academy of Forestry Sciences. According to the completely randomized block design, 3 blocks were set, with 6 plants per plot for each germplasm, and the plant spacing was 40×50 cm;

[0048] 1.2 Investigation and Resistance Identification of Leaf Rust in Poplar Cultivated Germplasm

[0049] In March 2022, all poplar cultivated germplasms were stubbled. In September 2022, the degree of leaf rust infection of different poplar cultivated germplasms in the first year was investigated; about 20 leaves were investigated for each plant, and the grading was carried out according to the proportion of uredinia on the diseased leaves in the total leaf area. As shown in Table 1, the disease grade, representative values, and grading standards are listed;

[0050] Table 1

[0051]

[0052] According to the above survey results, calculate the disease index of different poplar cultivation germplasms. The formula for calculating the disease index is: ∑(representative value of each damage level × number of leaves at this level) × 100 / (representative value of the highest damage level × total number of sampled leaves). Subsequently, identify its resistance level based on the disease index, as shown in Table 2 below;

[0053] Table 2

[0054]

[0055] 1.3 Whole-genome resequencing

[0056] Using the improved CTAB method, total DNA was extracted from the leaves of poplar germplasm resources for constructing an Illumina sequencing library. Sequencing was performed with Illumina HiSeqPE150 to obtain paired-end reads with a read length of 150 bp and a sequencing depth of approximately 30×. Perl scripts were used to perform quality control on the original reads. Subsequently, using BWA v.0.7.8 software, the high-quality paired-end sequencing reads were aligned to the Populus trichocarpa reference genome v4.1 (https: / / phytozome-next.jgi.doe.gov / info / Ptrichocarpa_v4_1); SNP calling was performed using the default parameters of Genome Analysis Toolkit (GATK) v.4.0.4.0;

[0057] 1.4 Multi-model GWAS analysis

[0058] Using Vcftools v.0.1.16, quality control was performed on the original file with the filtering criteria: maf 0.05, Geno 0.02. Subsequently, using Plink v2.0, linkage disequilibrium (LD) analysis was performed on the pre-screened SNPs, and the screening criteria were set as LD 50-1-0.8 to screen out SNPs with high linkage disequilibrium for subsequent genome-wide association analysis (GWAS). Among them, four single-locus models were included, namely the MLM model, CMLM model, SUPER model based on GAPIT3 software, and the LMM model based on GEMMA software. The significance threshold was set as p < 1 / N, where N is the number of SNPs. Subsequently, candidate genes were detected within the 20 kb region upstream and downstream of them;

[0059] 2. Results

[0060] 2.1 Phenotypic identification results of leaf rust resistance of poplar cultivated germplasm resources

[0061] As Figure 1 shown, the following are representative front and back images of poplar leaves with different degrees of leaf rust infection selected from the leaf rust resistance survey results of 135 poplar cultivated germplasms in Example 1 of the present invention: from left to right are 1. Populus 'Qinbaiyang5'; 2. Populus deltoides 'Zhongcheng2'; 3. Populus deltoides×Populus simonii '96 - 64 - 046'; 4. Populus 'Jingtong2'; 5. Populus deltoides '16 - 18'.

[0062] As Figure 2 and Table 3 show, among the identified materials, there are 29 highly resistant germplasms, accounting for 21.48% of the identified materials; 67 resistant germplasms, accounting for 49.63%; 29 susceptible germplasms, accounting for 21.48%; 10 highly susceptible germplasms, accounting for 7.41%; germplasms of the same section or different sections show great differences in leaf rust resistance; as shown in Table 3, 14 germplasms in the section Populus are all highly resistant, and most germplasms in the section Aigeiros show resistance (such as Populus×canadensis 'Guariento', Populus deltoides 'Zhongcheng2', Populus deltoides 'Huanghuai1', etc.); compared with the germplasms of the sections Populus and Aigeiros, there are more susceptible germplasms among the inter - sectional hybrids, and most of them are hybrids between the section Aigeiros and the section Tacamahaca (such as Populus 'Jingtong2', Populus 'Beijingensis82', Populus 'Senhai2', etc.); and the phenomenon of resistance differentiation to leaf rust is found within the section Aigeiros or in the same hybrid combination between the section Aigeiros and the section Tacamahaca; according to the seedling - stage resistance identification results, the overall resistance level of 135 poplar cultivated germplasms to leaf rust is relatively high;

[0063] Table 3

[0064]

[0065] 2.2 Multi - model GWAS analysis of leaf rust resistance

[0066] As Figure 3-1 shown, the Manhattan plot of single - locus GWAS analysis in Example 1 of the present invention, where the abscissa is the chromosome, the red dashed line represents the significance threshold, and the points exceeding the red dashed line are significant SNP loci, and the SNP loci co - located in each model are indicated by the red arrows; AsFigure 3-2 As shown, it is the QQ plot of single-site GWAS analysis in Example 1 of the present invention. Among them, the abscissa represents the -log transformation of the theoretical p-value 10 transformation, and the ordinate represents the -log transformation of the actually observed p-value 10 transformation. The red diagonal line represents the situation where the theoretical expected p-value distribution and the actually observed p-value distribution are exactly the same. The green dots represent SNP loci; finally, 4 significant SNP loci are detected by two or more GWAS models; to further seek stable genetic loci related to leaf rust, the co-localized significant SNP loci are compared and analyzed, and it is found that Chr13_14699534(T / C) is detected in both the SUPER model and the GEMMA model. It is worth noting that although this locus does not reach the significance level in the MLM model and the CMLM model, its p-value is close to the significant range;

[0067] In order to reveal whether this marker is related to poplar leaf rust resistance, the present invention Figure 4 As shown, it is the haplotype analysis of the SNP locus (Chr13_14699534(T / C)) in Example 1 of the present invention. Among them, the abscissa represents different genotypes (TT, TC, and CC) of this locus, and the ordinate represents the disease index; as Figure 5 shown, it is the Sanger sequencing of the genotypes of the SNP locus (Chr13_14699534(T / C)) in different germplasms in Example 1 of the present invention. Among them, the left side is the Sanger sequencing results of disease-resistant germplasms (1, Populus 'Qinbaiyang2'; 2, Populus 'Qinbaiyang4'; 3, Populus 'Qinbaiyang5'; 4, Populus 'Xibeiyang1'), and the right side is the Sanger sequencing results of disease-susceptible germplasms (1, P. deltoides '27-8'; 2, P. deltoides '278-1'; 3, P. deltoides '16-18'; 4, P.×canadensis 'J2'); the genotype TT of the Chr13_14699534(T / C) locus mainly appears in highly resistant and a few disease-resistant germplasms, while highly susceptible germplasms and only a few disease-susceptible germplasms are genotype CC, and the corresponding phenotypic differences between the two genotypes are significant;

[0068] As Figure 6As shown in the figure, the qRT-PCR analysis of the annotated genes of the SNP locus (Chr13_14699534(T / C)) in the resistant and susceptible cultivated germplasms in Example 1 of the present invention: Among them, the resistant germplasms are: 1. Populus alba 'Yx-6'; 2. Populus 'Qinbaiyang2'; 3. Populus bolleana; 4. Populus 'Qinbaiyang3'; 5. Populus 'Qinbaiyang4'; 6. Populus 'Xifeng25'; 7. Populus deltoides 'Huanghuai1'; 8. Populus 'Xifeng77'; 9. Populus 'Qinbaiyang1'; 10. Populus × canadensis 'Guariento'; The susceptible germplasms are: 1. Populus deltoides '278-1'; 2. Populus 'wq84'; 3. Populus deltoides 'Lux'; 4. Populus 'Beijingensis82'; 5. Populus 'wq91'; 6. Populus deltoides '2025'; 7. Populus deltoides 'Shan599'; 8. Populus deltoides '27-4'; 9. Populus 'Qinheiqingyang1'; 10. Populus 'Qinheiqingyang2'; The red font represents the significant analysis results (T-test) of the relative expression level differences of the gene (BBX29) between the resistant germplasm and the susceptible germplasm; The relative expression level of the key candidate gene BBX29 in the resistant germplasm (TT genotype) within the interval where this locus is located is significantly lower than that in the susceptible germplasm (CC genotype) (p = 0.006).

[0069] From the above, it can be concluded that at the 14,699,534bp locus on chromosome 13 of the reference sequence of the 4.1 version of the Populus trichocarpa genome, the mutation of the T / C base affects the expression of the genes related to Populus leaf rust, and this mutation affects the resistance difference to leaf rust. This SNP locus can be used as a molecular marker for screening the resistance phenotype of Populus leaf rust and applied in the process of Populus molecular breeding.

[0070] The present invention uses poplar cultivated germplasm resources such as the Leuce section, Aigeiros section, and inter-sectional hybrids as experimental materials, determines the key regulatory loci affecting the resistance of Populus leaf rust through multi-model GWAS analysis, and regulates the expression of its related genes, providing a new choice for breeding Populus germplasm resistant to leaf rust, and having important application value in accelerating the process of Populus breeding.

[0071] Although the above has described the purpose, concept and embodiments of the present invention in detail, those of ordinary skill in the art can recognize that various improvements and transformations can still be made to the present invention without departing from the scope defined by the claims, and such improvements and transformations should still fall within the protection scope of the present invention.

Claims

1. An SNP molecular marker related to poplar leaf rust resistance, which is located at 14,699,534 bp on chromosome 13 of the reference sequence of Populus trichocarpa genome version 4.1, with T / C polymorphism.

2. The SNP molecular marker related to poplar leaf rust resistance according to claim 1, characterized in that: The alleles of the SNP molecular marker are T and C.

3. The SNP molecular marker related to poplar leaf rust resistance according to claim 1, characterized in that: The genotypes corresponding to the alleles of the SNP molecular marker are TT genotype, TC genotype, and CC genotype, respectively.

4. The method for obtaining the SNP molecular marker related to poplar leaf rust resistance according to any one of claims 1-3, comprising the following steps: Step 1, extract the leaf DNA of poplar cultivated germplasm resources and perform whole-genome resequencing; Step 2, determine the phenotypic traits of poplar leaf rust resistance; Step 3, perform multi-model GWAS analysis to screen for key loci.

5. The method for obtaining the SNP molecular marker related to poplar leaf rust resistance according to claim 4, characterized in that: In Step 1, the poplar cultivated germplasm resources are germplasms of the Populus alba section, Populus nigra section, inter-sectional hybrids, etc.

6. The method for obtaining SNP molecular markers related to poplar leaf rust resistance according to claim 4, characterized in that: Step 1 is specifically as follows: Adopt the improved CTAB method to extract total DNA from the leaves of poplar germplasm resources for constructing an Illumina sequencing library, sequence with Illumina HiSeqPE150, obtain paired-end reads with a read length of 150 bp, with a sequencing depth of about 30×. Use Perl scripts to perform quality control on the original reads. Subsequently, use the BWA v.0.7.8 software to align the high-quality paired-end sequencing reads to the Populus trichocarpa v4.1 reference genome; use the Genome Analysis Toolkit (GATK) v.4.0.4.0 with default parameters for SNP calling.

7. The method for obtaining SNP molecular markers related to poplar leaf rust resistance according to claim 4, characterized in that: Step 2 is specifically as follows: Grade according to the proportion of uredinia on diseased leaves in the total leaf area. Subsequently, calculate the disease index of each cultivated germplasm according to the grading and identification results.

8. The method for obtaining SNP molecular markers related to poplar leaf rust resistance according to claim 4, characterized in that: Step 3 is specifically as follows: Use Vcftools v.0.1.16 to perform quality control on the original file, with filtering criteria: maf 0.05, Geno 0.02; subsequently, use Plink v2.0 to perform linkage disequilibrium analysis on the pre-screened SNPs, with the screening criterion set as LD 50-1-0.8 to screen out SNPs with high linkage disequilibrium. Finally, obtain 1,348,536 SNPs for association analysis; among them, there are 4 single-locus genome-wide association study models: the MLM model, CMLM model, SUPER model based on the GAPIT3 software, and the LMM model based on the GEMMA software. Set the significance threshold as p < 1 / N, where N is the number of SNPs. Subsequently, detect candidate genes in the 20 kb regions upstream and downstream of the significant loci.

9. The method for obtaining the SNP molecular marker related to poplar leaf rust resistance according to claim 8, characterized in that: Among the 4 single-locus GWAS models, the MLM model and the CMLM model detected the fewest significant loci. The LMM model of the GEMMA software detected 12 significant loci, and the SUPER model detected the most significant loci, which is 24. The MLM model, CMLM model, and SUPER model produced similar correlation results. Among them, 1 significant locus was detected by all 4 GWAS models, and 3 significant loci were detected by two models: the GEMMA model and the SUPER model. To further search for stable genetic loci related to leaf rust, comparison and analysis were carried out, and it was found that the Chr13_14699534(T / C) locus was detected in both the SUPER model and the GEMMA model. Although this locus did not reach the significance level in the MLM model and CMLM model, its p-value was close to the significant range, indicating that these models produced more statistically significant results for the core locus. The genotype TT of the Chr13_14699534(T / C) locus mainly appeared in highly resistant and a few resistant germplasms, while the highly susceptible germplasms and only a few susceptible germplasms were genotype CC. The corresponding phenotypes of the two genotypes were significantly different. Subsequently, Sanger sequencing was used to verify this locus, and the results were consistent with the GWAS analysis results. A gene BBX29 (Potri.013G150500) was annotated in the upstream region of the Chr13_14699534(T / C) locus. Through qRT-PCR, it was found that the relative expression level of BBX29 in resistant germplasms was significantly lower than that in susceptible germplasms.

10. Application of the method for obtaining the SNP molecular marker related to poplar leaf rust resistance according to any one of claims 4-9 in regulating poplar leaf rust resistance.

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