Molecular marker for screening drought-resistant poplar based on interspecific introgression theory and method and application thereof

Abstract

The invention discloses a molecular marker for screening drought-resistant poplars based on an interspecific introgression theory and a method and application thereof, the molecular marker is located at the 1022nd basic group of the upstream of a PtoTCP19 gene and has T / C polymorphism, the genotype of the molecular marker is a CC individual, and the drought resistance of the molecular marker is the strongest. The molecular marker provided by the invention is obtained based on an interspecific introgression theory, is high in accuracy, can be used for accurately evaluating the drought resistance of the poplar at a molecular level, accurately and efficiently screening drought-resistant tree species at a seedling stage and shortening a breeding period, and is of great significance in enhancing plant stress resistance and relieving damage of drought stress to forestry yield and quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular breeding, and particularly relates to a molecular marker for screening drought-resistant poplars, a method and an application thereof, and more particularly to a molecular marker for screening drought-resistant poplars based on the theory of interspecific introgression, 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. During the historical evolution of many animals and plants, extensive hybridization has occurred. Although the introgressed genes brought about are largely harmful, it is also possible to obtain adaptive variations of related species. These variations tend to be positively selected and can spread rapidly in the recipient species, thereby rapidly improving the environmental adaptability. As an un-domesticated perennial plant, forest trees have high genetic diversity and more extensive gene flow among species, and thus have important genomic research value.

[0003] After the occurrence of gene introgression, the introgressed genetic information usually has a genetic background more similar to that of the donor. Based on the above principle, the traces of introgression can be detected on the genome to locate the specific introgressed region. Adaptive introgressed genes often play an important role during a certain stress stage such as the historical evolution process or climate environmental change to help the species survive the complex external environment. Genome-wide association study is an important method for identifying key genes. However, due to the high heterozygosity of forest trees and complex quantitative traits, etc., gene association analysis often cannot directly obtain satisfactory results.

[0004] With the continuous deterioration of the global climate, forest trees are more vulnerable to abiotic stresses such as drought in the natural environment, which has an adverse impact on growth and development, wood yield and wood quality. In the past tens of thousands of years, there have been many major droughts in Asia. For example, during the Quaternary glaciation period (60,000 - 70,000 years ago), the Asian monsoon system weakened significantly, and the precipitation in East Asia and South Asia decreased; in the mid-Holocene (4,200 years ago), the solar activity weakened, resulting in significant drought in Asia, aggravated desertification, and a major impact on plant survival. Poplars may improve the drought resistance of the population through interspecific gene introgression, successfully survive the drought climate period, and be widely distributed globally. As an important ecological and economic tree species in China, poplars are not only widely used in artificial forests and vegetation restoration, but also provide sufficient raw materials for the paper industry and the wood industry.

[0005] However, at present, the arid and semi-arid regions in China severely restrict the growth, development and spatial distribution of poplars. With the continuous development of the application of molecular breeding technology, using genetic engineering technology to improve the stress resistance of forest trees provides an economical and efficient way to develop the production potential of arid zones. Therefore, it is necessary to provide an efficient and accurate adaptive introgression gene related to the drought resistance trait of poplars and related molecular markers to improve the selection speed and accuracy of drought-resistant tree species and effectively shorten the breeding cycle of poplars. Summary of the Invention

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

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

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

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

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

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

[0012] In the fifth aspect, a primer pair for amplifying the molecular marker described in the second aspect is provided, and 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] Sixth aspect, a poplar genetic improvement method is provided, the method comprising the steps of successively selecting poplar individuals with the CC genotype of the molecular marker described in the second aspect or the molecular marker obtained by the method described in the third aspect, and eliminating individuals with other types of genotypes.

[0014] Seventh aspect, a method for identifying or screening poplars with strong drought resistance is provided, the method comprising the following steps:

[0015] Step I, extracting genomic DNA of the poplar to be tested;

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

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

[0018] Step IV, determining the drought resistance of the poplar to be tested according to the genotype detection result.

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

[0020] (1) The PtoTCP19 gene provided by the present invention is significantly related to poplar drought resistance, and is of great significance for enhancing plant stress resistance and reducing the damage caused by drought stress to forestry yield and quality;

[0021] (2) The molecular marker 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 at the seedling stage of poplars, 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 the molecular marker provided by the present invention can more precisely locate the ancestral introgressed genes, which is beneficial to better understanding the mixed history between populations, identifying the advantages of certain populations under specific environmental pressures, and mining certain functional stress-resistant genes beneficial to adapting to specific environments. Description of the Drawings

[0023] Figure 1 Showing the phylogenetic tree of the poplar introgression population described in Example 1 of the present invention; Figure 2 Showing the candidate ancestral introgression regions of multiple populations of Populus alba described in Example 1 of the present invention; Figures 3 - 5 Respectively showing the genotype effect diagrams of different genotypes of the SNP marker locus described in Example 1 of the present invention on proline content, abscisic acid content and total antioxidant enzyme activity; Figure 6 In which a, b, and c respectively show the genotype effect diagrams of proline content, abscisic acid content and total antioxidant enzyme activity corresponding to the molecular marker. Detailed Embodiments

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

[0025] The special term "exemplary" herein means "serving as an example, embodiment, or illustration". Any embodiment described as "exemplary" herein need not be construed as superior to or better than other embodiments.

[0026] In a first aspect of the present invention, there is provided a gene related to the drought resistance of poplar, and the gene is PtoTCP19, and its nucleotide sequence includes the sequence shown in SEQ ID NO: 1. Preferably, its nucleotide sequence 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 is divided into sections such as Populus cathayana, Populus alba, Populus nigra, Populus euphratica, and Populus lasiocarpa. Among them, Populus tomentosa in the section Populus alba is a native tree species unique to China, with a wide distribution. It is centered in the middle and lower reaches of the Yellow River Basin and occupies an important position in the forestry production and ecological environment construction in northern China. It is a pioneer tree species for forest cultivation in northern regions. 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 the regulation of plant growth or development processes. TCP transcription factors share an atypical bHLH secondary structure of a basic region-helix-loop-helix composed of 55-60 amino acid residues, that is, the TCP conserved domain. This domain can bind to DNA and can also interact with other proteins, thereby regulating the transcription of target genes.

[0030] According to a preferred embodiment of the present invention, for the gene PtoTCP19 related to the drought resistance of poplar, the amino acid sequence encoded by it includes 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 plant tolerance to abiotic stresses. In Arabidopsis thaliana, overexpression of OsTCP19 enhances the drought and salt tolerance of transgenic plants by reducing the water loss rate and reactive oxygen species accumulation.

[0032] The inventor of the present invention has found through a large number of studies that the Populus tomentosa TCP19 gene (PtoTCP19) has experienced introgression from the ancestors of poplar and adaptive selection, and is significantly correlated with drought traits. Moreover, there is a REF6 binding site regulating ABA in the promoter region of this gene, indicating that this gene is likely to play an important role in the process of drought stress response in Populus tomentosa.

[0033] In the second aspect of the present invention, there is provided 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 T / C polymorphism.

[0034] In the present invention, the version of the poplar genome is the Populus tomentosa reference genome (published in the 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 the 979th position of the nucleotide sequence shown in SEQ ID NO: 3.

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

[0037] Among them, the CC genotype indicates that the locus of the poplar is a homozygote of C, the TT genotype indicates that the locus of the poplar is a homozygote of T, and the CT genotype indicates that the locus of the poplar is a heterozygote of C and T.

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

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

[0040] The inventor of the present invention has found through research that by detecting whether the genomic DNA of poplar individuals has the above-mentioned molecular markers, the drought resistance ability of poplar can be effectively determined. Specifically, among poplar individuals with the genotype CC at the corresponding locus of the above-mentioned molecular marker, their drought resistance ability is the strongest; among poplar individuals with the genotype CT at the corresponding locus of the above-mentioned molecular marker, their drought resistance ability is weaker; among poplar individuals with the genotype TT at the corresponding locus of the above-mentioned molecular marker, their drought resistance ability is the weakest. Therefore, early selection of poplar can be carried out according to actual needs to further improve the efficiency and accuracy of drought-resistant breeding.

[0041] In the third aspect of the present invention, a method for obtaining the molecular marker described in the second aspect is provided, and the method includes the following steps:

[0042] Step 1, obtaining the adaptive ancestral ancient introgression genes of poplar.

[0043] In the process of poplar molecular breeding, identifying the segments in the poplar genome that may have experienced ancestral ancient introgression, and then mining the candidate ancestral ancient introgression genes with potential functions, can mine more genes with important functions based on the evolutionary history of the poplar lineage.

[0044] The inventor of the present invention has found through research that by analyzing the local adaptation mechanism under climate change from the perspective of the evolutionary history process and combining the association analysis of adaptive introgression genes with target traits, the target genes can be more accurately located, thereby accelerating the process of poplar molecular breeding. Among them, ancestral ancient introgression refers to the gene introgression that occurred in the common ancestor before species divergence.

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

[0046] Step 1-1, obtaining the SNP data set 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 poplar from all over the world, preferably including Populus euphratica, Populus alba, Populus adenopoda, Populus davidiana, Populus tremula, Populus deltoides, Populus trichocarpa, Populus simonii and Populus koreana.

[0048] In a further preferred embodiment, the re-sequencing data of the poplar introgression population is composed of the re-sequencing data of 834 poplar individuals, preferably composed of the re-sequencing data of 66 Populus alba, 62 Populus adenopoda, 111 Populus tremula, 93 Populus simonii, 97 Populus deltoides, 96 Populus davidiana, 80 Populus euphratica, 95 Populus koreana and 134 Populus trichocarpa.

[0049] The present inventors considered that the ancestral introgression events of species occurred before species divergence, and the introgression regions generally remained in the genomes of different species after divergence. Therefore, multiple poplar populations were selected to obtain the ancestral ancient introgression regions, and multiple populations could represent different subclades to which they belonged during the identification process. In addition, since there are few studies on the number of the Aigeiros and Leuce sections in Populus, the present invention selected representative tree populations from the other four most common subclades (i.e., the Populus, Aigeiros, Tacamahaca, and Turanga sections), which could basically represent the genetic information of Populus.

[0050] Among them, in the poplar introgression population of the present invention, Populus adenopoda, Populus alba, Populus davidiana, and Populus tremula belong to the Populus section; Populus koreana, Populus simonii, and Populus trichocarpa belong to the Tacamahaca section; Populus deltoides belongs to the Aigeiros section.

[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 ), and SNP sites are identified at the whole-genome level and genotypes of the SNP sites are obtained.

[0052] Genome-wide single nucleotide polymorphism sites are identified, and quality control and filtering are performed on the SNP dataset. Genotype imputation is performed on the quality-controlled genome-wide SNPs, preferably using the beagle v5.4 software with conventional software parameters. Among them, the genotype imputation refers to supplementing missing data based on known genotype data to increase the marker density and improve the result accuracy. In the present invention, a total of 3,434,667 SNP sites are obtained for the high-quality SNP dataset of the poplar introgression population after screening.

[0053] Step 1-2: Obtain the ancestral ancient introgression regions of the poplar introgression population.

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

[0055] Step 1-2-1: Initially locate the introgression regions of the poplar introgression population.

[0056] First, calculate the population structure, and cluster the poplar introgression population 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 the Admixture v1.3.0 software, and the minimum cross-validation error value is selected as the best clustering.

[0058] Then, according to the clustering results of the subpopulations, an outgroup is determined, and whether gene flow occurs between different triplets of the subpopulations is judged, where the triplet refers to a combination formed by three different subpopulations, which is called a triplet.

[0059] In the present invention, preferably, Populus euphratica is 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 the D value (i.e., the D-statistic, which is used to judge whether there is gene introgression) between different triplets in the subpopulations. When D>0, it is determined that gene flow may occur in the subpopulations. And the subpopulation combination with significant gene flow in the triplet is the combination that may have introgression.

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

[0062] Finally, the introgression combinations of the obtained poplar introgression population are detected by the method of sliding window analysis to obtain the introgression regions of the poplar introgression population.

[0063] Preferably, the Dinvestigate program in the D-suite software is used to obtain the introgression regions of all poplar introgression population combinations. Among them, the sliding window analysis uses 50 SNPs as the window and 20 SNPs as the step size.

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

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

[0066] The inventors of the present invention have found that first determining the combinations with significant introgression signals in the poplar introgression population and then determining the introgression regions for these combinations can effectively avoid the analysis of wrong combinations, reduce false positives, reduce the computational amount, and effectively improve the identification speed. According to the preferred embodiment of the present invention, during the process of determining the introgression regions, setting the window size in the sliding window analysis to 50 SNPs is beneficial to accurately identify the poplar introgression regions, so that the introgression genes can be more precisely located.

[0067] According to a preferred embodiment of the present invention, during the process of obtaining the poplar introgression population introgression regions, the top 5% of the windows with the largest detection results of each introgression combination are set as the introgression regions of this combination.

[0068] The present invention has found through research that the target ancestral introgression occurred before the divergence of the target species, and unique changes are very likely to occur during the long historical evolution after divergence, resulting in the weakening of the introgression signal. Therefore, in the present invention, a relatively loose threshold of 5% is selected in the process of screening introgression regions by fdM, rather than the commonly used 1%-3%, that is, even the detection of weaker signals is included, which is conducive to further screening of ancestral ancient introgression regions subsequently.

[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 subgroups.

[0072] Among them, the poplar subgroups are the poplar subgroups obtained in Step 1-2-1.

[0073] In the present invention, the subgroup topological structure of poplar can be obtained by using commonly used software in the prior art, such as SNPhylo software. Preferably, taking Populus euphratica as the outgroup to generate a phylogenetic tree to obtain the topological relationship among different poplar populations.

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

[0075] Step 1-2-2-2: Group the poplar subgroups according to the topological structure.

[0076] According to a preferred embodiment of the present invention, different five-taxon combinations of poplar subgroups are obtained according to the topological structure; preferably, the five-taxon is expressed as (((P1, P2), (P3, P4)), O), where the divergence time of P3 and P4 populations is not later than that of P1 and P2 populations, and O represents the outgroup;

[0077] Perform introgression analysis on the above-mentioned five-taxon, that is, determine whether the ancestors of P1 and P2 populations have introgressed with P3 or P4 populations.

[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 to identify the ancestral introgression regions shared by multiple populations of Populus alba.

[0079] Step 1-2-2-3: Obtain the ancestral introgression regions of each combination.

[0080] First, select the re-sequenced individuals with the deepest sequencing depth in each poplar population in the combination; then, split the chromosomes of the selected re-sequenced individuals into genomic segments; and then perform ancestral introgression detection on each genomic segment.

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

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

[0083] The inventor of the present invention has found through research 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, preferably, the present invention detects the candidate ancestral introgression regions of the five-taxa poplar combination according to the method including 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-taxa (((P1, P2), (P3, P4)), O), keep the first group of P1 and P2 unchanged, denoted as $P1 and $P2 respectively, and then change different combinations of P3 and P4 to obtain multiple different introgression results for the first group of $P1 and $P2.

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

[0087] Then, obtain all possible ancestral introgression regions for the first group of $P1 and $P2. Preferably, obtain all possible ancestral introgression regions for the first group of $P1 and $P2 by taking the union of multiple different introgression results, where the union is preferably denoted as U1.

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

[0089] Among them, n represents the number of combinations of P1 and P2, that is, one group of P1 and P2 is denoted as: n = 1.

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

[0091] Step ii, confirm the candidate ancestral introgression regions of 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 overlap degree of U1 to Un is obtained by taking the intersection, that is, taking the intersection of U1, U2, ..., 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 populations of poplar.

[0095] For example, when n=6, when 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 rate can be significantly improved by obtaining the gene flow of the common ancestors of multiple subpopulations through the above method.

[0097] Preferably, after the above steps, the ancestral introgression regions of all P1 and P2 combinations of 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 initially 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 more precisely located. 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 for obtaining the ancestral ancient introgression area of ​​the poplar introgression population of the present invention first determines the introgression combination and preliminarily locates the poplar introgression area, and then accurately obtains the common ancestral introgression area of ​​multiple populations according to the topological result, and then combines the two results and verifies each other, which can further reduce false positives and more accurately locate the ancestral ancient introgression area, which is conducive to excavating more genes that play important functions on the basis of 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 mine 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 alba and Populus nigra factions were not completely separated on the phylogenetic evolutionary tree, and therefore regarded Populus alba and Populus nigra as one group.

[0110] In a further preferred embodiment, XP-CLR is used to detect two subgroups to evaluate the frequency variation of different loci between populations, wherein the parameters of XR-CLR are conventionally set, and the composite likelihood ratio is used to evaluate 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, and the sliding window size is 10 kb and the step size is 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 region obtained in step 1-3-1 and the ancestral paleo-introgression region obtained in step 1-2-3 is used as the adaptive ancestral paleo-introgression region 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, gene annotation is performed on the adaptive ancestral ancient introgression region of the poplar introgression population obtained in Step 1-3-2. Preferably, in the present invention, the gene region and the 2000-bp promoter region upstream of the gene are used as the gene annotation file for the adaptive ancestral ancient introgression region of poplar.

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

[0118] In the present invention, preferably, a germplasm resource population composed of naturally growing Populus tomentosa individuals is selected and used as the analysis population for obtaining molecular markers after drought treatment, denoted as the poplar drought population. It preferably includes 303 genotypic individuals. The drought treatment steps are as follows: In 2018, in Guanxian, Shandong, asexual propagation is carried out through root cuttings. Each genotypic material has 3 replicates. When the sixth leaf grows at the top, drought treatment is started, maintaining the soil water 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 phenotype data includes proline content, total antioxidant enzyme activity, and abscisic acid content; among them, 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] Among them, 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] Obtain the DNA of each individual in the Populus tomentosa drought population and perform resequencing. Then, align the resequencing data to the Populus tomentosa reference genome, identify SNPs at the whole-genome level, and simultaneously obtain the genotypes of the SNP sites.

[0124] Preferably, according to the method described in Step 1-1, identify the SNP sites of the Populus tomentosa drought population and adopt the same quality control standards as in the above steps to obtain the SNP dataset of the Populus tomentosa drought population.

[0125] Step 3-2: Extract the SNP sites within the range of the adaptive ancestral ancient introgression genes 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 the present invention, since the population used in Step 1 contains different species of various sections of poplar, the genome of Populus trichocarpa, which has been the most studied, is selected as the reference genome. In Step 2, there is only Populus tomentosa. To reduce the alignment bias of the reference genome, the genome of Populus tomentosa is selected as the reference genome. To correspond the genes obtained in the two parts, the present invention performs homologous inference on each gene of the two reference genomes, and obtains the one-to-one best 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 subjected to alignment analysis to obtain the corresponding adaptive ancestral ancient introgression genes on the Populus tomentosa genome.

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

[0132] Step 3-3: Perform association analysis between the SNP sites of the adaptive ancestral ancient introgression genes in Step 3-2 and the drought phenotype to obtain SNP sites significantly associated with the poplar drought resistance phenotype.

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

[0134] In a further preferred embodiment, the SNP site significantly associated with the poplar drought resistance phenotype is located at the 979th position 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 poplar drought resistance phenotype, the significance of the differences in the phenotypes of poplar individuals with different genotype combinations is further examined.

[0136] In the present invention, the Student's t-test (P≤0.05) is preferably used to test the significance of differences in the phenotypes of poplars with different genotype combinations.

[0137] Preferably, when the genotype of the above-mentioned molecular marker of an individual of Populus tomentosa is CC, the drought resistance is the strongest; when the genotype of the above-mentioned molecular marker of an individual of Populus tomentosa is CT, the drought resistance is weaker; when the genotype of the above-mentioned molecular marker of an individual of Populus tomentosa is TT, the drought resistance is the weakest.

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

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

[0140] In the fifth aspect of the present invention, there is provided a primer pair for amplifying the molecular marker for screening drought-resistant poplars described in the second aspect. The primer pair includes primer P3 and primer P4. Among them, 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.

[0141] Preferably, the nucleotide sequence of primer P3 is as shown in SEQ ID NO: 6, and the nucleotide sequence of primer P4 is as shown in 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 the same DNA fragment (the DNA sequence between the upstream and downstream primers is the same) are all included in the primer pairs of the present invention.

[0143] In the present invention, the above-mentioned primer pair can be used to perform PCR amplification on the nucleotide fragment where the molecular marker significantly related to the drought-resistant trait of the to-be-tested Populus tomentosa is located. Then, through methods such as direct sequencing, the detection of this molecular marker can be effectively achieved, and then it can be determined whether the to-be-tested Populus tomentosa has this molecular marker. Specifically, individuals of Populus tomentosa with the genotype CC of the above-mentioned SNP marker have stronger drought resistance; individuals of Populus tomentosa with the genotype CT of the above-mentioned SNP marker have weaker drought resistance; individuals of Populus tomentosa with the genotype TT of the above-mentioned SNP marker have the weakest drought resistance.

[0144] The primer pair provided by the present invention can quickly, at low cost, and with high accuracy identify the drought resistance ability of poplar at an early stage, effectively shortening the drought resistance breeding cycle of poplar.

[0145] In the sixth aspect of the present invention, a method for genetic improvement of poplar is provided. The method includes the steps of successive selection of poplar individuals with the CC genotype of the molecular marker described in the second aspect or the molecular marker obtained by the method described in the third aspect, and elimination of individuals with other types of genotypes.

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

[0147] In the seventh aspect of the present invention, a method for identifying or screening poplar with strong drought resistance ability is provided. The method includes the following steps:

[0148] Step I: Extract the genomic DNA of the poplar to be tested;

[0149] Step II: Using the extracted genomic DNA as a template, perform PCR amplification;

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

[0151] Step IV: Determine the drought resistance ability of the poplar to be tested according to the genotype detection result.

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

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

[0154] In Step III, the method for determining the molecular marker genotype includes but is not limited to sequencing.

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

[0156] Examples

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

[0158] 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.

[0159] Example 1: Obtaining Molecular Markers for Screening Drought-Resistant Poplars

[0160] 1. Obtain the adaptive ancestral introgression genes of poplar

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

[0162] Table 1

[0163]

[0164]

[0165]

[0166]

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

[0168] Use Genome Analysis Toolkit (GATK) v4.0 to identify single nucleotide polymorphisms (SNPs) at the whole genome level. The parameters are as follows: SNP: QD < 2.0 || MQ < 20.0 || FS > 60.0 || SOR > 3.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0, to obtain the original SNP dataset of the poplar introgression population.

[0169] Perform quality control and filtering on the above original SNP dataset using Vcftools_0.1.16. The criteria are: ①biallelic sites; ②maximum missing rate < 0.2; ③minor allele frequency (MAF) > 0.05. And use the beagle v5.4 software to impute the filtered SNPs set. Finally, a high-quality SNP dataset of the poplar introgression population with a total of 3,434,667 SNP sites is obtained. (2)

[0171] (2.1) Based on the SNPs dataset obtained in step 1, the population structure of the poplar introgression population was obtained using the Admixture v1.3.0 software, and the minimum cross-validation error value was selected as the best population clustering. The clustering results are: Populus euphratica, Populus tremula, Populus davidiana, Populus alba, Populus adenopoda, Populus deltoides, Populus simonii, Populus koreana, Populus trichocarpa.

[0172] According to the subgroup clustering results, using Populus euphratica as the outgroup, the D value was calculated between different trios of the 8 subgroups using the Dtrios program in the Dsuite v0.5 software. The Dtrios results are shown in Table 2:

[0173] Table 2

[0174]

[0175]

[0176] It can be seen from Table 2 that the trios corresponding to Z-scores >= 3 are the subgroup combinations with significant gene flow. For example, in the first row of Table 2, the Z-score is 41.8332, indicating that there is gene flow between the Populus simonii subgroup and the Populus trichocarpa subgroup in the trio composed of Populus simonii, Populus trichocarpa, and Populus deltoides.

[0177] For the combinations with significant gene flow, the Dinvestigate program was further used to locate the introgression regions through a sliding window algorithm. The window size was set to 50 SNPs, and the step size was 20 SNPs to identify the introgression regions between the introgression population combinations. Finally, all candidate introgression regions between the Populus alba, Populus cathayana, and Populus nigra introgression combinations were obtained. Among them, the introgression region was defined as the window with the top 5% of the largest fdM in the results of Dinvestigate.

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

[0179] To identify the ancestral gene introgression regions shared by multiple populations of Populus alba, further based on the topological results, the five-taxon group (((P1, P2), (P3, P4)), O) was determined, where P1 and P2 were designated as Populus alba populations, P3 and P4 were designated as Populus cathayana and Populus nigra populations, and the outgroup O was designated as Populus euphratica. Among them, the Populus alba populations include Populus adenopoda, Populus alba, Populus davidiana, Populus tremula, the Populus cathayana populations include Populus koreana, Populus simonii, Populus trichocarpa, and the Populus nigra population includes Populus deltoides.

[0180] Keep P1 and P2 unchanged, change different combinations of P3 and P4, select the re-sequenced individuals with the deepest sequencing depth in each poplar population, convert them into fasta format and then split them into 100-kb genomic segments. After alignment, use the DFOIL method to calculate the introgression results for each combination respectively. As shown in Table 3, there are a total of 6 pairs of P1 and P2 combinations. Each pair of P1 and P2 combinations yields 6 results. Denote the union of the 6 results as U1 (i.e., the union of the 6 results in the first row), which represents the candidate ancestral introgression segments for this pair of P1 and P2 combinations. Among them, the five-class poplar group includes 6 white poplar combinations (i.e., 6 pairs of P1 and P2 combinations), and the total number of introgression combinations is 36. For example, the first row is the first pair of P1 and P2 combinations. The first introgression combination in it is: P1 is Populus adenopoda, P2 is Populus alba, P3 is Populus deltoides, P4 is Populus koreana, and O is Populus euphratica. The other combinations follow this pattern. Table 2 has a total of six rows, representing 6 pairs of P1 and P2 combinations.

[0181] Table 3

[0182]

[0183]

[0184] Change the population categories of P1 and P2, repeat the above steps, and 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, 6 groups of ancestral introgression segments are obtained for the 36 five-class poplar combinations, namely U1, U2, U3, U4, U5, and U6.

[0186] Take the intersection of each group of ancestral introgression segments (i.e., U1 - U6). When a region is present in 5 white poplar combinations (P1, P2 combinations) simultaneously, it is considered as the candidate ancestral introgression region for multiple poplar populations. The results are as Figure 2 shown, where the solid triangle markers represent the results where the same region is present in ≥5 combinations, that is, the candidate ancestral introgression regions for multiple poplar populations.

[0187] From Figure 2 it can be seen that: the number of introgression regions present in all six white 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% (i.e., ≥5) of the combinations are also considered as ancestral ancient introgression regions because during the long historical evolution process, some regions may have undergone unique changes in individual species and thus cannot be recognized, but are still detected as ancestral ancient introgression regions in other species. Therefore, these regions are also identified as ancestral ancient introgression regions.

[0188] (2.3) Take the intersection of the introgression segments of each significant triplet result obtained in step (2.1) and the Populus alba candidate ancestral introgression regions obtained in step (2.2) to obtain the final Populus alba ancestral paleo-introgression segments.

[0189] (3) Consider the Tacamahaca and Aigeiros sections as one sub-group and all Populus alba as another sub-group, and use XP-CLR to detect the adaptive regions of the two sub-groups. The size of the sliding window is 10 kb and the step size is 5 kb. Take the top 5% of the windows with the most significant detection results as the final adaptive regions.

[0190] Combine the above-obtained adaptive regions with the ancestral paleo-introgression regions obtained in step (2.3), that is: when a region belongs to both the ancestral paleo-introgression regions and the adaptive regions, it is considered an adaptive ancestral paleo-introgression region, and thus obtain the adaptive ancestral paleo-introgression regions of the Populus introgression population.

[0191] Take each gene region and the 2000-bp upstream promoter region of the gene in the genome annotation file of the reference genome as the gene annotation file of the adaptive ancestral paleo-introgression regions of Populus, and use the BEDTools intersect command to annotate the adaptive ancestral paleo-introgression genes of the Populus introgression population.

[0192] 2. Select a Populus population to obtain the drought phenotype data of Populus.

[0193] Select a germplasm resource population consisting of 303 naturally growing Populus tomentosa individuals covering the entire suitable growth area of Populus tomentosa. After drought treatment, it is used as the analysis population for obtaining molecular markers. The drought treatment steps are as follows: In 2018, conduct asexual propagation through root cuttings in Guanxian, Shandong. Each genotype material has 3 replicates. When the sixth leaf grows at the top, start the drought treatment, maintain the soil water content at 10%, continue for 30 days, and the average air humidity is 67.5%.

[0194] Collect the functional leaves (i.e., the 4th to 6th leaves at the top of the stem) of each genotype from 303 Populus tomentosa individuals through 3 cloning replicates, immediately immerse them in liquid nitrogen, store them at -80 °C, and then perform vacuum freeze-drying for measuring the drought resistance index.

[0195] 3. Obtain molecular markers related to Populus drought resistance

[0196] (3.1) Use the FastPure Plant DNA Isolation Mini Kit (Nanjing Novoprotein Biological Technology Co., Ltd.) kit to extract the genomic DNA of 303 genotype individuals in the Populus tomentosa drought population. Use a UV spectrophotometer and gel electrophoresis to detect the DNA quality, and store the qualified DNA at -20 °C.

[0197] Resequence the DNA of each individual in the Populus tomentosa drought population (using common methods in existing technologies), and use Burrows-Wheeler Aligner v0.7.5a-r405 (default parameters) to align each resequencing data to the Populus tomentosa reference genome (published in the China National GeneBank Sequence Archive (CNGB Sequence Archive, CNSA), project number CNP0004290), and identify single nucleotide polymorphism sites at the whole genome level and obtain the genotypes of SNP sites.

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

[0199] (3.2) Infer homologous genes between the Populus trichocarpa reference genome used in the Populus hybridization introgression population and the Populus tomentosa reference genome used in the Populus tomentosa drought population. Use the Genetribe v1.0 software to perform reciprocal best hit (RBH) analysis of genes, obtain the corresponding adaptive ancestral ancient introgressed genes on the Populus tomentosa genome, and extract SNP sites within the range of adaptive ancestral ancient introgressed genes among the SNP sites of the Populus tomentosa drought population in step (3.1).

[0200] The obtained adaptive ancestral ancient introgressed gene of Populus tomentosa is PtoTCP19, whose nucleotide sequence 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 is obtained according to the following steps: Use the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Novoprotein Biological Technology Co., Ltd.) kit to extract RNA from the leaves of 300 genotype individuals of Populus tomentosa. Use the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novoprotein Biological Technology Co., Ltd.) to reverse transcribe RNA into cDNA. Refer to the Populus tomentosa reference genome as a template, and comprehensively consider the principles of primer design to design primers using the primer blast tool (NCBI, https: / / blast.ncbi.nlm.nih.gov). The primers are P1 and 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. Use primers P1 and P2 to perform PCR amplification with the obtained genomic cDNA as a template to obtain the gene sequence.

[0202] The kinship of the Populus tomentosa population was calculated using the Efficient Mixed-Model Association eXpedited (EMMAX) software, and the population structure of the Populus tomentosa population was calculated using the Admixture software (the lowest cverror was obtained when K = 3). The kinship and population structure were used as covariates, and candidate gene association analysis was performed on the SNPs set of the adaptive ancestral ancient introgression genes of Populus tomentosa and the drought phenotype data in step 2 through the Mixed linear Model (MLM). After correcting the P values by False Discovery Rate (FDR), SNP loci (threshold: P < 0.05) significantly associated with Populus tomentosa drought and their genotypes were obtained.

[0203] After the above screening, 1 SNP locus was significantly associated with the drought-resistant traits of the Populus tomentosa drought population, with P < 0.05. This locus is located at the 1022nd base upstream of the PtoTCP19 gene in the Populus tomentosa reference genome, that is, at the 979th position of the nucleotide sequence shown in SEQ ID NO: 3, with T / C polymorphism.

[0204] The significance of the differences in the phenotypes of Populus tomentosa with different genotype combinations was tested using the Student’s t-test (P ≤ 0.05) method. It was found that Populus tomentosa individuals with the CC genotype at the above molecular marker had higher drought resistance (higher proline content, abscisic acid content, and total antioxidant enzyme activity) compared to Populus tomentosa individuals with the TT genotype.

[0205] The genotypes and phenotype data of the molecular markers related to the 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 the proline content, abscisic acid content, and total antioxidant enzyme activity of Populus tomentosa corresponding to different genotypes are respectively as Figures 3 - 5 shown.

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

[0213] Example 2: Verification of the Effectiveness of Molecular Markers

[0214] In the early spring of 2022, 500 individuals of Populus tomentosa with different genotype combinations were randomly selected from the Populus tomentosa germplasm resource population (1,047 plants, Guaxian base, Shandong). Using the above method, 9 functional leaves (the 3rd to 5th mature leaves at the upper end of the branch) were collected from 9:00 to 11:00 in the morning. Six leaves were used for the determination of stomatal area, and 3 leaves were immediately placed in a liquid nitrogen environment (-196 °C) for preservation to extract genomic DNA. Primers P3 and P4 were used to determine the genotype of the 1022nd base upstream of the TCP19 gene. The results of the drought resistance ability (total antioxidant enzyme activity, abscisic acid content, proline content) corresponding to different individuals are shown in Table 5 and Figure 6 as follows.

[0215] Table 5

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] As can be seen from Table 5 and Figure 6 it can be seen that among the 500 randomly selected individuals of Populus tomentosa in this example, there are significant differences in the three drought resistance indexes of different genotypes. The individuals with the genotype CC have the highest proline content, abscisic acid content and total antioxidant enzyme activity, while the individuals with the genotype TT have the lowest proline content, abscisic acid content and total antioxidant enzyme activity, further proving that the CC genotype has stronger drought resistance ability.

[0224] The present invention has been described in detail above in connection with specific embodiments and exemplary examples, but these descriptions should not be construed as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications or improvements can be made to the technical solutions and their implementation manners of the present invention, and all of these fall within the scope of the present invention.

Claims

1. A gene related to drought resistance of poplar, characterized in that: The gene is PtoTCP19, and its nucleotide sequence includes the sequence shown in SEQ ID NO:

1.

2. A molecular marker 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.

3. A method for obtaining the molecular marker according to claim 2, characterized in that: The method comprises the following steps: Step 1, obtaining the adaptive ancestral ancient introgression genes of poplar; Step 2, selecting a poplar population and obtaining drought phenotype data of the poplar; Step 3, obtaining molecular markers related to drought resistance of poplar.

4. The method according to claim 3, characterized in that Step 1 includes the following sub-steps: Step 1-1, obtaining a SNP data set of a poplar introgression population; Step 1-2, obtaining the ancestral paleo-introgression region of the poplar introgression population; Steps 1-3, obtain the adaptive ancestral ancient introgression genes of the poplar introgression population.

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

5.

6. A primer pair for amplifying the molecular marker according to claim 2, characterized in that: 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.

7. A method for genetic improvement of poplars, characterized in that: The method comprises the steps of successive breeding of poplar individuals with CC genotypes using the molecular markers described in claim 2 or the molecular markers obtained by the method described in claim 3, and eliminating individuals with other genotypes.

8. The improved method according to claim 7, characterized in that: The poplar is Populus tomentosa.

9. A method for identifying or screening poplars with strong drought resistance, characterized in that: The method comprises the following steps: Step I, extracting genomic DNA of the poplar to be tested; Step II, using the above-extracted genomic DNA as a template, performing PCR amplification; Step III, determining the genotype of the molecular marker of claim 2 of the poplar to be tested; Step IV, determining the drought resistance of the tested poplar according to the genotype detection results.

10. The method according to claim 9, characterized in that In step IV, if the genotype of the tested poplar molecular marker is CC, its drought resistance is the strongest; if the genotype of the tested poplar molecular marker is CT, its drought resistance is relatively weak; if the genotype of the tested poplar molecular marker is TT, its drought resistance is the weakest.

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