A molecular marker for accurately predicting the spring bud break time of Populus tomentosa and its application

Abstract

The present invention discloses a molecular marker for accurately predicting the spring bud break time of Populus tomentosa. The molecular marker is located at position 395 of the nucleotide sequence shown in SEQ ID NO: 1 and has a G / T polymorphism. Populus tomentosa individuals with a genotype of GG have a late spring bud break time, Populus tomentosa individuals with a genotype of TT have an early spring bud break time, and Populus tomentosa individuals with a genotype of GT have a moderate spring bud break time. The molecular marker for accurately predicting the spring bud break time provided by the present invention has a clear function and significant effect, and can be directly applied to molecular marker-assisted breeding of poplars, significantly improving breeding efficiency and shortening the breeding cycle of poplars.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular breeding, and specifically relates to a molecular marker, related kits and applications, and in particular to a molecular marker, primer set, kit, prediction method and application that can accurately predict the spring budding time of Populus tomentosa. Background Art

[0002] The precise synchronization of the inter-annual growth and development patterns of forest trees with periodic seasonal changes can prevent plants from being harmed by environmental stresses such as "late spring cold snaps", which is crucial for their growth and development. The budding time of different tree germplasms has a high heritability, and the budding time is mainly affected by the adaptive selection of environmental climatic factors in the provenance area. The end of tree dormancy and the germination of spring buds can ensure their normal growth in a specific regional environment. Especially in the current context of global warming and unstable extreme temperatures, effectively predicting the spring budding time of trees will help select germplasm individuals with different breeding needs according to the climatic conditions of the cultivation area, and will also help to protect and defend early-sprouting materials in advance due to extremely unstable climates.

[0003] Populus tomentosa is an important plantation species in northern China and a unique native species. It is widely distributed and cultivated in large areas in provinces such as Beijing, Hebei, Shandong, Shanxi, Henan, Shaanxi, Gansu, and Ningxia. Its distribution is concentrated in the middle and lower reaches of the Yellow River. It plays a crucial role in forestry production and ecological environment development in northern my country and is a pioneering species in forest cultivation in the region. Therefore, predicting the spring bud break time of Populus tomentosa is of great significance to ecological and environmental development.

[0004] However, Populus tomentosa is a perennial species, and genetic improvement through traditional hybridization takes a long time. Furthermore, existing research focuses primarily on functional studies of candidate genes, such as genetic transformation, at the individual level. Studies using germplasm resources to investigate the spring bud break period across different ecotypes are rare.

[0005] Therefore, it is necessary to explore universal, highly effective molecular markers at the molecular level that can accurately predict the spring bud break time of Populus tomentosa in order to solve the above problems. Summary of the Invention

[0006] In order to overcome the above problems, the inventors conducted intensive research and, by integrating the methods of whole-genome association analysis and selective elimination, obtained a SNP molecular marker that can accurately predict the spring bud break time of Populus tomentosa at the molecular level. This molecular marker has environmental adaptability, clear functions, and significant effects. It can scientifically predict the spring bud break time of Populus tomentosa, and select different germplasm breeding methods such as early bud break / late bud break according to the requirements of the cultivation environment, significantly improving breeding efficiency and shortening the breeding cycle of poplars, thereby completing the present invention.

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

[0008] In a first aspect, a molecular marker for accurately predicting the spring bud break time of Populus tomentosa is provided. The molecular marker is located at position 395 of the nucleotide sequence shown in SEQ ID NO: 1 and has a G / T polymorphism.

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

[0010] Step 1, obtaining the phenotypic traits of the Populus tomentosa population;

[0011] Step 2, obtaining molecular markers related to the spring bud break time of Populus tomentosa;

[0012] Wherein, 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, identifying single nucleotide polymorphism sites and their genotypes at the genome-wide level;

[0015] Step 2-3: Screening SNPs at the whole genome level to obtain a high-quality SNP marker set;

[0016] Steps 2-4, obtain SNP markers that are significantly associated with the spring bud break time of Populus tomentosa.

[0017] In a third aspect, the present invention provides the use of the molecular marker for accurately predicting the spring bud break time of Populus tomentosa according to the first aspect or the molecular marker obtained by the method according to the second aspect in any of the following aspects (1) to (3):

[0018] (1) Prediction of spring bud break time of Populus tomentosa; (2) Molecular marker-assisted breeding of Populus tomentosa; (3) Germplasm selection of Populus tomentosa.

[0019] In a fourth aspect, a method for identifying or assisting in identifying the spring bud break time of Populus tomentosa is provided, characterized in that the method comprises the following steps:

[0020] Step 1, extracting genomic DNA of the tested Populus tomentosa;

[0021] Step II, performing PCR amplification using the above-extracted genomic DNA as a template;

[0022] Step III, determining the genotype of the SNP molecular marker of the tested Populus tomentosa;

[0023] Step IV: determining the spring bud break time of the tested Populus tomentosa according to the genotype results.

[0024] The beneficial effects of the present invention include:

[0025] (1) The molecular markers provided by the present invention for accurately predicting the spring bud break time of Populus tomentosa have clear functions and significant effects, and can be directly applied to molecular marker-assisted breeding of poplars;

[0026] (2) The molecular markers provided by the present invention for accurately predicting the spring bud break time of Populus tomentosa are SNP sites that are significantly associated with the spring bud break time trait in the natural distribution area of ​​Populus tomentosa at the whole genome level. They can accurately predict the bud break period of poplar trees at the molecular level, and based on this, genotype germplasm individuals with different breeding requirements can be selected according to the climatic conditions of the cultivation area. This is also conducive to the early protection and defense of early bud break materials due to extremely unstable environmental climates.

[0027] (3) The method provided by the present invention for obtaining molecular markers for accurately predicting the spring bud break time of Populus tomentosa integrates the use of whole genome association analysis and selective elimination methods, so that the obtained molecular markers have environmental adaptability;

[0028] (4) The molecular markers obtained by the method provided by the present invention can accurately predict the spring bud break time of Populus tomentosa based on the genotype effect of the SNP site of the molecular marker and the differentiation status in different subpopulations, thereby selecting different germplasm breeding methods such as early bud break / late bud break according to the requirements of the cultivation environment;

[0029] (5) The method for identifying or assisting in identifying the spring budding time of Populus tomentosa provided by the present invention is easy to operate and highly accurate, and can significantly improve breeding efficiency and shorten the breeding cycle of poplars. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A graph showing the genotype effects of different genotypes of the SNP loci described in Example 1 of the present invention on the budding period of Populus tomentosa at different latitudes in 2019 is shown;

[0031] Figure 2 The genotype effect of different SNP sites according to Example 1 of the present invention on the genotype of Populus tomentosa populations at different latitudes during the budding period in 2020 is shown. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the characteristics and advantages of the present invention will become more clear and distinct.

[0033] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0034] A first aspect of the present invention provides a molecular marker for accurately predicting the spring bud break time of Populus tomentosa, wherein the molecular marker is located at the 395th base of the AGL80 gene in the genomic DNA of Populus tomentosa.

[0035] The nucleotide sequence of the AGL80 gene in the Populus tomentosa genomic DNA is shown in SEQ ID NO: 1.

[0036] According to a preferred embodiment of the present invention, the molecular marker for accurately predicting the spring bud break time of Populus tomentosa is located at position 395 of the nucleotide sequence shown in SEQ ID NO: 1 and has a G / T polymorphism.

[0037] In a further preferred embodiment, the amino acid sequence encoded by the AGL80 protein is shown in SEQ ID NO: 2.

[0038] The AGL80 gene belongs to the M-type MADS-box transcription factor family. In plants, genes in this family are primarily involved in floral development, fruit ripening, flowering timing, bud dormancy, and responses to abiotic stresses. Similar to the bud dormancy-related genes DAM, SVP, and AGL24 within the family, AGL80 is primarily expressed in vegetative tissues. Overexpression of the DAM gene inhibits bud germination, with DAM gradually upregulated during physiological dormancy induction and downregulated during ecological dormancy release. Studies on Euphorbia oleracea, strawberry trees, poplar, potato, and grapevine have shown that changes in the expression of MADS-box family genes are associated with the initiation, maintenance, and release of dormancy in plants.

[0039] Preferably, positions 2 to 60 of the sequence shown in SEQ ID NO: 2 are the conserved domain of the MADS transcription factor.

[0040] In a further preferred embodiment, the genotypes of the molecular markers are GG, TT and GT, wherein,

[0041] The Populus tomentosa individuals with the genotype GG of the molecular marker had a late budding time in spring.

[0042] The Populus tomentosa individuals with the genotype of the molecular marker being TT have an earlier budding time in spring;

[0043] The genotype of the molecular marker is GT for the Populus tomentosa individual, and the spring budding time is moderate.

[0044] In the present invention, the emergence of leaf tips in the terminal buds of individual plants starting from the early spring month of late February is defined as terminal bud germination, i.e., the spring germination time.

[0045] Preferably, the spring bud break time of the Populus tomentosa individuals with the molecular marker genotype of GG differs from that of the Populus tomentosa individuals with the molecular marker genotype of TT by 22 to 28 days, preferably by 25 days.

[0046] In the present invention, the GG genotype is the homozygous type of Populus tomentosa with the molecular marker G, the TT genotype is the homozygous type of Populus tomentosa with the molecular marker T, and the GT genotype is the heterozygous type of Populus tomentosa with the molecular markers G and T.

[0047] According to an embodiment of the present invention, Populus tomentosa individuals with a genotype of GG of the above-mentioned molecular marker are distributed in high latitudes (latitude range of 37°N to 42°N), Populus tomentosa individuals with a genotype of TT are distributed in low latitudes (latitude range of 30°N to 35°N), and Populus tomentosa individuals with a genotype of GT are distributed in mid-latitudes (latitude range of 35°N to 37°N).

[0048] The molecular markers described in the present invention can accurately predict the budding period of poplar trees at the molecular level, and on this basis can select genotype germplasm individuals with different breeding needs according to the climatic conditions of the cultivation area. It is also beneficial to provide early protection and defense for early budding materials due to extremely unstable environmental climate.

[0049] The 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:

[0050] Step 1: Obtain the phenotypic traits of the Populus tomentosa population.

[0051] According to an embodiment of the present invention, the Populus tomentosa population consists of 199 naturally grown individuals covering the entire suitable habitat of Populus tomentosa (from three latitude zones, namely 61 individuals from high latitudes, 64 individuals from mid-latitudes, and 74 individuals from low latitudes).

[0052] Preferably, in the spring of two consecutive years from 2019 to 2020, the terminal bud germination time of 199 individuals of Populus tomentosa is counted in the field, and at least 3 biological replicates are performed for each individual.

[0053] Specifically, starting from the early spring month of late February, the emergence of leaf tips from the terminal buds of individual plants was defined as terminal bud initiation, and the initiation was counted using Julian dates (i.e., January 1 was counted as JD 1, and December 31 was counted as JD 365). Finally, the spring terminal bud initiation time data for 199 individuals of Populus tomentosa from three different latitudes were obtained.

[0054] Step 2: Obtain molecular markers related to the spring bud break time of Populus tomentosa.

[0055] Preferably, step 2 includes the following sub-steps:

[0056] Step 2-1: resequence the DNA of each individual in the Populus tomentosa population and perform quality control on the obtained raw data.

[0057] Preferably, the DNA resequencing is double-end sequencing with a sequencing depth of 30×, and the sequencing is performed using the Illumina GA2 resequencing platform.

[0058] The quality control criteria are as follows: (i) removal of reads containing ≥10% unidentified nucleotides; (ii) removal of reads with a base quality <5 and a number >50%; (iii) removal of reads with >10 nt aligned to the adapter, allowing ≤10% mismatch; and (iv) deletion of putative PCR duplicates (two identical paired reads 1 and 2) generated by PCR amplification during library construction.

[0059] Step 2-2, identify single nucleotide polymorphism sites and their genotypes at the genome-wide level.

[0060] The quality-controlled data were aligned to the Populus reference genome v3.0 (http: / / popgenie.org), and single nucleotide polymorphisms (SNPs) were identified at the genome-wide level to obtain the genotypes of the SNPs.

[0061] Step 2-3: Screen SNPs at the whole genome level to obtain a high-quality SNP marker set.

[0062] Preferably, VCFTOOLS software is used to screen SNPs at the whole genome level, and the screening conditions are: minor allele frequency (MAF)>0.05, missing genotype (MG)<0.2.

[0063] Steps 2-4, obtain SNP markers that are significantly associated with the spring bud break time of Populus tomentosa.

[0064] Among them, Efficient Mixed-Model Association eXpedited (EMMAX) and Admixture software were used to calculate the kinship of Populus tomentosa populations, and Admixture software was used to calculate the population structure of Populus tomentosa populations (when K=3, the lowest cv error was obtained). With kinship and population structure as covariates, a mixed linear model (MLM) was used to perform association analysis between the whole-genome high-quality SNPs set and the budding period of Populus tomentosa, and SNP sites significantly associated with the budding time of Populus tomentosa were obtained.

[0065] The site is located in the AGL80 gene, preferably at position 395 of the nucleotide sequence shown in SEQ ID NO: 1, and has a G / T polymorphism.

[0066] Steps 2-5: Perform selective elimination analysis on the obtained significantly associated SNP markers.

[0067] Selective sweep refers to a phenomenon in which the frequency of certain dominant alleles in a population increases during natural selection or artificial selection, and the polymorphism of the surrounding chromosomal regions linked to them decreases due to the hitchhiking effect.

[0068] 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 were calculated using VCFTOOLS software through a sliding window algorithm.

[0069] The sliding window size is 2kb and the step size is 1kb.

[0070] After removing the windows with π<1E-03, the top 5% windows with the most significant π and Fst values ​​were selected as the most significant selective clearance regions, and single scan regions with intervals less than 5 kb were merged. Genes overlapping with the scan regions were defined as selective clearance genes.

[0071] In a further preferred embodiment, it is determined whether the obtained SNP site significantly associated with the spring bud break time of Populus tomentosa is located within the selective elimination region obtained above.

[0072] Among them, if the SNP marker is located in the selective clearance area, then the marker is a marker that is subject to natural selection and has environmental adaptability; if the SNP marker is not located in the selective clearance area, then the marker is a marker that is not subject to natural selection and has no environmental adaptability.

[0073] According to a preferred embodiment of the present invention, whole genome-environment association analysis was used to obtain SNP molecular markers that were significantly associated with the growth environment of Populus tomentosa.

[0074] Preferably, the environmental characteristics include latitude, annual average temperature, annual average precipitation and annual average sunshine hours.

[0075] In a further preferred embodiment, based on the analysis results of the population structure in steps 2-4, the natural population consisting of 199 individuals covering the entire suitable habitat range of Populus tomentosa is divided into 3 subpopulations. The division of the subpopulations is consistent with the subpopulations divided according to latitude, including 61 high-latitude (H) individuals, 64 mid-latitude (M) individuals and 74 low-latitude (L) individuals.

[0076] Among them, the latitude information data of Populus tomentosa population (199 individuals) were obtained through the National Geographic Information Public Service Platform (https: / / www.tianditu.gov.cn / );

[0077] The environmental data of annual average temperature, annual average precipitation, and annual average sunshine hours of 199 individuals of Populus tomentosa for 50 consecutive years from 1971 to 2020 were collected through the China Meteorological Data Network (http: / / data.cma.cn / ) and the U.S. Centers for Environmental Information (https: / / www.ncei.noaa.gov / ).

[0078] In a further preferred embodiment, based on the environmental trait information of the Populus tomentosa population and the genomic data in step 2-1, a genome-wide-environment association analysis is performed using the latent factor mixed models (LFMM) in the LEA software to detect SNP sites that are significantly associated with environmental traits.

[0079] According to an embodiment of the present invention, it was found that the SNP site significantly associated with the spring bud break time of Populus tomentosa was also significantly correlated with the latitude of Populus tomentosa, and the site was located in a selective clearance section and was subject to natural selection.

[0080] In the present invention, the spring budding time of Populus tomentosa can be accurately predicted based on the genotype effect of the molecular marker SNP site and the differentiation in different subpopulations, thereby selecting different germplasm breeding methods such as early budding / late budding according to the cultivation environment requirements.

[0081] In the present invention, whole-genome-phenotype association analysis and whole-genome-environment association analysis were used to obtain SNP sites that were significantly associated with the spring bud break time and latitude of Populus tomentosa, and the population differentiation coefficient (Fixation statistic value, Fst) and nucleotide diversity index (π) were used to determine whether the site was located in the selective clearance segment.

[0082] The above molecular marker acquisition method, combined with a strategy of selective elimination and genome-wide association analysis, enabled the precise discovery of key alleles for analyzing the adaptive genetic mechanisms of Populus tomentosa, providing insights into the prediction of adaptive genetic variation in germplasm resources and the analysis of local adaptation mechanisms under global climate change. Furthermore, the selected alleles may determine the significant differences in spring bud break dates between populations in different habitats, leading to divergent patterns in the allele frequencies of intragenic molecular markers between populations in different geographic and climatic zones.

[0083] The third aspect of the present invention provides a use of the molecular marker described in the first aspect or the molecular marker obtained by the method described in the second aspect in any one of the following aspects (1) to (3):

[0084] (1) Prediction of spring bud break time of Populus tomentosa; (2) Molecular marker-assisted breeding of Populus tomentosa; (3) Germplasm selection of Populus tomentosa.

[0085] According to a preferred embodiment of the present invention, the application includes the steps of performing PCR amplification on the genomic DNA of Populus tomentosa and determining the genotype of the molecular markers described in the first aspect of Populus tomentosa.

[0086] The primer pair used in the PCR amplification includes primer P1 and primer P2.

[0087] 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.

[0088] 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: 3 and SEQ ID NO: 4, respectively, and can produce substantially identical DNA fragments (the DNA sequences between the upstream and downstream primers are identical) are all included in the primer pairs of the present invention.

[0089] Preferably, the nucleotide sequence of the primer P1 is shown as SEQ ID NO: 3, and the nucleotide sequence of the primer P2 is shown as SEQ ID NO: 4.

[0090] In the present invention, the primer pair used in the PCR amplification can be used to detect the molecular markers described in the first aspect.

[0091] In a further preferred embodiment, the method for determining the genotype of the molecular markers of Populus tomentosa in the first aspect includes but is not limited to sequencing.

[0092] A fourth aspect of the present invention provides a method for identifying or assisting in identifying the spring bud break time of Populus tomentosa, the method comprising the following steps:

[0093] Step I: extracting the genomic DNA of the Populus tomentosa to be tested.

[0094] Step II: PCR amplification is performed using the genomic DNA extracted above as a template.

[0095] The primer pair used in the PCR amplification includes primer P1 and primer P2.

[0096] 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.

[0097] Preferably, the nucleotide sequence of the primer P1 is shown as SEQ ID NO: 3, and the nucleotide sequence of the primer P2 is shown as SEQ ID NO: 4.

[0098] Step III, determining the genotype of the SNP molecular markers of the tested Populus tomentosa.

[0099] Among them, methods for determining molecular marker genotypes include but are not limited to sequencing.

[0100] Step IV: determining the spring bud break time of the tested Populus tomentosa according to the genotype results.

[0101] Among them, the Populus tomentosa individuals with the genotype of the molecular marker being GG have a late spring budding time, the Populus tomentosa individuals with the genotype of the molecular marker being TT have an early spring budding time; and the Populus tomentosa individuals with the genotype of the molecular marker being GT have a moderate spring budding time.

[0102] The method for identifying or assisting in identifying the spring bud break time of Populus tomentosa provided by the present invention is easy to operate and highly accurate. It can accurately predict the bud break period of poplars at the molecular level. Based on this, genotype germplasm individuals with different breeding requirements can be selected according to the climatic conditions of the cultivation area, thereby effectively shortening the breeding cycle of poplars.

[0103] Example

[0104] The present invention is further described below through specific examples. However, these examples are merely exemplary and do not constitute any limitation to the scope of protection of the present invention.

[0105] Example 1 Acquisition of molecular markers for accurately predicting the spring bud break time of Populus tomentosa

[0106] 1. Acquisition of phenotypic traits:

[0107] The timing of terminal bud initiation was recorded in a natural field population of Populus tomentosa (199 individuals, each with at least three biological replicates) during the spring of 2019-2020. Starting in early spring in late February, terminal bud initiation was defined as the appearance of the leaf tip in the terminal bud of an individual plant. Counting was performed using Julian dates (i.e., January 1 as JD 1, December 31 as JD 365). Data on the timing of spring terminal bud initiation were collected for 199 individuals of Populus tomentosa from three different latitudes.

[0108] The natural population of Populus tomentosa consists of 199 naturally grown individuals covering the entire suitable habitat of Populus tomentosa (from three latitude zones, 61 from high latitudes, 64 from mid-latitudes, and 74 from low latitudes).

[0109] The latitude range of high latitude areas is 37°N~42°N, the latitude range of mid-latitude areas is 35°N~37°N, and the latitude range of low latitude areas is 30°N~35°N.

[0110] 2. Obtain molecular markers related to the spring bud break time of Populus tomentosa:

[0111] 2.1. Genomic DNA was extracted from leaf tissues of 240 individuals in the experimental population using the DNeasy Plant Mini Kit (Qiagen, Shanghai, China);

[0112] DNA quality was tested by UV spectrophotometer and gel electrophoresis, and qualified DNA was stored at -20°C.

[0113] DNA from each individual was resequenced to obtain raw data. After quality control, the data were aligned to the Populus reference genome v3.0 (http: / / popgenie.org). Single nucleotide polymorphisms (SNPs) were identified and genotyped at the genome-wide level. The quality control criteria were: (i) removal of reads containing ≥10% unidentified nucleotides; (ii) removal of reads with >50% base quality <5; (iii) removal of reads with >10 nt aligned to the adapter, with ≤10% mismatch allowed; and (iv) removal of putative PCR duplicates (two identical paired reads 1 and 2) generated by PCR amplification during library construction.

[0114] 2.2. The quality-controlled data were aligned to the Populus reference genome v3.0 (http: / / popgenie.org), and single nucleotide polymorphisms (SNPs) were identified at the genome-wide level and the genotypes of the SNPs were obtained.

[0115] 2.3. VCFTOOLS software was used to screen SNPs at the whole genome level. The screening conditions were: minor allele frequency (MAF)>0.05, missing genotype (MG)<0.2.

[0116] 2.4. Efficient Mixed-Model Association eXpedited (EMMAX) and Admixture software were used to calculate the kinship of the Populus tomentosa population. Admixture software was also used to calculate the population structure of the Populus tomentosa population (when K = 3, the lowest cv error was achieved). Using kinship and population structure as covariates, a mixed linear model (MLM) was used to analyze the association between the whole-genome high-quality SNPs set and the budding period of Populus tomentosa, and SNP sites significantly associated with the budding time of Populus tomentosa were obtained.

[0117] The site is located in the AGL80 gene, specifically at position 395 of the nucleotide sequence shown in SEQ ID NO: 1, and has a G / T polymorphism.

[0118] Among them, the amino acid sequence of the protein encoded by the AGL80 gene is shown in SEQ ID NO: 2.

[0119] Furthermore, through the online conserved domain alignment tool on the website (https: / / www.ncbi.nlm.nih.gov / ), it was found that the amino acid sequence of the protein encoded by the AGL80 gene (SEQ ID NO: 2) from positions 2 to 60 constituted the MADS transcription factor conserved domain.

[0120] 2.5. Perform selective elimination analysis on the significantly associated SNP markers obtained.

[0121] The population differentiation coefficient (Fixation statistic value, Fst) and nucleotide diversity index (π) of the three subpopulations were calculated using the sliding window algorithm using VCFTOOLS software.

[0122] The sliding window size is 2kb and the step size is 1kb.

[0123] After removing the windows with π<1E-03, the top 5% windows with the most significant π and Fst values ​​were selected as the most significant selective clearance regions, and single scan regions with intervals less than 5 kb were merged. Genes overlapping with the scan regions were defined as selective clearance genes.

[0124] Genome-wide association analysis was used to identify SNP molecular markers significantly associated with the growth environment traits of Populus tomentosa (latitude, mean annual temperature, mean annual precipitation, and mean annual sunshine hours). Based on the population structure analysis results in step 2.4, a natural population of 199 individuals covering the entire suitable habitat of Populus tomentosa was divided into three subpopulations. The division of these subpopulations was consistent with the subpopulation division based on latitude, including 61 high-latitude (H) individuals, 64 mid-latitude (M) individuals, and 74 low-latitude (L) individuals.

[0125] The latitude information data of the Populus tomentosa population (199 individuals) was obtained through the National Geographic Information Public Service Platform (https: / / www.tianditu.gov.cn / );

[0126] The environmental data of annual average temperature, annual average precipitation, and annual average sunshine hours of 199 individuals of Populus tomentosa for 50 consecutive years from 1971 to 2020 were collected through the China Meteorological Data Network (http: / / data.cma.cn / ) and the U.S. Centers for Environmental Information (https: / / www.ncei.noaa.gov / ).

[0127] Based on the environmental trait information of the Populus tomentosa population and the genomic data in 2.1, a genome-wide association analysis was performed using the latent factor mixed models (LFMM) in the LEA software to detect SNP sites that were significantly associated with environmental traits.

[0128] After the above steps, it was determined that the SNP site significantly associated with the spring bud break time of Populus tomentosa was also significantly correlated with the latitude of Populus tomentosa, and the site was located in the selective clearance area and was significantly subject to natural selection.

[0129] Specifically, the genotypes of the above-mentioned SNP sites in 199 individuals of Populus tomentosa and the corresponding latitude distribution, as well as the spring bud break time results for two consecutive years are shown in Table 1. Figure 1 、 2 As shown:

[0130] Table 1

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] As can be seen from Table 1, three genotypes were detected at base 395 of the AGL80 gene in three different latitude populations of Populus tomentosa (H: 61, M: 64, L: 74): GG, GT and TT. Among them, TT was the dominant genotype, mainly concentrated in low-latitude populations. Moreover, the spring budding time of Populus tomentosa individuals with the GG genotype was later, the spring budding time of Populus tomentosa individuals with the TT genotype was earlier, and the spring budding time of Populus tomentosa individuals with the GT genotype was moderate.

[0138] Among them, the spring bud break time of Populus tomentosa individuals with genotype GG and Populus tomentosa individuals with genotype TT differs by about 28 days.

[0139] Furthermore, genotype analysis was performed on the 395th base of the AGL80 gene in the three different latitude populations of Populus tomentosa. The distribution results of allele and genotype frequencies are shown in Table 2.

[0140] Table 2

[0141]

[0142] As shown in Table 2, three genotypes were detected at the 395th base of the AGL80 gene in the Populus tomentosa population: GG, GT and TT. TT was the dominant genotype, mainly concentrated in the low-latitude population. The frequency of the TT genotype in the low-latitude population (0.62) was significantly higher than that in the high-latitude population (0.07) and the mid-latitude population (0.14).

[0143] Example 2 Verification of the effectiveness of molecular markers

[0144] In the early spring of 2022, 60 individuals of Populus tomentosa were randomly selected from a natural population of 300 three-year-old Populus tomentosa (300 plants, located in Guanxian County, Shandong Province), including 20 individuals from high latitudes, 20 from mid-latitudes, and 20 from low latitudes. The genomes of the 60 individuals were used as templates for amplification using primers P1 and P2. The genotypes at base 395 of SEQ ID NO: 1 of the 60 individuals were then determined by direct sequencing. The genotypes were then compared with the spring bud break times of the 60 individuals that had been counted. The results are shown in Table 3.

[0145] Table 3

[0146]

[0147]

[0148] As shown in Table 3, the 60 randomly selected Populus tomentosa individuals in the high-latitude population mainly contained the GG genotype, with an average budding period of the 100th day under natural conditions; the mid-latitude population mainly contained the GT genotype, with an average budding period of the 93rd day under natural conditions; and the low-latitude population mainly contained the TT genotype, with an average budding period of the 82nd day under natural conditions.

[0149] From the above, it can be seen that the molecular markers for accurately predicting the spring bud break time of Populus tomentosa provided by the present invention are accurate and effective, and can accurately and quickly identify or predict the spring bud break time (bud break period) of Populus tomentosa at the molecular level, thereby selecting genotype germplasm individuals with different breeding requirements according to the climatic conditions of the cultivation area, effectively shortening the breeding cycle of poplar.

[0150] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions are not to be construed as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention.

Claims

1. Application of a molecular marker for accurately predicting the spring bud break time of Populus tomentosa in any of the following aspects (1) to (3): (1) Predicting the spring bud break time of Populus tomentosa; (2) Molecular marker-assisted breeding of Populus tomentosa; (3) Germplasm selection of Populus tomentosa; The molecular marker is located at position 395 of the nucleotide sequence shown in SEQ ID NO: 1 and has a G / T polymorphism. The genotypes of the molecular markers are GG, TT and GT, The Populus tomentosa individuals with the genotype GG of the molecular marker had a late budding time in spring. The Populus tomentosa individuals with the genotype of the molecular marker being TT have an earlier budding time in spring; The genotype of the molecular marker is GT for the Populus tomentosa individual, and the spring budding time is moderate.

2. The use according to claim 1, characterized in that The method for obtaining the molecular marker comprises the following steps: Step 1, obtaining the phenotypic traits of the Populus tomentosa population; Step 2, obtaining molecular markers related to the spring bud break time of Populus tomentosa; Wherein, step 2 includes the following sub-steps: Step 2-1: resequencing the DNA of each individual in the Populus tomentosa population and performing quality control on the obtained raw data; Step 2-2, identifying single nucleotide polymorphism sites and their genotypes at the genome-wide level; Step 2-3: Screening SNPs at the whole genome level to obtain a high-quality SNP marker set; Steps 2-4, obtain SNP markers that are significantly associated with the spring bud break time of Populus tomentosa.

3. The use according to claim 2, characterized in that The method further comprises steps 2-5, performing selective elimination analysis on the obtained significantly associated SNP markers.

4. The use according to claim 1, characterized in that The application includes the steps of performing PCR amplification on the genomic DNA of Populus tomentosa and determining the genotype of the molecular marker of Populus tomentosa. The primer pair used in the PCR amplification includes primer P1 and primer P2. Primer P1 has a nucleotide sequence as shown in SEQ ID NO: 3, and primer P2 has a nucleotide sequence as shown in SEQ ID NO: 4.

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