A molecular marker for screening drought-resistant poplars based on interspecific introgression theory, and its method and application

Abstract

The present invention discloses a molecular marker for screening drought-resistant poplars based on the interspecific introgression theory, as well as a method and application thereof. The molecular marker is located at the 1022nd base upstream of the PtoTCP19 gene and has a T / C polymorphism. Individuals with the CC genotype of the molecular marker have the strongest drought resistance. The molecular marker provided by the present invention, obtained based on the interspecific introgression theory, has high accuracy and can accurately evaluate the drought resistance of poplars at the molecular level. It can accurately and efficiently screen drought-resistant tree species during the seedling stage, shortening the breeding cycle, and is of great significance for enhancing plant stress resistance and mitigating the damage caused by drought adversity to forestry yield and quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular breeding, and specifically relates to a molecular marker for screening drought-resistant poplars, a method and an application thereof, and especially relates to a molecular marker for screening drought-resistant poplars based on interspecific introgression theory, a method and an application thereof. Background Art

[0002] Gene introgression refers to the transfer of alleles from a donor species or population to a recipient species or population through hybridization or backcrossing. Many plants and animals have experienced extensive hybridization throughout their evolutionary history. Although the introgressed genes are largely deleterious, they can also acquire adaptive variants from related species. These variants tend to be positively selected and can spread rapidly within the recipient species, rapidly improving their ability to adapt to the environment. As undomesticated perennials, forest trees have high genetic diversity and more extensive interspecific gene flow, making them of great value for genomic research.

[0003] After gene introgression occurs, the introgressed genetic information typically has a genetic background more similar to that of the donor. Based on this principle, traces of introgression can be detected on the genome, locating the specific introgressed area. Adaptive introgressed genes often play an important role in historical evolution or during certain stressful phases such as climate change, helping species survive complex external environments. Whole-genome association analysis is an important method for identifying key genes, but due to the high heterozygosity and complex quantitative traits of forest trees, gene association analysis often cannot directly obtain satisfactory results.

[0004] As the global climate continues to deteriorate, forests in their natural environments are more susceptible to abiotic stresses such as drought, which adversely affects growth, development, timber yield, and material quality. Over the past tens of thousands of years, Asia has experienced numerous major droughts. For example, during the Quaternary Ice Age (60,000 to 70,000 years ago), the Asian monsoon system weakened significantly, leading to reduced precipitation in East and South Asia. During the mid-Holocene (4,200 years ago), solar activity weakened, leading to significant droughts and intensified desertification in Asia, significantly impacting plant survival. Poplars may have improved their population's drought resistance through interspecific genetic introgression, successfully surviving periods of drought and achieving widespread global distribution. As an important ecological and economic tree species in my country, poplars are not only widely used in plantations and vegetation restoration, but also provide sufficient raw materials for the paper and timber industries.

[0005] However, the current arid and semi-arid regions of my country severely restrict the growth, development, and spatial distribution of poplars. With the continuous advancement of molecular breeding techniques, using genetic engineering to improve the stress tolerance of trees offers a cost-effective approach to unlocking the production potential of arid regions. Therefore, it is necessary to provide efficient and precise adaptive introgression genes and related molecular markers associated with drought resistance in poplars to increase the speed and accuracy of breeding drought-resistant species and effectively shorten the poplar breeding cycle. Summary of the Invention

[0006] In order to overcome the above problems, the inventors conducted intensive research and provided adaptive introgression genes and related molecular markers for screening drought-resistant poplars. These genes are obtained based on the theory of interspecific introgression, have high accuracy, and can accurately evaluate the drought resistance of poplars at the molecular level. They can accurately and efficiently screen drought-resistant tree species in the seedling stage, shorten the breeding cycle, and are of great significance for enhancing plant stress resistance and alleviating the damage caused by drought adversity to forestry yield and quality, thereby completing the present invention.

[0007] Specifically, the purpose of the present invention is to provide the following aspects:

[0008] In a first aspect, a gene related to drought resistance of poplar is provided, wherein the gene is PtoTCP19, and the nucleotide sequence thereof includes the sequence shown in SEQ ID NO: 1.

[0009] In a second aspect, a molecular marker for screening drought-resistant poplars is provided, wherein the molecular marker is located at the 1022nd base upstream of the PtoTCP19 gene and has a T / C polymorphism.

[0010] In the third aspect, a method for obtaining the molecular markers described in the second aspect is provided, the method comprising the following steps: step 1, obtaining the adaptive ancestral ancient introgression gene of poplar; step 2, selecting a poplar population and obtaining drought phenotypic data of poplar; step 3, obtaining molecular markers related to drought resistance of poplar.

[0011] In a fourth aspect, a primer pair for amplifying the gene of the first aspect is provided, the primer pair comprising primer P1 and primer P2, primer P1 comprising the nucleotide sequence shown in SEQ ID NO: 4, and primer P2 comprising the nucleotide sequence shown in SEQ ID NO: 5.

[0012] In a fifth aspect, a primer pair for amplifying the molecular marker described in the second aspect is provided, wherein the primer pair includes primer P3 and primer P4, primer P3 includes the nucleotide sequence shown in SEQ ID NO: 6, and primer P4 includes the nucleotide sequence shown in SEQ ID NO: 7.

[0013] In the sixth aspect, a method for genetic improvement of poplars is provided, comprising the steps of successive breeding of poplar individuals with CC genotypes obtained by the molecular markers described in the second aspect or the molecular markers obtained by the method described in the third aspect, and eliminating individuals with other types of genotypes.

[0014] A seventh aspect provides a method for identifying or screening poplars with strong drought resistance, the method comprising the following steps:

[0015] Step 1, extracting genomic DNA from the poplar to be tested;

[0016] Step II, performing PCR amplification using the above-extracted genomic DNA as a template;

[0017] Step III, determining the genotype of the molecular markers of the second aspect of the poplar to be tested;

[0018] Step IV: determining the drought resistance of the tested poplar trees based on the genotype detection results.

[0019] The beneficial effects of the present invention include:

[0020] (1) The PtoTCP19 gene provided by the present invention is significantly associated with drought resistance in poplars and is of great significance for enhancing plant stress resistance and alleviating the damage caused by drought stress to forestry yield and quality;

[0021] (2) The molecular markers for screening drought-resistant poplars provided by the present invention can accurately evaluate the drought resistance of poplars at the molecular level, accurately and efficiently screen drought-resistant tree species during the poplar seedling stage, shorten the poplar breeding cycle, and provide an effective means for molecular marker-assisted selection breeding of Populus tomentosa.

[0022] (3) The method for obtaining molecular markers provided by the present invention can more precisely locate ancestral introgressed genes, which is conducive to better understanding the mixing history between populations, identifying the advantages of certain populations under specific environmental pressures, and exploring certain functional stress-resistant genes that are conducive to adapting to specific environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The phylogenetic tree of the poplar introgression population described in Example 1 of the present invention is shown; Figure 2 The candidate ancestral introgression regions of multiple populations of Populus white poplar described in Example 1 of the present invention are shown; Figures 3-5 The genotype effect diagrams of different genotypes of the SNP marker sites described in Example 1 of the present invention on proline content, abscisic acid content and total antioxidant enzyme activity are shown respectively; Figure 6 a, b, and c in the figure respectively show the genotype effect diagrams of proline content, abscisic acid content, and total antioxidant enzyme activity corresponding to the molecular markers. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the characteristics and advantages of the present invention will become more clear and distinct.

[0025] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0026] In a first aspect, the present invention provides a gene related to drought resistance in poplar trees, wherein the gene is PtoTCP19, and the nucleotide sequence thereof includes the sequence shown in SEQ ID NO: 1. Preferably, the nucleotide sequence thereof is as shown in SEQ ID NO: 1.

[0027] In the present invention, the poplar is preferably Populus tomentosa.

[0028] Poplar is a model species of woody plants, which are divided into the Populus alba, Populus nigra, Populus euphratica and Populus macrophylla schools. Among them, Populus tomentosa in the Populus tomentosa school is a native tree species unique to China and is widely distributed, with the middle and lower reaches of the Yellow River as the central distribution area. It occupies an important position in forestry production and ecological environment construction in northern my country and is a pioneer tree species for forest cultivation in the northern region. Therefore, Populus tomentosa is preferably selected as the research object in the present invention.

[0029] TCP (TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR1) is a plant-specific transcription factor that is widely involved in regulating plant growth and development. TCP transcription factors share an atypical bHLH secondary structure consisting of 55-60 amino acid residues: a basic region-helix-loop-helix (BHLH). This conserved TCP domain binds to DNA and interacts with other proteins, thereby regulating the transcription of target genes.

[0030] According to a preferred embodiment of the present invention, the gene PtoTCP19 related to poplar drought resistance encodes an amino acid sequence including the sequence shown in SEQ ID NO: 2. Preferably, the amino acid sequence encoded by it is as shown in SEQ ID NO: 2.

[0031] TCP proteins are widely involved in plant growth and development and tolerance to abiotic stresses. In Arabidopsis thaliana, overexpression of OsTCP19 enhanced drought and salt tolerance in transgenic plants by reducing water loss and reactive oxygen species accumulation.

[0032] After extensive research, the inventors discovered that the Populus tomentosa TCP19 gene (PtoTCP19) has undergone ancestral introgression and adaptive selection in poplars and is significantly associated with drought resistance. Furthermore, the promoter of this gene contains a REF6 binding site, which regulates ABA, suggesting that this gene likely plays a key role in Populus tomentosa's drought stress response.

[0033] The second aspect of the present invention provides a molecular marker for screening drought-resistant poplars. The molecular marker is located at the 1022nd base upstream of the PtoTCP19 gene in the poplar genome and has a T / C polymorphism.

[0034] In the present invention, the version of the poplar genome is the Populus tomentosa reference genome (published in the National Gene Bank Sequence Archive (CNGB Sequence Archive, CNSA), project number CNP0004290).

[0035] According to a preferred embodiment of the present invention, the molecular marker for screening drought-resistant poplars is located at position 979 of the nucleotide sequence shown in SEQ ID NO: 3.

[0036] In a further preferred embodiment, the genotypes of the molecular markers for screening drought-resistant poplars are CC, TT and CT, among which the poplar individuals with the genotype of the molecular marker CC have the strongest drought resistance, followed by the poplar individuals with the genotype CT, and the poplar individuals with the genotype TT have the weakest drought resistance.

[0037] Among them, the CC genotype indicates that the poplar is homozygous for C at this site, the TT genotype indicates that the poplar is homozygous for T at this site, and the CT genotype indicates that the poplar is a heterozygote of C and T at this site.

[0038] In a further preferred embodiment, the phenotype of poplar drought resistance is proline content, abscisic acid content and total antioxidant enzyme activity; wherein the total antioxidant enzyme activity includes catalase activity, peroxidase activity and superoxide dismutase activity.

[0039] In a further preferred embodiment, the poplar individual whose genotype of the molecular marker is CC has the highest proline content and abscisic acid content, and the highest total antioxidant enzyme activity; the poplar individual whose genotype of the molecular marker is CT has lower proline content and abscisic acid content, and lower total antioxidant enzyme activity; and the poplar individual whose genotype of the molecular marker is TT has the lowest proline content and abscisic acid content, and the lowest total antioxidant enzyme activity.

[0040] The inventors have discovered that by testing the genomic DNA of individual poplar trees for the presence of these molecular markers, their drought resistance can be effectively determined. Specifically, poplar trees with a CC genotype at the locus corresponding to the molecular markers have the strongest drought resistance; those with a CT genotype at the locus corresponding to the molecular markers have weaker drought resistance; and those with a TT genotype at the locus corresponding to the molecular markers have the weakest drought resistance. Therefore, early selection of poplar trees can be carried out according to actual needs, further improving the efficiency and accuracy of drought-resistant breeding.

[0041] The third aspect of the present invention provides a method for obtaining the molecular marker according to the second aspect, the method comprising the following steps:

[0042] Step 1: Obtain the adaptive ancestral ancient introgression genes of poplar.

[0043] In the process of poplar molecular breeding, identifying the segments in the poplar genome where ancestral ancient introgression may have occurred, and then exploring candidate ancestral ancient introgression genes with potential functions, can help to discover more genes that play important functions based on the evolutionary history of the poplar lineage.

[0044] The inventors have discovered that by analyzing local adaptation mechanisms under climate change from the perspective of evolutionary history and combining association analysis of adaptive introgressed genes with target traits, they can more accurately locate target genes, thereby accelerating the progress of molecular breeding in poplars. Ancestral introgression refers to gene introgression that occurred in a common ancestor before species diverged.

[0045] Preferably, step 1 includes the following sub-steps:

[0046] Step 1-1, obtain the SNP dataset of the poplar introgression population.

[0047] According to a preferred embodiment of the present invention, the poplar introgression population is composed of different species of poplars around the world, preferably including Populus euphratica, Populus alba, Populus roxburghii, Populus tremula, Populus tremula, Populus nigra, Populus trichocarpa, Populus simonii and Populus odoratus.

[0048] In a further preferred embodiment, the resequencing data of the poplar introgression population consists of resequencing data of 834 poplar individuals, preferably consisting of resequencing data of 66 alba Populus, 62 clapperwood Populus, 111 European Populus, 93 small-leaved Populus, 97 American black Populus, 96 Chinese Populus, 80 Populus euphratica, 95 Fragrant Populus and 134 Trichocarpa Populus.

[0049] The inventors considered that the ancestral paleointrogression event of a species occurred before species divergence, and that the introgression region is generally retained in the genomes of the diverging species. Therefore, they selected multiple poplar populations to obtain the ancestral paleointrogression region. These multiple populations can represent the different subclades to which they belong during the identification process. In addition, because the number and research of the Populus genus are relatively small, the present invention selected representative tree populations from the other four most common subclades (i.e., Populus alba, Populus nigra, Populus tsagaca, and Populus euphratica) to provide a basic representation of the genetic information of the genus.

[0050] Among them, in the poplar introgression population of the present invention, Populus rapa, Populus alba, Populus tremula, and Populus tremula belong to the Populus school; Populus odoratus, Populus simonii, and Populus trichocarpa belong to the Populus alba school; and Populus nigra belongs to the Populus nigra school.

[0051] According to a preferred embodiment of the present invention, resequencing data of each individual in the poplar introgression population is obtained, and the resequencing data is aligned to the Populus trichocarpa reference genome ( https: / / phytozome-next.jgi.doe.gov / info / Ptrichocarpa_v4_1 ) to identify SNP sites at the whole genome level and obtain the genotype of the SNP sites.

[0052] The whole genome SNP sites were identified and the SNP data set was quality controlled and filtered. The whole genome SNPs after quality control were genotyped, preferably using Beagle v5.4 software with conventional software parameters. The genotype filling refers to supplementing the missing data based on the known genotype data to increase the marker density and improve the accuracy of the results. In the present invention, a high-quality SNP data set of 3,434,667 SNP sites was obtained from the poplar introgression population after screening.

[0053] Steps 1-2, obtain the ancestral paleointrogression region of the poplar introgression population.

[0054] Preferably, the steps 1-2 include the following sub-steps:

[0055] Step 1-2-1, preliminarily locate the introgression area of ​​the poplar introgression population.

[0056] First, the population structure was calculated and the poplar introgression population was divided into groups to obtain subpopulations of the poplar introgression population.

[0057] Preferably, based on the SNP dataset of the poplar introgression population obtained in step 1-1, the population structure of the poplar introgression population is obtained by Admixture v1.3.0 software, and the minimum cross-validation error value is selected as the optimal clustering.

[0058] Then, based on the subgrouping results, the outgroup is determined to determine whether gene exchange occurs between different triplets of the subgroups, wherein the triplets refer to a combination of three different subgroups, which is called a triplet.

[0059] In the present invention, Populus euphratica is preferably used as the outgroup.

[0060] Preferably, the Dtrios program in the Dsuite v0.5 software commonly used in the prior art is used to obtain D values ​​(i.e., D-statistics, used to determine whether gene introgression occurs) between different triplets in the subpopulation. When D>0, it is determined that gene exchange may occur in the subpopulation. Subpopulation combinations with significant gene exchange in the triplets are combinations that may have introgressed.

[0061] Furthermore, combinations with Z-scores>=3 were selected as introgression combinations with significant gene exchange, where Z-scores is the standardized result of D value (Z=D / std_err(D)).

[0062] Finally, the introgression combinations of the poplar introgression population obtained above were detected by sliding window analysis to obtain the introgression areas of the poplar introgression population.

[0063] Preferably, the introgression regions of all combinations of poplar introgression populations are obtained using the Dinvestigate program in the D-suite software, wherein the sliding window analysis uses 50 SNPs as a window and 20 SNPs as a step size.

[0064] Through the above steps, all the introgression regions between the introgression combinations of Populus alba, Populus cathayana and Populus nigra were finally obtained.

[0065] In the present invention, the poplar introgression region obtained through this step includes the ancestral introgression region and the non-ancestral introgression region.

[0066] The present inventors have discovered that by first identifying combinations with significant introgression signals within the poplar introgression population and then determining introgression regions based on these combinations, analysis of incorrect combinations can be effectively avoided, false positives can be reduced, computational effort can be reduced, and identification speed can be effectively increased. According to a preferred embodiment of the present invention, in the process of determining introgression regions, the window size in the sliding window analysis is set to 50 SNPs, which facilitates accurate identification of poplar introgression regions, thereby enabling more precise localization of introgressed genes.

[0067] According to a preferred embodiment of the present invention, in the process of obtaining the introgression region of the poplar introgression population, the largest top 5% window of the detection results of each introgression combination is set as the introgression region of the combination.

[0068] The present study found that the target ancestral introgression occurred before the divergence of the target species. During the long evolutionary history after the divergence, unique changes likely occurred, resulting in a weakened introgression signal. Therefore, the present invention selected a more relaxed threshold of 5% when screening for introgressed regions using fdM, rather than the commonly used 1%-3%. This threshold also includes weaker signals, thus facilitating the subsequent screening of ancestral paleointrogressed regions.

[0069] Step 1-2-2, locate the candidate ancestral introgression regions of the poplar introgression population.

[0070] Among them, step 1-2-2 includes the following sub-steps:

[0071] Step 1-2-2-1, obtain the topological structure among poplar subpopulations.

[0072] Wherein, the poplar subpopulation is the poplar subpopulation obtained in step 1-2-1.

[0073] In the present invention, the subpopulation topology of poplar can be obtained using software commonly used in the prior art, such as SNPhylo software. Preferably, a phylogenetic tree is generated using Populus euphratica as an outgroup to obtain the topological relationship between different populations of poplar.

[0074] According to a preferred embodiment of the present invention, the phylogenetic tree is as follows Figure 1 shown.

[0075] Step 1-2-2-2, group the poplar subpopulations according to the topological structure.

[0076] According to a preferred embodiment of the present invention, different five-taxon group combinations of poplar subpopulations are obtained based on the topological structure; preferably, the five-taxon group is represented as (((P1, P2), (P3, P4)), O), wherein the differentiation time of the P3 and P4 populations cannot be later than that of the P1 and P2 populations, and O represents an outgroup;

[0077] Introgression analysis was performed on the five taxa composed above, that is, to determine whether the ancestors of the two populations P1 and P2 had introgressed with the P3 or P4 population.

[0078] In a further preferred embodiment, P1 and P2 are set as Populus alba populations, P3 and P4 are set as Populus cathayana and Populus nigra populations, and O is set as Populus euphratica, so as to identify the ancestral introgression regions common to multiple populations of Populus alba.

[0079] Steps 1-2-2-3, obtain the ancestral introgression region of each combination.

[0080] First, the resequenced individuals with the deepest sequencing depth in each poplar population in the combination were selected; then, the chromosomes of the selected resequenced individuals were split into genomic segments; and then, ancestral introgression detection was performed on each genomic segment.

[0081] The sequencing depth refers to the ratio of the total number of bases (bp) obtained by sequencing to the genome size (Genome), and is one of the indicators for evaluating the sequencing amount.

[0082] Preferably, the chromosomes are split into 100 kb genomic fragments for detection.

[0083] The inventors have found that the DFOIL method in the prior art cannot directly detect the common ancestral gene flow of multiple populations. After a large number of experimental studies, the present invention preferably detects candidate ancestral introgression regions of the five taxonomic groups of poplars according to a method comprising the following steps:

[0084] Step i, obtain all possible ancestral introgression regions for each pair of P1 and P2.

[0085] According to a preferred embodiment of the present invention, in step i, first, for the five taxonomic groups (((P1, P2), (P3, P4)), O), the first group P1 and P2 are controlled to remain unchanged, expressed as $P1 and $P2 respectively, and then different combinations of P3 and P4 are changed to obtain multiple different introgression results of the first group $P1 and $P2.

[0086] Among them, each time the combination of P3 and P4 is changed, a gradual introgression result is obtained. By changing different combinations of P3 and P4, multiple different gradual introgression results of the first group $P1 and $P2 are obtained.

[0087] Then, all possible ancestral introgression regions of the first group $P1 and $P2 are obtained, preferably by taking the union of multiple different introgression results, wherein the union is preferably recorded as U1.

[0088] According to a preferred embodiment of the present invention, the combination of P1 and P2 is changed, and step i is repeated to obtain all possible ancestral introgression regions corresponding to n different combinations of P1 and P2.

[0089] Here, n represents the number of combinations of P1 and P2, that is, a group of P1 and P2 is recorded as: n=1.

[0090] Preferably, all possible ancestral introgression regions of each pair P1 and P2 are obtained by taking the union of multiple different introgression results, and the union is preferably recorded as U2, U3, ..., Un respectively.

[0091] Step ii, confirm candidate ancestral introgression regions in multiple populations.

[0092] According to a preferred embodiment of the present invention, the overlap of all possible ancestral introgression regions of each pair of P1 and P2 is obtained, and the candidate ancestral introgression regions of multiple populations are confirmed based on the overlap.

[0093] In a further preferred embodiment, the degree of overlap of U1 to Un is obtained by taking the intersection, that is, taking the intersection of U1, U2, ..., and Un.

[0094] In a further preferred embodiment, the overlap is 80%, that is, when a region exists in ≥80% of the Un union results, the region is considered to be a candidate ancestral introgression region of multiple poplar populations.

[0095] For example, when n=6, if a region exists in greater than or equal to 4.8 combinations (i.e., 5 and 6 combinations), the region is considered to be a candidate ancestral introgression region of multiple populations.

[0096] The inventors have found that the accuracy of obtaining the gene flow of the common ancestors of multiple subpopulations through the above method can be significantly improved.

[0097] Preferably, after the above steps, the ancestral introgression regions of the P1 and P2 combinations of all poplars can be obtained, and the candidate ancestral introgression regions of the poplar faction can be obtained with an overlap of 80% as a threshold.

[0098] Step 1-2-3, obtain the ancestral paleointrogression region of the poplar introgression population.

[0099] According to a preferred embodiment of the present invention, the introgression region detected simultaneously in step 1-2-1 and step 1-2-2 is the final ancestral introgression region of the poplar introgression population, that is, the ancestral ancient introgression region of the poplar introgression population.

[0100] In a further preferred embodiment, the intersection of the introgression region of the poplar introgression population preliminarily located in step 1-2-1 and the candidate ancestral introgression region of the poplar introgression population located in step 1-2-2 is taken to obtain the ancestral ancient introgression region of the poplar introgression population.

[0101] In the present invention, it is preferred that the above steps are followed to finally obtain the ancestral ancient introgression area between Populus alba and Populus cathayana and Populus nigra.

[0102] In the present invention, by combining the introgression region located by the 50 SNPs region in step 1-2-1 with the ancestral introgression region located in step 4, the size of the ancestral introgression segment can be reduced, false positives can be reduced, and the ancestral introgression gene can be located more precisely. It is also beneficial to better understand the mixing history between populations, identify the advantages of certain populations under specific environmental pressures, and explore certain functional stress-resistant genes that are beneficial to adapting to specific environments, laying the foundation for the later development of molecular technology-assisted breeding.

[0103] The method of the present invention for obtaining the ancestral ancient introgression region of a poplar introgression population comprises the following steps: firstly determining the introgression combination and preliminarily locating the poplar introgression region; then accurately obtaining the common ancestral introgression region of multiple populations based on the topological results; and then combining the two results and verifying each other, thereby further reducing false positives and more precisely locating the ancestral ancient introgression region. This is conducive to discovering more genes that play an important role based on the evolutionary history of the poplar lineage, thereby laying the foundation for molecular design breeding of poplar.

[0104] Steps 1-3, obtain the adaptive ancestral ancient introgression genes of the poplar introgression population.

[0105] The present invention finds that the accuracy of tree molecular breeding can be effectively improved by comprehensively utilizing introgression detection and adaptability detection to jointly analyze the adaptive introgression mechanism of trees and to explore adaptive introgression genes.

[0106] Preferably, steps 1-3 include the following sub-steps:

[0107] Step 1-3-1, obtain the adaptive region of the poplar introgression population.

[0108] According to a preferred embodiment of the present invention, the green poplar and black poplar branches are regarded as one group, and all white poplars are regarded as another group, and the adaptability sections of the two groups are detected.

[0109] The inventors considered that the Populus dahurica and Populus nigra factions were not completely separated on the phylogenetic tree, and therefore regarded Populus dahurica and Populus nigra as one group.

[0110] In a further preferred embodiment, XP-CLR is used to detect two populations to assess the frequency variation of different loci between populations. The parameters of the XR-CLR are conventionally set, and the composite likelihood ratio is used to assess the frequency variation of different loci between populations.

[0111] In a further preferred embodiment, a sliding window method is used to detect the adaptive segments of the two groups, with a sliding window size of 10 kb and a step size of 5 kb.

[0112] Preferably, the most significant top 5% windows in the detection results are taken as the final adaptive regions.

[0113] Step 1-3-2, obtain the adaptive ancestral paleointrogression region of the poplar introgression population.

[0114] Among them, the area that belongs to both the final adaptive area obtained in step 1-3-1 and the ancestral ancient introgression area obtained in step 1-2-3 is used as the adaptive ancestral ancient introgression area of ​​the poplar introgression population.

[0115] Step 1-3-3, obtain the adaptive ancestral ancient introgression genes of the poplar introgression population.

[0116] According to a preferred embodiment of the present invention, the adaptive ancestral ancient introgression region of the poplar introgression population obtained in step 1-3-2 is subjected to gene annotation. Preferably, in the present invention, the gene region and the promoter region 2000bp upstream of the gene are taken as the gene annotation file of the adaptive ancestral ancient introgression region of poplar.

[0117] Step 2: Select a poplar population and obtain drought phenotypic data of the poplar.

[0118] In the present invention, a germplasm resource group composed of naturally grown white poplar individuals is preferably selected, and after drought treatment, it is used as an analysis group for obtaining molecular markers, recorded as a poplar drought group, which preferably includes 303 genotype individuals. The drought treatment steps are: asexual reproduction through root segments in Guan County, Shandong in 2018, each genotype material has 3 replicates, and drought treatment is started when the sixth leaf grows at the top, maintaining the soil moisture content at 10% for 30 days, and the average air humidity is 67.5%.

[0119] According to a preferred embodiment of the present invention, the drought phenotypic data include proline content, total antioxidant enzyme activity and abscisic acid content; wherein the total antioxidant enzyme activity includes catalase activity, peroxidase activity and superoxide dismutase activity.

[0120] Step 3: Obtain molecular markers related to poplar drought resistance.

[0121] The poplar is preferably Populus tomentosa. Preferably, step 3 includes the following sub-steps:

[0122] Step 3-1, obtain the SNP dataset of the poplar drought population described in step 2.

[0123] The DNA of each individual in the drought-resistant population of Populus tomentosa was obtained and resequenced. The resequencing data was then aligned to the Populus tomentosa reference genome, SNPs were identified at the whole genome level, and the genotypes of the SNP sites were obtained.

[0124] Preferably, the SNP sites of the drought-resistant population of Populus tomentosa are identified according to the method described in step 1-1, and the same quality control standards as in the above steps are adopted to obtain the SNP data set of the drought-resistant population of Populus tomentosa.

[0125] Step 3-2, extract SNP sites within the adaptive ancestral ancient introgression gene range from the SNP dataset of the poplar drought population.

[0126] Preferably, step 3-2 includes the following sub-steps:

[0127] Step 3-2-1, infer homologous genes between the reference genome used in the poplar introgression population in step 1 and the reference genome used in the poplar drought population in step 2.

[0128] In this study, because the population used in step 1 encompasses diverse species from various poplar families, the most studied genome, Populus trichocarpa, was selected as the reference genome. In step 2, only Populus tomentosa was used; to reduce alignment bias, the Populus tomentosa genome was selected as the reference genome. To align the genes obtained from the two reference genomes, the present invention performed homology inference on each gene in the two reference genomes, resulting in a one-to-one optimal match between the genes of Populus trichocarpa and Populus tomentosa.

[0129] Step 3-2-2, obtain the SNP sites of the adaptive ancestral ancient introgression genes of the poplar drought population.

[0130] According to a preferred embodiment of the present invention, the adaptive ancestral ancient introgression genes of the poplar introgression population obtained in step 1 are compared and analyzed to obtain the corresponding adaptive ancestral ancient introgression genes on the Populus tomentosa genome.

[0131] After obtaining the corresponding adaptive ancestral ancient introgression gene on the Populus tomentosa genome, SNP sites within the adaptive ancestral ancient introgression gene range are extracted from the SNP dataset of the Populus tomentosa drought population obtained in step 3-1.

[0132] Step 3-3, performing association analysis between the SNP sites of the adaptive ancestral ancient introgressed genes in step 3-2 and the drought phenotype, and obtaining SNP sites that are significantly associated with the drought resistance phenotype of poplar.

[0133] According to a preferred embodiment of the present invention, the SNP site significantly associated with the drought resistance phenotype of poplar is located at the 1022nd base upstream of the TCP19 gene (PtoTCP19) of Populus tomentosa, wherein the nucleotide sequence of the TCP19 gene is shown in SEQ ID NO: 1, and the amino acid sequence encoded by it is shown in SEQ ID NO: 2.

[0134] In a further preferred embodiment, the SNP site significantly associated with the drought-resistant phenotype of poplar is located at position 979 of the nucleotide sequence shown in SEQ ID NO:3.

[0135] According to a preferred embodiment of the present invention, after obtaining the SNP sites significantly associated with the drought resistance phenotype of poplar, the significance of the difference in phenotype of individual poplars with different genotype combinations is further tested.

[0136] In the present invention, it is preferred to use the Student's t-test (P≤0.05) method to test the significance of the difference in poplar phenotypes of different genotype combinations.

[0137] Preferably, when the genotype of the above molecular markers of Populus tomentosa individuals is CC, the drought resistance is the strongest; when the genotype of the above molecular markers of Populus tomentosa individuals is CT, the drought resistance is weaker; when the genotype of the above molecular markers of Populus tomentosa individuals is TT, the drought resistance is the weakest.

[0138] In a fourth aspect of the present invention, a primer pair is provided for amplifying the drought resistance-related gene described in the first aspect, the primer pair comprising primer P1 and primer P2, wherein primer P1 comprises the nucleotide sequence shown in SEQ ID NO: 4, and primer P2 comprises the nucleotide sequence shown in SEQ ID NO: 5.

[0139] Preferably, the nucleotide sequence of the primer P1 is shown as SEQ ID NO: 4, and the nucleotide sequence of the primer P2 is shown as SEQ ID NO: 5.

[0140] In a fifth aspect of the present invention, a primer pair is provided for amplifying the molecular marker for screening drought-resistant poplars as described in the second aspect, the primer pair comprising primer P3 and primer P4, wherein primer P3 comprises the nucleotide sequence shown in SEQ ID NO: 6, and primer P4 comprises the nucleotide sequence shown in SEQ ID NO: 7.

[0141] Preferably, the nucleotide sequence of the primer P3 is shown as SEQ ID NO: 6, and the nucleotide sequence of the primer P4 is shown as SEQ ID NO: 7.

[0142] In the present invention, primer pairs that add 1 to 20 bases to the 5' end and 3' end of the nucleotide sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and can obtain substantially identical DNA fragments (the DNA sequences between the upstream and downstream primers are the same) are all included in the primer pairs of the present invention.

[0143] In the present invention, the above-mentioned primers are used to perform PCR amplification on the nucleotide fragment containing the molecular marker that is significantly associated with the drought resistance trait of the tested white poplar, and then the molecular marker can be effectively detected by direct sequencing and other methods to determine whether the tested white poplar has the molecular marker. Specifically, individuals of the white poplar with a genotype of CC at the above-mentioned SNP marker have a stronger drought resistance; individuals of the white poplar with a genotype of CT at the above-mentioned SNP marker have a weaker drought resistance; and individuals of the white poplar with a genotype of TT at the above-mentioned SNP marker have the weakest drought resistance.

[0144] The primer pair provided by the present invention can realize rapid, low-cost and high-accuracy identification of poplar drought resistance at an early stage, effectively shortening the poplar drought resistance breeding cycle.

[0145] The sixth aspect of the present invention provides a method for genetic improvement of poplars, which includes the steps of successive breeding of poplar individuals with CC genotypes using the molecular markers described in the second aspect or the molecular markers obtained by the method described in the third aspect, and eliminating individuals with other genotypes.

[0146] Among them, the poplar is preferably Populus tomentosa.

[0147] A seventh aspect of the present invention provides a method for identifying or screening poplars with strong drought resistance, the method comprising the following steps:

[0148] Step 1, extracting genomic DNA from the poplar to be tested;

[0149] Step II, performing PCR amplification using the above-extracted genomic DNA as a template;

[0150] Step III, determining the genotype of the SNP molecular marker of the poplar to be tested;

[0151] Step IV: determining the drought resistance of the tested poplar trees based on the genotype detection results.

[0152] Among them, the poplar is preferably Populus tomentosa.

[0153] In step II, the primers used for PCR amplification are the primers P3 and P4 described in the fifth aspect.

[0154] In step III, methods for determining the genotype of molecular markers include but are not limited to sequencing.

[0155] In step IV, if the genotype of the SNP molecular marker of the poplar to be tested is CC, then its drought resistance is the strongest; if the genotype of the SNP molecular marker of the poplar to be tested is CT, then its drought resistance is relatively weak; if the genotype of the SNP molecular marker of the poplar to be tested is TT, then its drought resistance is the weakest.

[0156] Example

[0157] The present invention is further described below through specific examples. However, these examples are merely exemplary and do not constitute any limitation to the scope of protection of the present invention.

[0158] Unless otherwise specified, the reagents involved in the following examples are all conventional reagents available on the market, and the methods used are all commonly used methods in this technical field.

[0159] Example 1 Obtaining molecular markers for screening drought-resistant poplars

[0160] 1. Obtaining ancient introgression genes from poplar's adaptive ancestors

[0161] (1) Resequencing data of 834 poplar individuals were obtained from the databases of the National Center for Biotechnology Information (NCBI) and the China National Center for Bioinformation (CNCB). The specific composition is shown in Table 1.

[0162] Table 1

[0163]

[0164]

[0165]

[0166]

[0167] The bam file of each sample was aligned to the reference genome of Populus trichocarpa using Burrows-Wheeler Aligner v0.7.5a-r405 (default parameters). https: / / phytozome-next.jgi.doe.gov / info / Ptrichocarpa_ v4_1 ) to identify single nucleotide polymorphism sites at the genome-wide level and obtain the genotype of the SNP sites.

[0168] Genome Analysis Toolkit (GATK) v4.0 was used to identify genome-wide single nucleotide polymorphism (SNP) sites with the following parameters: SNP: QD < 2.0 || MQ < 20.0 || FS > 60.0 || SOR > 3.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0, and the original SNP dataset of the poplar introgression population was obtained.

[0169] The raw SNP dataset was quality-controlled and filtered using Vcftools_0.1.16, with the following criteria: ① biallelic sites; ② maximum missing rate (Maximum Missing Rate) <0.2; and ③ minor allele frequency (MAF) >0.05. The filtered SNPs were then imputed using Beagle v5.4 software, resulting in a high-quality SNP dataset of 3,434,667 SNPs for the poplar introgression population. (2)

[0171] (2.1) Based on the SNP dataset obtained in step 1, the population structure of the poplar introgression population was determined using Admixture v1.3.0 software. The optimal clustering was selected with the minimum cross-validation error. The clustering results were: Populus euphratica, Populus tremula, Populus tremula, Populus alba, Populus siliqua, Populus deltoides, Populus simonii, Populus odorifera, and Populus trichocarpa.

[0172] Based on the subpopulation results, using Populus euphratica as the outgroup, the D values ​​of the different triple groups of the eight subpopulations were calculated using the Dtrios program in Dsuite v0.5 software. The Dtrios results are shown in Table 2:

[0173] Table 2

[0174]

[0175]

[0176] As can be seen from Table 2, the triplet groups corresponding to Z-scores >= 3 are subpopulation combinations with significant gene exchange. For example, the Z-score of the first row in Table 2 is 41.8332, indicating that there is gene exchange between the Populus simonii, Populus trichocarpa, and Populus deltoides subpopulations and the Populus deltoides subpopulations.

[0177] For combinations with significant gene exchange, we further used the Dinvestigate program to locate introgression regions using a sliding window algorithm. The window size was set to 50 SNPs and the step size was 20 SNPs. This algorithm identified introgression regions between introgression population combinations, ultimately obtaining all candidate introgression regions between introgression combinations of Populus alba, Populus cathayana, and Populus nigra. Introgression regions were defined as windows with the top 5% maximum fdM values ​​in the Dinvestigate results.

[0178] (2.2) Based on the above subpopulation results, the SNPhylo software was used to generate a phylogenetic tree with Populus euphratica as the outgroup to obtain the topological relationship between different Populus populations. The results are as follows Figure 1 shown.

[0179] To identify ancestral introgression regions shared by multiple populations of Populus alba, five taxonomic groups (((P1, P2), (P3, P4)), and O) were further defined based on the topological results. P1 and P2 were designated as Populus alba populations, P3 and P4 as Populus cathayana and Populus nigra populations, and outgroup O as Populus euphratica. The Populus alba populations include Populus siliqua, Populus alba, Populus tremula, and Populus tremula; the Populus cathayana populations include Populus odorifera, Populus simonii, and Populus trichocarpa; and the Populus nigra populations include Populus deliciosa.

[0180] While keeping P1 and P2 constant, we changed different combinations of P3 and P4. We selected the resequenced individuals with the deepest sequencing depth in each poplar population, converted them into fasta format, and split them into 100 kb genomic segments. After alignment, we calculated the introgression results of each combination using the DFOIL method. As shown in Table 3, there are 6 pairs of P1 and P2 combinations in total. Each pair of P1 and P2 combination obtained 6 results. The union of the 6 results is denoted as U1 (i.e., the union of the 6 results in the first column). Set), which represents the candidate ancestral introgression segment of the pair of P1 and P2 combinations. Among them, the five taxonomic groups of poplars include 6 pairs of poplar combinations (i.e., 6 pairs of P1 and P2 combinations), and their introgression combinations total 36. For example, the first horizontal column is the first pair of P1 and P2 combinations, and the first introgression combination is: P1 is Populus scolopendra, P2 is Populus alba, P3 is Populus nigra, P4 is Populus odoratus, and O is Populus euphratica. The same applies to other combinations. Table 2 has a total of six horizontal columns, representing 6 pairs of P1 and P2 combinations.

[0181] Table 3

[0182]

[0183]

[0184] By changing the population categories of P1 and P2 and repeating the above steps, we can obtain the ancestral introgression segments U2, U3, U4, U5 and U6 corresponding to different combinations of P1 and P2, respectively.

[0185] After the above steps, a total of 6 groups of ancestral introgression segments were obtained from the 36 combinations of the five taxonomic groups of poplars, namely U1, U2, U3, U4, U5 and U6.

[0186] Take the intersection of each group of ancestral introgression segments (i.e., U1 to U6) and determine that when a region exists in all five pairs of poplar combinations (P1 and P2 combinations), it is considered to be a candidate ancestral introgression region for multiple poplar populations. The results are as follows: Figure 2 As shown, the solid triangle marks indicate that the same region exists in ≥5 pairs of combinations, that is, the candidate ancestral introgression region of multiple poplar populations.

[0187] Depend on Figure 2 It can be seen that the number of introgression regions present in all six pairs of poplar combinations is the largest, indicating that a considerable number of regions left by ancestral ancient introgression can still be detected in each combination; and the regions present in ≥80% or ≥5 combinations are also considered to be ancestral ancient introgression regions, because in the long historical evolution process, some regions may have undergone unique changes in individual species and cannot be identified, but are still detected as ancestral ancient introgression regions in other species, so these areas are also identified as ancestral ancient introgression regions.

[0188] (2.3) The final ancient introgression segment of the poplar ancestor is obtained by intersecting the introgression segment of each significant triplet result obtained in step (2.1) with the introgression region of the candidate poplar ancestor obtained in step (2.2).

[0189] (3) Considering the green and black poplars as one cluster and all white poplars as another cluster, XP-CLR was used to detect the adaptive segments of the two clusters, with a sliding window size of 10 kb and a step size of 5 kb. The top 5% of the most significant windows in the detection results were used as the final adaptive regions.

[0190] Combine the adaptive regions obtained above with the ancestral paleo-introgression regions obtained in step (2.3), that is, when a region belongs to both the ancestral paleo-introgression region and the adaptive region, it is considered to be the adaptive ancestral paleo-introgression region, thereby obtaining the adaptive ancestral paleo-introgression region of the poplar introgression population.

[0191] Each gene region and the promoter region 2000bp upstream of the gene in the genome annotation file of the reference genome were taken as the gene annotation file of the adaptive ancestral ancient introgression region of poplar. The BEDToolsintersect command was used to annotate the adaptive ancestral ancient introgression genes of the poplar introgression population.

[0192] 2. Select a poplar population and obtain drought phenotypic data of poplars.

[0193] A germplasm resource population consisting of 303 naturally grown Populus tomentosa individuals covering the entire suitable habitat of Populus tomentosa was selected and used as the analysis population for obtaining molecular markers after drought treatment. The drought treatment steps were as follows: asexual propagation through root segments in Guanxian County, Shandong Province in 2018, with three replicates for each genotype material. Drought treatment began when the sixth leaf grew at the top, maintaining the soil moisture content at 10% for 30 days, and the average air humidity was 67.5%.

[0194] Functional leaves (i.e., the 4th to 6th leaves at the top of the stem) of 303 Populus tomentosa individuals were collected for each genotype through three clone repetitions, immediately immersed in liquid nitrogen, stored at -80°C, and then vacuum freeze-dried for the measurement of drought resistance index.

[0195] 3. Obtaining molecular markers related to drought resistance in poplars

[0196] (3.1) Genomic DNA from 303 genotyped individuals of the drought-prone population of Populus tomentosa was extracted using the FastPure Plant DNA Isolation Mini Kit (Nanjing Novozymes Biotech Co., Ltd.). DNA quality was tested using a UV spectrophotometer and gel electrophoresis. DNA that passed the test was stored at −20°C.

[0197] The DNA of each individual in the drought-resistant population of Populus tomentosa was resequenced (using commonly used methods in the existing technology). Each resequencing data was aligned to the Populus tomentosa reference genome (published in the National GeneBank Sequence Archive (CNGB Sequence Archive, CNSA), project number CNP0004290) using Burrows-Wheeler Aligner v0.7.5a-r405 (default parameters). Single nucleotide polymorphism sites were identified at the genome-wide level and the genotypes of the SNP sites were obtained.

[0198] According to the quality control and filtering method described in step 1 (1), a high-quality SNP dataset of the drought-resistant population of Populus tomentosa was finally obtained, with a total of 13,063,406 SNP sites.

[0199] (3.2) Homologous genes were inferred between the Populus trichocarpa reference genome used in the poplar introgression population and the Populus tomentosa reference genome used in the Populus tomentosa drought population. Reciprocal best hit (RBH) analysis was performed using Genetribe v1.0 software to identify the corresponding adaptive ancestral ancient introgressed genes on the Populus tomentosa genome. SNPs within the adaptive ancestral ancient introgressed genes were then extracted from the SNP loci in the Populus tomentosa drought population in step (3.1).

[0200] The obtained ancient introgression gene of the adaptive ancestor of Populus tomentosa is PtoTCP19, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence encoded by it is shown in SEQ ID NO: 2.

[0201] Specifically, the gene sequence of PtoTCP19 was obtained according to the following steps: RNA was extracted from leaves of 300 individual genotypes of Populus tomentosa using the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Novozymes Biotech Co., Ltd.). RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novozymes Biotech Co., Ltd.). Primers were designed using the Primer Blast tool (NCBI, https: / / blast.ncbi.nlm.nih.gov) with reference to the Populus tomentosa reference genome as a template, taking into account various principles of primer design. Primers P1 and P2 were designated. The nucleotide sequence of primer P1 is shown in SEQ ID NO: 4, and the nucleotide sequence of primer P2 is shown in SEQ ID NO: 5. PCR amplification was performed using primers P1 and P2 using the obtained genomic cDNA as a template to obtain the gene sequence.

[0202] Efficient Mixed-Model Association eXpedited (EMMAX) software was used to calculate the kinship of Populus tomentosa populations, and Admixture software was used to calculate the population structure of Populus tomentosa populations (when K = 3, the lowest cverror was obtained). Phylogenetic relationship and population structure were used as covariates, and candidate gene association analysis was performed on the SNPs set of ancient introgressed genes of Populus tomentosa adaptive ancestors and the drought phenotype data in step 2 using a mixed linear model (MLM). After False Discovery Rate (FDR) correction was performed on the P values, SNP sites significantly associated with Populus tomentosa drought (threshold P < 0.05) and their genotypes were obtained.

[0203] The above screening revealed a single SNP site that was significantly associated with drought resistance in the P. tomentosa population (P < 0.05). This site is located at base 1022 upstream of the PtoTCP19 gene in the P. tomentosa reference genome, i.e., at position 979 of the nucleotide sequence shown in SEQ ID NO: 3, and exhibits a T / C polymorphism.

[0204] The Student's t-test (P≤0.05) was used to test the significance of the phenotypic differences among different genotype combinations of Populus tomentosa. It was found that Populus tomentosa individuals with the CC genotype at the above-mentioned molecular markers had higher drought resistance (higher proline and abscisic acid content, and higher total antioxidant enzyme activity) than those with the TT genotype.

[0205] The genotype and phenotype data of molecular markers related to drought resistance of Populus tomentosa obtained according to the above steps are shown in Table 4:

[0206] Table 4

[0207]

[0208]

[0209]

[0210]

[0211] Furthermore, the genotype effects of different genotypes on proline content, abscisic acid content and total antioxidant enzyme activity of Populus tomentosa were as follows: Figures 3-5 shown.

[0212] From Table 4 and Figures 3-5It can be seen that the individuals with CC genotype of the above molecular markers of Populus tomentosa have the highest proline content, abscisic acid content and total antioxidant enzyme activity, and the individuals with TT genotype of the above molecular markers of Populus tomentosa have the lowest proline content, abscisic acid content and total antioxidant enzyme activity.

[0213] Example 2 Verification of the effectiveness of molecular markers

[0214] In the early spring of 2022, 500 Populus tomentosa individuals with different genotype combinations were randomly selected from the Populus tomentosa germplasm resource population (1047 plants, Guanxian base, Shandong Province). Nine functional leaves (the third to fifth mature leaves on the upper end of the branches) were collected between 9:00 and 11:00 in the morning using the above method. Six leaves were used to measure the stomatal area. After the three leaves were collected, they were immediately stored in a liquid nitrogen environment (-196°C) to extract genomic DNA. The genotype of the 1022nd base upstream of the TCP19 gene was determined using primers P3 and P4. The drought resistance (total antioxidant enzyme activity, abscisic acid content, and proline content) of different individuals are shown in Tables 5 and 6. Figure 6 shown.

[0215] Table 5

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] From Table 5 and Figure 6 It can be seen that among the 500 randomly selected Populus tomentosa individuals in this example, the three drought resistance indicators of different genotypes showed significant differences. Individuals with CC genotype had the highest proline content, abscisic acid content, and total antioxidant enzyme activity, while individuals with genotype TT had the lowest proline content, abscisic acid content, and total antioxidant enzyme activity, further demonstrating that the CC genotype has stronger drought resistance.

[0224] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions are not to be construed as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention.

Claims

1. A method for obtaining molecular markers for screening drought-resistant poplars, characterized in that: The molecular marker is located at the 1022nd base upstream of the PtoTCP19 gene and has a T / C polymorphism; the nucleotide sequence of the PtoTCP19 gene is shown in SEQ ID NO: 1; The poplar is Populus tomentosa; The method comprises the following steps: Step 1: Obtain the adaptive ancestral ancient introgression genes of poplar; Step 2, selecting a poplar population and obtaining drought phenotypic data of the poplar; Step 3, obtaining molecular markers related to drought resistance of poplar; Step 1 includes the following sub-steps: Step 1-1, obtain the SNP dataset of the poplar introgression population; The poplar introgression population includes Populus euphratica, Populus alba, Populus siliqua, Populus tremula, Populus tremula, Populus nigra, Populus trichocarpa, Populus simonii, and Populus odorifera; Obtain resequencing data for each individual in the poplar introgression population and align the resequencing data to the Populus trichocarpa reference genome to identify SNPs at the genome-wide level and obtain genotypes of the SNPs. Steps 1-2, obtaining the ancestral paleointrogression region of the poplar introgression population; Step 1-2 includes the following sub-steps: Step 1-2-1, preliminarily locate the introgression area of ​​the poplar introgression population; First, the population structure was calculated and the poplar introgression population was divided into groups to obtain subpopulations of the poplar introgression population; Then, based on the subpopulation clustering results, the outgroup was determined to determine whether gene exchange occurred between different triplet groups of the subpopulation; Finally, the introgression combinations of the poplar introgression population obtained above were detected by sliding window analysis to obtain the introgression regions of the poplar introgression population; Step 1-2-2, locate the candidate ancestral introgression region of the poplar introgression population; Among them, step 1-2-2 includes the following sub-steps: Step 1-2-2-1, obtain the topological structure among poplar subpopulations; Step 1-2-2-2, group the poplar subpopulations according to the topological structure; Step 1-2-2-3, obtain the ancestral introgression region of each combination; Step 1-2-3, obtain the ancestral paleointrogression region of the poplar introgression population; The introgression regions detected simultaneously in steps 1-2-1 and 1-2-2 are taken as the final ancestral introgression regions of the poplar introgression population; Steps 1-3, obtain the adaptive ancestral ancient introgression genes of the poplar introgression population.

2. The method according to claim 1, characterized in that The primer pair for amplifying the gene includes primer P1 and primer P2, The nucleotide sequence of primer P1 is shown in SEQ ID NO: 4, and the nucleotide sequence of primer P2 is shown in SEQ ID NO:

5.

3. The method according to claim 1, characterized in that The primer pair for amplifying the molecular marker includes primer P3 and primer P4, The nucleotide sequence of primer P3 is shown in SEQ ID NO: 6, and the nucleotide sequence of primer P4 is shown in SEQ ID NO:

7.

4. A method for genetic improvement of poplars, characterized in that: The method comprises the steps of successive breeding of poplar individuals with CC genotypes obtained by the molecular marker according to the method of claim 1, and eliminating individuals with other genotypes; the poplar is Populus tomentosa.

5. A method for identifying or screening poplars with strong drought resistance, characterized in that: The method comprises the following steps: Step 1, extracting genomic DNA from the poplar to be tested; Step II, performing PCR amplification using the above-extracted genomic DNA as a template; Step III, determining the genotype of the molecular marker of claim 1 of the poplar to be tested; Step IV, determining the drought resistance of the tested poplar trees based on the genotype test results; In step IV, if the genotype of the tested poplar molecular marker is CC, then its drought resistance is the strongest; if the genotype of the tested poplar molecular marker is CT, then its drought resistance is relatively weak; if the genotype of the tested poplar molecular marker is TT, then its drought resistance is the weakest; The poplar is Populus tomentosa.

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