Molecular marker for identifying geographical origin of populus tomentosa and application thereof

Abstract

The application discloses a molecular marker for identifying geographical origin of Populus tomentosa and application thereof, and the molecular marker is located at the 42th base of FPA gene in a poplar genome and has C / A polymorphism, wherein a P. tomentosa individual with a genotype AA of the molecular marker has a shorter annual average sunshine duration, and a P. tomentosa individual with a genotype CC of the molecular marker has a longer annual average sunshine duration. The molecular marker provided by the application has a clear function and a significant effect, and can be directly applied to molecular marker assisted breeding of the poplar; and the molecular marker has environmental adaptability and is suitable for directional introduction of the poplar.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically involving molecular markers, primer sets, reagent kits, and their applications for accurately identifying the germplasm origin of poplar trees. Background Technology

[0002] Tree introduction is an important means of effectively utilizing, transforming, and protecting tree species resources. Introducing suitable exotic tree species can enrich the variety of local afforestation and greening tree species, improve tree species distribution and natural landscape, and provide new genetic resources for further breeding of superior varieties. However, my country's tree introduction work is currently in its initial stage, and there are still problems such as unclear provenance of introduced materials and low introduction efficiency.

[0003] Poplar (Populus tomentosa) is a commonly used native tree species in artificial forest construction in northern my country. It is widely distributed in Liaoning (southern part), Hebei, Shandong, Shanxi, and Shaanxi provinces, with its central distribution area in the middle and lower reaches of the Yellow River. It plays an important role in forestry production and ecological environment construction in northern my country and is a pioneer species for forest cultivation in the region. However, poplars are perennial species, and genetic improvement through traditional hybridization breeding methods is time-consuming. With the continuous development of modern molecular breeding technology, it has become possible to accurately identify the germplasm origin of Poplar tomentosa at the molecular level.

[0004] However, there is currently a lack of molecular markers that can accurately identify the germplasm origin of Populus tomentosa, making it impossible to quickly and accurately determine the germplasm origin of unknown Populus tomentosa species.

[0005] Therefore, it is necessary to discover universal, high-impact, and precise molecular markers for identifying the geographical origin of Populus tomentosa germplasm in order to solve the above problems. Summary of the Invention

[0006] To overcome the above problems, the inventors conducted intensive research and provided a molecular marker for identifying the germplasm geographical origin of Populus tomentosa. This marker is obtained by integrating genotype-environment association analysis and selective removal strategies. By screening the gene marker types of Populus tomentosa under site conditions, the germplasm geographical origin of unknown Populus tomentosa species can be accurately and efficiently determined, shortening the breeding cycle. Furthermore, it allows for targeted introduction of the species based on its biological characteristics, ensuring that site conditions and species characteristics are mutually adapted, thus completing this invention.

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

[0008] In one aspect, a molecular marker is provided for identifying the geographical origin of Populus tomentosa germplasm. The molecular marker is located at the 42nd base of the FPA gene in the Populus genome and has C / A polymorphism.

[0009] In a second aspect, a method for obtaining the molecular marker described in the first aspect is provided, the method comprising the following steps:

[0010] Step 1: Obtain the geographical and climatic information of the origin of the Populus tomentosa population;

[0011] Step 2: Obtain molecular markers related to the geographical origin of Populus tomentosa germplasm;

[0012] Preferably, step 2 includes the following sub-steps:

[0013] Step 2-1: Resequencing the DNA of each individual in the Populus tomentosa population and performing quality control on the obtained raw data;

[0014] Step 2-2: Identify single nucleotide polymorphism sites and their genotypes at the whole-genome level;

[0015] Steps 2-3 involve screening SNPs at the whole genome level to obtain a high-quality set of SNP markers;

[0016] Steps 2-4: Obtain SNP markers that are significantly associated with the annual average sunshine duration of Populus tomentosa seed source areas;

[0017] Steps 2-5 involve selectively removing SNP markers that show significant associations.

[0018] Thirdly, a primer pair is provided for amplifying the molecular markers used in the first aspect to identify the geographical origin of Populus tomentosa germplasm, the primer pair comprising primer P1 and primer P2.

[0019] Primer P1 includes the nucleotide sequence shown in SEQ ID NO: 3, and primer P2 includes the nucleotide sequence shown in SEQ ID NO: 4.

[0020] Fourthly, a detection kit is provided, preferably for detecting the molecular markers used in the first aspect to identify the geographical origin of Populus tomentosa germplasm, the detection kit comprising the primer pairs described in the third aspect, and further comprising PCR amplification reagents.

[0021] Fifthly, the application of the molecular markers for identifying the geographical origin of Populus tomentosa described in the first aspect, the molecular markers obtained by the method described in the second aspect, the primer pairs described in the third aspect, or the detection kits described in the fourth aspect in marker-assisted breeding of Populus tomentosa or targeted introduction of Populus tomentosa.

[0022] Sixthly, a method for identifying or assisting in the identification of the geographical origin of Populus tomentosa germplasm is provided, characterized in that the method includes the following steps:

[0023] Step 1: Extract genomic DNA from the Populus tomentosa species to be tested;

[0024] Step II: Using the extracted genomic DNA as a template, perform PCR amplification;

[0025] Step III: Determine the polymorphism or genotype of the SNP marker described in the first aspect of the Populus to be tested;

[0026] Step IV: Determine the germplasm geographical origin of the Populus to be tested based on the genotype results.

[0027] The beneficial effects of this invention include:

[0028] (1) The molecular markers provided by this invention for identifying the geographical origin of Populus tomentosa germplasm have clear functions and significant effects, and can be directly applied to molecular marker-assisted breeding of poplar.

[0029] (2) The molecular markers provided by this invention for identifying the geographical origin of Populus tomentosa germplasm are SNP loci that are significantly associated with the "sunshine hours in the source area" trait in the natural distribution area of ​​Populus tomentosa at the whole genome level, and have significantly differentiated allele frequencies among different geographical populations. They can accurately and quickly identify the geographical origin of Populus tomentosa germplasm at the molecular level, which is beneficial for seed regionalization and effectively shortens the breeding cycle of Populus tomentosa.

[0030] (3) The method for obtaining molecular markers for identifying the geographical origin of Populus tomentosa provided by this invention integrates genome-wide association analysis and selective clearance methods, so that the obtained molecular markers have stable adaptability and are suitable for the targeted introduction of poplar trees;

[0031] (4) The method for identifying or assisting in the identification of the geographical origin of Populus tomentosa provided by the present invention is easy to operate and significantly improves breeding efficiency. Attached Figure Description

[0032] Figure 1 The diagram illustrates the genotypic effect of different genotypes of the SNP marker sites described in Embodiment 1 of the present invention on the latitude of the source region of Populus tomentosa germplasm. Detailed Implementation

[0033] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the features and advantages of the present invention will become clearer and more apparent.

[0034] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0035] Based on the above research, in a first aspect, the present invention provides a molecular marker for identifying the geographical origin of Populus tomentosa germplasm, wherein the molecular marker is an SNP marker located at the 42nd base of the FPA gene in the Populus tomentosa genome and exhibits C / A polymorphism.

[0036] In this invention, the sequence of the FPA gene is shown in SEQ ID NO: 1.

[0037] The FPA gene is an autonomously regulated gene independent of day length. Encoding an RNA-binding protein, it may have post-transcriptional regulatory mechanisms and possesses a SPOC functional domain at its C-terminus, participating in plant developmental signal transduction. The FPA gene is highly expressed in developing tissues and can promote earlier flowering; artificially constructed fpa mutants also exhibit the desired late-flowering trait. This gene not only plays a role in inducing flowering but may also play a broader role in plant growth and development.

[0038] According to a preferred embodiment of the present invention, the molecular marker for identifying the geographical origin of Populus tomentosa germplasm is located at position 42 of the nucleotide sequence shown in SEQ ID NO: 1.

[0039] In a further preferred embodiment, the amino acid sequence encoding the FPA protein is shown in SEQ ID NO: 2.

[0040] Among them, positions 483-607 of the sequence shown in SEQ ID NO:2 are SPOC functional structural domains.

[0041] In a further preferred embodiment, the molecular marker is significantly correlated with the average annual sunshine duration trait of the Populus tomentosa seed origin.

[0042] The average annual sunshine duration is the average amount of sunlight received by the ground within a year. Sunlight is an energy requirement for poplar growth and a necessary condition for photosynthesis. Through extensive research, the inventors have discovered that different environmental pressures shape the genetic diversity at the genome level of Populus tomentosa populations. The duration of sunshine in different provenance locations also influences the distribution of Populus tomentosa across different latitudes and spaces, and alters the allele frequency distribution among different subpopulations. Therefore, in-depth analysis of the genetic structure related to the duration of sunshine in the provenance locations of Populus tomentosa can effectively predict the germplasm origin of Populus tomentosa, providing technical reference for the introduction and breeding of Populus tomentosa, and has significant biological value.

[0043] According to a preferred embodiment of the present invention, the genotypes of the molecular marker are AA, CA, and CC, wherein,

[0044] Poplar individuals with the genotype AA, identified by the molecular marker, are distributed in high-latitude regions with shorter average annual sunshine hours.

[0045] Poplar individuals with the genotype CC, identified by the molecular marker, are distributed in low-latitude regions with longer average annual sunshine hours.

[0046] Populus tomentosa individuals with the molecular marker genotype CA are distributed in mid-latitude regions with moderate annual average sunshine hours.

[0047] In this invention, the AA genotype is a homozygous type of Populus tomentosa with the molecular marker A, the CC genotype is a homozygous type of Populus tomentosa with the molecular marker C, and the CA genotype is a heterozygous type of Populus tomentosa with the molecular markers C and A.

[0048] Among them, Populus tomentosa individuals with genotype AA are mostly distributed in high-latitude regions with shorter average annual sunshine hours; while Populus tomentosa individuals with genotype CC are mostly distributed in low-latitude regions with longer average annual sunshine hours.

[0049] In this invention, the annual average sunshine duration is an inherent attribute of the Populus tomentosa seed origin, not a unique phenotype of individual Populus tomentosa individuals. Information on the annual average sunshine duration and latitude of the Populus tomentosa populations was obtained from the China Climate Data Network and the National Geographic Information Public Platform, respectively, revealing a significant negative correlation between the two. According to an embodiment of this invention, as shown in Table 1, this indicates that the higher the latitude, the lower the annual average sunshine duration.

[0050] Preferably, the high latitude region is 37.00–42.00°N, the mid latitude region is 35.00–37.00°N, and the low latitude region is 30.00–35.00°N.

[0051] The molecular markers described in this invention are SNP loci that are significantly associated with the "average annual sunshine hours" trait in the natural distribution area of ​​Populus tomentosa at the whole genome level, and have significantly differentiated allele frequencies among different geographical populations. They can accurately and quickly identify the geographical origin of poplar germplasm at the molecular level, which is beneficial for seed regionalization and effectively shortens the breeding cycle of poplar.

[0052] A second aspect of the present invention provides a method for obtaining the molecular marker described in the first aspect, the method comprising the following steps:

[0053] Step 1: Obtain the geographical and climatic information of the origin of the Populus tomentosa population.

[0054] According to an embodiment of the present invention, the poplar population consists of 240 individuals covering the entire suitable growing area of ​​the poplar.

[0055] Preferably, data is obtained through the National Geographic Information Public Service Platform (https: / / www.tianditu.gov.cn / ), the China Meteorological Data Network (http: / / data.cma.cn / ), and the U.S. Environmental Information Center (...). https: / / www.ncei.noaa.gov / We collected latitude information of the seed origin of 240 individuals of Populus tomentosa and climate data of the average annual sunshine hours for 50 consecutive years from 1971 to 2020.

[0056] Climate data from 1971 to 2010 were collected using the China Meteorological Data Network, and climate data from 2011 to 2020 were collected using the U.S. Environmental Information Center.

[0057] Step 2: Obtain molecular markers related to the geographical origin of Populus tomentosa germplasm.

[0058] Since the length of sunshine in different provenance areas also affects the distribution of Populus tomentosa at different latitudes and in different spaces, in this invention, it is preferred that the molecular markers related to the average annual sunshine hours of Populus tomentosa are the same molecular markers related to the geographical origin of Populus tomentosa germplasm. At the same time, the latitude of the provenance area can be determined based on the average annual sunshine hours. The trend is that Populus tomentosa individuals with shorter average annual sunshine hours are distributed in high-latitude areas, while Populus tomentosa individuals with longer average annual sunshine hours are distributed in low-latitude areas.

[0059] Preferably, step 2 includes the following sub-steps:

[0060] Step 2-1: Resequencing the DNA of each individual in the Populus tomentosa population and performing quality control on the obtained raw data.

[0061] Preferably, the DNA resequencing is paired-end sequencing with a sequencing depth of 30×, and the sequencing is performed using the Illumina GA2 resequencing platform.

[0062] The quality control standards are as follows: (i) removing reads containing ≥10% unidentified nucleotides; (ii) removing reads with a base mass <5 >50%; (iii) removing reads >10nt aligned to the adapter, allowing ≤10% mismatches; and (iv) deleting putative PCR repeats (two identical paired reads 1 and 2) generated by PCR amplification during library construction.

[0063] Step 2-2: Identify single nucleotide polymorphism sites and their genotypes at the whole genome level.

[0064] The data, after quality control, will be compared to the poplar reference genome v3.0 (http: / / popgenie.org) to identify single nucleotide polymorphism sites and obtain the genotypes of SNP sites at the whole genome level.

[0065] Steps 2-3 involve screening SNPs at the whole genome level to obtain a high-quality set of SNP markers.

[0066] Preferably, SNPs at the whole genome level are screened using VCFTOOLS software, with the following screening conditions: minimum allele frequency (MAF) > 0.05 and missing genotype (MG) < 0.2.

[0067] Steps 2-4: Obtain SNP markers that are significantly associated with the annual average sunshine hours of Populus tomentosa seed source areas.

[0068] Among them, the Latent Factor Mixed Models (LFMM) in LEA software was preferred to be used to conduct genome-environmental variable association analysis on the high-quality SNP set of whole genome of Populus tomentosa. At the same time, the population structure of Populus tomentosa population was calculated. Based on the whole genome SNP markers, the natural Populus tomentosa population was divided into 3 main subpopulations (i.e., K=3) to correct the false positive associations caused by them. Finally, SNP loci that were significantly associated with the annual average sunshine hours of Populus tomentosa seed source area were obtained.

[0069] This site is located within the FPA gene, preferably at position 42 of the nucleotide sequence shown in SEQ ID NO: 1, and exhibits C / A polymorphism.

[0070] Steps 2-5 involve selectively removing SNP markers that show significant associations.

[0071] In this invention, based on the analysis results of the population structure in steps 2-4, the natural population of 240 individuals covering the entire suitable habitat of Populus tomentosa is divided into 3 subgroups. The division of these subgroups is consistent with the division of subgroups according to latitude, including 86 high-latitude (H) individuals, 63 mid-latitude (M) individuals and 91 low-latitude (L) individuals.

[0072] Among them, high-latitude individuals refer to those distributed at latitudes of 37.00–42.00°N, mid-latitude individuals refer to those distributed at latitudes of 35.00–37.00°N, and low-latitude individuals refer to those distributed at latitudes of 30.00–35.00°N.

[0073] Among them, there is a significant negative correlation between the latitude of the source area of ​​Populus tomentosa and the average annual sunshine hours of the source area.

[0074] Selective elimination refers to a phenomenon in which, during natural or artificial selection, the frequency of certain dominant alleles in a population increases, while the polymorphism of the surrounding chromosomal regions linked to them decreases due to the hitch effect.

[0075] The strategy of integrating selective sweeping and genotype-environment association analysis can identify marker loci in the genome that exhibit genetic adaptation characteristics, with allelic frequencies showing significant differentiation signals among different provenance populations. This strategy can effectively detect how allelic markers change with environmental variations and can be widely applied to analyze adaptive selection patterns in perennial trees, providing new insights into predicting adaptive genetic variation and elucidating local adaptation mechanisms under global change.

[0076] According to a preferred embodiment of the present invention, the population differentiation coefficient (Fixation statistic value, Fst) and nucleotide diversity index (π) of the three subpopulations are calculated using the VCFTOOLS software via a sliding window algorithm.

[0077] The sliding window has a size of 2kb and a step size of 1kb.

[0078] After removing windows with π < 1E-03, the top 5% of windows with the most significant π and Fst values ​​are selected as the most significant selective clearance regions and merged with single scan regions with an interval of less than 5kb. Genes that overlap with the scan regions are defined as selective clearance genes.

[0079] In a further preferred embodiment, it is determined whether the SNP loci obtained that are significantly associated with the annual average sunshine hours of the Populus tomentosa seed source area are located within the selectively cleared region obtained above.

[0080] If an SNP marker is located within a selective removal area, then the marker is a marker that has undergone natural selection, is environmentally adaptable, and can be used for regional breeding; if an SNP marker is not located within a selective removal area, then the marker is a marker that has not undergone natural selection, is not environmentally adaptable, and cannot be used for regional breeding.

[0081] In this invention, the 42nd base of the FPA gene is significantly associated with the trait of average annual sunshine hours and is also located in a selectively eliminated region, indicating that it has been subject to significant natural selection. FPA is a gene subject to adaptive selection, exhibiting a significant pattern of germplasm differentiation, resulting in differences in the allele frequency of the target SNP across different latitudes and geographical regions. This can be used to identify the origin of Populus tomentosa germplasm. This invention obtains molecular markers related to the geographical origin of Populus tomentosa germplasm. It integrates genotype-environment association analysis with selective removal strategies to identify the germplasm origin of Populus tomentosa. First, the population structure of Populus tomentosa populations is calculated using a latent factor mixed-effects model in LEA software. Based on genome-wide SNP markers, the natural Populus tomentosa populations are divided into three main genetic subpopulations (i.e., K=3). Genome-environmental variable association analysis is performed on the average annual sunshine duration of the Populus tomentosa population's provenance and the set of high-quality SNPs from the genome. SNP loci significantly associated with the average annual sunshine duration of the provenance are screened out, and related genes are annotated. Based on the population structure results from the above steps, a natural population of 240 Populus tomentosa individuals covering the entire suitable growing area is divided into three subpopulations. It is found that dividing the populations according to latitude is consistent with dividing them by latitude, and a significant negative correlation is found between the latitude of the provenance and the average annual sunshine duration.

[0082] Using the molecular markers obtained above to predict the growth of Populus tomentosa under site conditions, and introducing the species in a targeted manner according to the principle of suitable species for suitable site, can effectively improve the disadvantage of the long time required for genetic improvement of poplar trees by traditional hybridization breeding methods.

[0083] A third aspect of the present invention provides a primer pair for amplifying the molecular markers used in the first aspect for identifying the geographical origin of Populus tomentosa germplasm, the primer pair comprising primer P1 and primer P2.

[0084] Primer P1 includes the nucleotide sequence shown in SEQ ID NO: 3, and primer P2 includes the nucleotide sequence shown in SEQ ID NO: 4.

[0085] In this invention, primer pairs that add 1 to 20 bases to the 5' and 3' ends of the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively and can produce substantially identical DNA fragments (the DNA sequences between the upstream and downstream primers are the same) are all included in the primer pairs of this invention.

[0086] Preferably, the nucleotide sequence of primer P1 is shown in SEQ ID NO: 3, and the nucleotide sequence of primer P2 is shown in SEQ ID NO: 4.

[0087] In this invention, the aforementioned primers are used to amplify the nucleotide fragment containing the molecular marker that is significantly associated with the annual average sunshine duration of the tested Populus tomentosa trait in its provenance region via PCR. Then, methods such as direct sequencing can be used to effectively detect this molecular marker and determine whether the tested Populus tomentosa possesses it. Specifically, individuals of Populus tomentosa with the genotype AA for the aforementioned SNP marker have shorter annual average sunshine durations and are distributed in high-latitude regions; individuals of Populus tomentosa with the genotype CC for the aforementioned SNP marker have longer annual average sunshine durations and are distributed in low-latitude regions.

[0088] Therefore, when the molecular marker of a Populus tomentosa individual is detected as AA genotype, it can be determined that the germplasm origin of the individual has a shorter average annual sunshine duration and is located in a high-latitude region; when the molecular marker of a Populus tomentosa individual is detected as CC genotype, it can be determined that the germplasm origin of the individual has a longer average annual sunshine duration and is located in a low-latitude region.

[0089] The primer pairs provided by this invention can achieve early, short-term, low-cost, and high-accuracy identification of the geographical origin of Populus tomentosa germplasm, which is beneficial for seed regionalization and effectively shortens the poplar breeding cycle.

[0090] In a fourth aspect, the present invention provides a detection kit, preferably for detecting the molecular markers described in the first aspect.

[0091] According to a preferred embodiment of the present invention, the detection kit includes the primer pair described in the third aspect, and also includes PCR amplification reagents.

[0092] The PCR amplification reagents include PCR buffer, dNTPs, and DNA polymerase.

[0093] In a fifth aspect, the present invention provides the application of the molecular markers described in the first aspect, the molecular markers obtained by the method described in the second aspect, the primer pairs described in the third aspect, or the detection kit described in the fourth aspect in marker-assisted breeding or directional introduction of poplar.

[0094] The poplar tree is preferably the white poplar.

[0095] A sixth aspect of the present invention provides a method for identifying or assisting in the identification of the geographical origin of Populus tomentosa germplasm, the method comprising the following steps:

[0096] Step 1: Extract genomic DNA from the Populus tomentosa species to be tested;

[0097] Step II: Using the extracted genomic DNA as a template, perform PCR amplification;

[0098] Step III: Determine the polymorphism or genotype of the above SNP markers in the Populus tomentosa to be tested;

[0099] Step IV: Determine the germplasm geographical origin of the Populus to be tested based on the genotype results.

[0100] In step II, the primers for PCR amplification are preferably the amplification primer pairs for identifying the geographical origin of Populus tomentosa germplasm as described in the third aspect.

[0101] In step III, the geographical origin of Populus tomentosa germplasm can be identified by determining the polymorphism or genotype of the above-mentioned SNP markers.

[0102] Preferably, the methods for determining molecular marker polymorphisms or genotypes include, but are not limited to, sequencing.

[0103] In step IV, if the genotype of the SNP marker of the Populus to be tested is AA, it has a shorter average annual sunshine duration and is distributed in high-latitude regions; if the genotype of the SNP marker of the Populus to be tested is CC, it has a longer average annual sunshine duration and is distributed in low-latitude regions.

[0104] Example

[0105] The present invention is further described below through specific examples; however, these examples are merely exemplary and do not constitute any limitation on the scope of protection of the present invention.

[0106] Example 1: Obtaining Molecular Markers for Identifying the Geographical Origin of Populus tomentosa Germplasm

[0107] 1. Experimental population selection: a natural population of Populus tomentosa under normal growing conditions, consisting of 240 naturally growing individuals covering the entire suitable growing area of ​​Populus tomentosa.

[0108] Genomic DNA was extracted from leaf tissues of 240 individuals in the experimental population using the DNeasy Plant Mini Kit (Qiagen China, Shanghai, China).

[0109] DNA quality was assessed using a UV spectrophotometer and gel electrophoresis, and DNA that passed the tests was stored at -20°C.

[0110] 2. Latitude information of the provenance of 240 individuals of Populus tomentosa was collected through the National Geographic Information Public Service Platform (https: / / www.tianditu.gov.cn / ), China Meteorological Data Network (http: / / data.cma.cn / ), and the Environmental Information Center of the United States (https: / / www.ncei.noaa.gov / ), as well as environmental data on the average annual sunshine hours for 50 consecutive years from 1971 to 2020.

[0111] 3. The DNA of each individual was resequencing to obtain the raw data. After quality control, the data was compared with the poplar reference genome v3.0 (http: / / popgenie.org) to identify single nucleotide polymorphism sites and obtain the genotype of SNP sites at the whole genome level.

[0112] The quality control standards are as follows: (i) removing reads containing ≥10% unidentified nucleotides; (ii) removing reads with a base mass <5 >50%; (iii) removing reads >10nt aligned to the adapter, allowing ≤10% mismatches; and (iv) deleting putative PCR repeats (two identical paired reads 1 and 2) generated by PCR amplification during library construction.

[0113] Genome-wide SNPs were screened to obtain a high-quality SNP marker set. The VCFTOOLS software was used to screen genome-wide SNPs, with the following criteria: minimum allele frequency (MAF) > 0.05 and missing genotype (MG) < 0.2.

[0114] We used the Latent Factor Mixed Models (LFMM) in LEA software to conduct genome-environmental association analysis on a high-quality set of SNPs from the whole genome of Populus tomentosa. At the same time, we calculated the population structure of Populus tomentosa populations. Based on the whole genome SNP markers, the natural Populus tomentosa populations were divided into three main genetic clusters (i.e., K=3) to correct for false positive associations. Finally, we obtained SNP loci that were significantly associated with the annual average sunshine hours of Populus tomentosa seed source areas.

[0115] The SNP site is located within the FPA gene, specifically at position 42 of the nucleotide sequence shown in SEQ ID NO: 1, and exhibits C / A polymorphism.

[0116] The amino acid sequence of the protein encoded by the FPA gene is shown in SEQ ID NO: 2.

[0117] Furthermore, using the online conserved domain alignment tool at (https: / / www.ncbi.nlm.nih.gov / ), it was discovered that positions 483-607 of the amino acid sequence (SEQ ID NO: 2) of the protein encoded by the FPA gene is a SPOC functional domain, which is a typical domain of the FPA gene.

[0118] 4. Based on the above analysis of the population structure, the natural population of 240 individuals covering the entire suitable habitat of Populus tomentosa was divided into 3 subgroups. The division of these subgroups is consistent with that of the subgroups divided by latitude, including 86 high-latitude (H) individuals, 63 mid-latitude (M) individuals and 91 low-latitude (L) individuals.

[0119] The population differentiation coefficient (Fixation statistic value, Fst) and nucleotide diversity index (π) of the three subpopulations were calculated using the sliding window algorithm in VCFTOOLS software.

[0120] The sliding window has a size of 2kb and a step size of 1kb.

[0121] After removing windows with π < 1E-03, the top 5% of windows with the most significant π and Fst values ​​are selected as the most significant selective clearance regions and merged with single scan regions with an interval of less than 5kb. Genes that overlap with the scan regions are defined as selective clearance genes.

[0122] Using the combination of genome-wide association analysis and selective clearance, the SNP site located at the 42nd base of the FPA gene was identified as being significantly associated with the annual average sunshine duration and also located in a selective clearance region, indicating that it has been subject to significant natural selection.

[0123] Specifically, the genotypes of the above-mentioned SNP sites in Populus tomentosa and the corresponding annual average sunshine durations in 240 individuals are shown in Table 1.

[0124] Table 1

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] Where H represents individuals at high latitudes; M represents individuals at mid latitudes; and L represents individuals at low latitudes.

[0133] As shown in Table 1, three genotypes were detected at the 42nd base of the FPA gene in the three subpopulations of Populus tomentosa (86 high-latitude individuals, 63 mid-latitude individuals, and 91 low-latitude individuals), namely AA, CA, and CC. The AA genotype was mainly concentrated in the short-day high-latitude population, while the CC genotype was mainly concentrated in the long-day low-latitude population.

[0134] Furthermore, genotypic analysis was performed on the 42nd base of the FPA gene in the three subpopulations of Populus tomentosa. The distribution results of allele and genotype frequencies are shown in Table 2.

[0135] Table 2

[0136]

[0137] As shown in Table 2, three genotypes were detected at the above SNP sites, namely AA, CA and CC. The AA genotype is only distributed in high-latitude populations, while the CC genotype is mainly concentrated in low-latitude populations.

[0138] Example 2: Validation of the effectiveness of molecular markers

[0139] Forty individuals were randomly selected from a two-year-old Populus tomentosa germplasm resource population (300 individuals, from a nursery in Guanxian County, Shandong Province). These included 20 individuals from high-latitude regions with shorter average annual sunshine hours and 20 individuals from low-latitude regions with longer average annual sunshine hours. Using the genomes of these 40 individuals as templates, amplification was performed using primers P1 and P2. The genotype at position 42 of the FPA gene in the 40 individuals was then determined by direct sequencing. The results were compared with the latitude of the individuals' germplasm origins. The results are shown in Table 3.

[0140] Table 3

[0141]

[0142]

[0143]

[0144] Table 3 shows that among the 40 randomly selected Populus tomentosa individuals, the genotype of the SNP marker was mainly AA in the high-latitude population and mainly CC in the low-latitude population. This indicates that the molecular markers described in this invention for identifying the geographical origin of Populus tomentosa germplasm are accurate and effective, enabling precise and rapid identification of the geographical origin of poplar germplasm at the molecular level. This is beneficial for seed regionalization and effectively shortens the poplar breeding cycle.

[0145] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.

Claims

1. The application of a molecular marker for identifying the geographical origin of Populus tomentosa germplasm in marker-assisted breeding or targeted introduction of Populus tomentosa, characterized in that, The molecular marker is located at position 42 of the nucleotide sequence shown in SEQ ID NO:

1. The molecular marker exhibits C / A polymorphism. The genotypes of the molecular markers are AA, CA, and CC, among which, Poplar individuals with the genotype AA, identified by the molecular marker, are distributed in high-latitude regions with shorter average annual sunshine hours, ranging from 37.00°N to 42.00°N. Populus tomentosa individuals with the genotype CC, as indicated by the molecular marker, are distributed in low-latitude regions with long average annual sunshine hours, ranging from 30.00 to 35.00°N.

2. A method for identifying or assisting in the identification of the geographical origin of Populus tomentosa germplasm, characterized in that, The method includes the following steps: Step 1: Extract genomic DNA from the Populus tomentosa species to be tested; Step II: Using the extracted genomic DNA as a template, perform PCR amplification; Step III: Determine the genotype of the molecular marker to be tested in Populus tomentosa; the molecular marker is located at position 42 of the nucleotide sequence shown in SEQ ID NO: 1; Step IV: Determine the germplasm geographical origin of the Populus to be tested based on the genotype results. In step III, the molecular marker exhibits C / A polymorphism, with genotypes of AA, CA, and CC, wherein... Poplar individuals with the genotype AA, identified by the molecular marker, are distributed in high-latitude regions with shorter average annual sunshine hours, ranging from 37.00°N to 42.00°N. Poplar individuals with the genotype CC, identified by the molecular marker, are distributed in low-latitude regions with long average annual sunshine hours, ranging from 30.00 to 35.00°N. In step IV, if the genotype of the SNP marker of the Populus to be tested is AA, then it is distributed in high-latitude regions with shorter average annual sunshine hours; if the genotype of the SNP marker of the Populus to be tested is CC, then it is distributed in low-latitude regions with longer average annual sunshine hours.

3. The method according to claim 2, characterized in that, In step II, the PCR amplification includes primers P1 and P2. Primer P1 is the nucleotide sequence shown in SEQ ID NO: 3, and primer P2 is the nucleotide sequence shown in SEQ ID NO: 4.

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