Molecular marker related to efficient utilization of slash phosphorus and application

Abstract

The invention discloses a molecular marker related to efficient utilization of slash phosphorus and application of the molecular marker, and belongs to the technical field of molecular biology. The molecular marker is two missense mutation SNP (Single Nucleotide Polymorphism) sites: SNP-1 (620th site, C-G) and SNP-2 (1897th site, AG-GC) located in a slash pine PeSTOP1 gene coding region. Specific genotypes (a GG type of SNP-1 and a GC / GC type of SNP-2) are obviously related to high phosphorus utilization efficiency and a high-low-phosphorus-resistance comprehensive evaluation value. The invention further provides a primer pair and a kit for detecting the marker and an application method of the primer pair and the kit in auxiliary screening of the slash pine with efficient phosphorus utilization. Through the molecular marker, rapid and accurate genotype identification can be realized in the seedling stage, the defects of long period and low efficiency of traditional phenotype screening are overcome, and a core tool is provided for efficient utilization and genetic improvement of slash pine phosphorus and molecular marker assisted breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a molecular marker and its application for efficient phosphorus utilization in slash pine. Background Technology

[0002] As an important fast-growing timber and ecological restoration species, the growth and productivity of slash pine are often constrained by the shortage of available phosphorus in the site soil, especially in acidic soil areas of the tropics and subtropics. Low phosphorus stress has become one of the key abiotic stress factors restricting the sustainable development of plantations. During long-term evolution, plants have developed a series of adaptive mechanisms, covering aspects such as root morphological plasticity, phosphorus absorption activation (such as secretion of acid phosphatase), and efficient phosphorus utilization within the plant.

[0003] Traditionally, the screening and breeding of low-phosphorus tolerant tree genotypes has primarily relied on phenotypic selection in the field or in pots, comparing the growth performance, biomass, and phosphorus content of different families or individuals under low phosphorus stress. However, this phenotypic-based screening method has significant drawbacks: the screening cycle is lengthy, typically taking several years; it is highly susceptible to interference from environmental factors (such as climate and soil heterogeneity), resulting in poor reproducibility and low screening efficiency; and it is difficult to make accurate predictions in the early stages of seedling growth, severely hindering the breeding process. In recent years, with the development of molecular biology, the use of molecular markers closely linked to or directly causally related to target traits for assisted selection has become a mainstream trend in crop genetic improvement. However, in the field of forestry, especially for the complex quantitative trait of phosphorus use efficiency in slash pine, there are still few reports on the discovery of key regulatory genes and practical molecular markers directly related to it that can be applied to breeding.

[0004] Therefore, developing a molecular marker system that can rapidly and accurately identify genotypes of high phosphorus utilization efficiency in slash pine in the early stages, and combining it with reliable seedling phenotypic identification methods, is of urgent theoretical and practical importance for achieving targeted genetic improvement of phosphorus utilization efficiency in slash pine and accelerating the breeding of superior varieties. Summary of the Invention To overcome the shortcomings of the existing technology, the present invention aims to provide a molecular marker and its application for efficient utilization of phosphorus in slash pine.

[0005] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a molecular marker related to the efficient utilization of phosphorus in slash pine, wherein the molecular marker is located in slash pine PeSTOP1 Single nucleotide polymorphism (SNP) sites in the coding region of a gene, wherein the SNP sites are SNP-1 and / or SNP-2; Wherein, the SNP-1 is located in the PeSTOP1The 620th nucleotide in the gene coding region has a reference allele of C and a variant allele of G. The SNP-2 is located in PeSTOP1 The 1897th nucleotide in the gene coding region has a reference allele sequence of AG and a variant allele sequence of GC.

[0006] Secondly, the present invention provides an application of the above-mentioned molecular markers related to the high-efficiency utilization of phosphorus in slash pine in identifying or assisting in the identification of the high-efficiency utilization capacity of slash pine.

[0007] Thirdly, the present invention provides a reagent for detecting molecular markers related to the efficient utilization of phosphorus in the above-mentioned slash pine.

[0008] Fourthly, the present invention provides the application of the above-mentioned reagent in the preparation of products for assisting in the screening or identification of families or individuals of phosphate-efficient pine.

[0009] Fifthly, the present invention provides a primer pair or probe for detecting the aforementioned molecular markers related to efficient phosphorus utilization in pine needles, wherein the primer pair or probe is capable of specifically amplifying or binding to sites containing the SNP-1 and / or SNP-2 sites. PeSTOP1 Gene fragments.

[0010] In a sixth aspect, the present invention provides a kit for assisting in the screening or identification of phosphorus-efficient slash pine, comprising the aforementioned primer pairs or probes.

[0011] In a seventh aspect, the present invention provides a method for screening phosphorus-efficient slash pine trees using molecular markers related to the high phosphorus utilization efficiency of slash pine, comprising the following steps: (1) Obtain total RNA from the individual pine trees to be tested and reverse transcribe it into cDNA; (2) Detect the individual PeSTOP1 The nucleotide type or allele at position 620 and / or position 1897 of the gene coding region; (3) Make a judgment based on the test results: a) If the 620th position is a homozygous G allele (GG) and / or the 1897th position is a homozygous GC allele (GC / GC), then the individual is judged to have the potential for efficient phosphorus utilization. b) If the individual contains both the G allele at position 620 and the GC allele at position 1897, then the individual is considered to have a higher potential for efficient phosphorus utilization.

[0012] The present invention has the following beneficial effects: Molecular markers directly correlated with phosphorus efficiency were provided: first identified in slash pine. PeSTOP1Two key single nucleotide polymorphism (SNP) sites (SNP-1 and SNP-2) within the gene. Specific genotypes at these sites (such as the GG type of SNP-1 and the GC / GC type of SNP-2) are significantly correlated with high phosphorus use efficiency and high overall evaluation value for low phosphorus tolerance.

[0013] Early, precise, and efficient screening has been achieved: With the help of the above molecular markers, it is possible to quickly and accurately identify slash pine families or individuals with high phosphorus utilization potential through DNA detection in the early stage of seedling growth (without needing to grow to maturity or experience long-term stress), overcoming the drawbacks of traditional phenotypic screening, such as long phenotype screening cycle, significant environmental influence, and huge workload.

[0014] It provides core tools for molecular-assisted breeding: the developed SNP markers and corresponding detection primers and kits can be directly applied to the genetic improvement breeding project of slash pine, accelerating the breeding process of phosphorus-efficient varieties and achieving the goal of targeted breeding.

[0015] A reliable seedling phenotypic identification system was established: through a systematic low phosphorus stress test method, phenotypic indicators with phosphorus use efficiency (PUE), root morphology, and acid phosphatase activity as the core were identified, and phosphorus efficiency was comprehensively evaluated by combining mathematical models (low phosphorus tolerance coefficient, membership function, and comprehensive evaluation value D), providing solid and reproducible phenotypic data support for genotype-phenotype association analysis.

[0016] The physiological mechanism of slash pine's adaptation to low phosphorus stress was revealed: Through examples, it was clarified that slash pine with high phosphorus utilization adapts to stress in low phosphorus environment by increasing root biomass, enhancing acid phosphatase activity, and optimizing phosphorus distribution and utilization in the body (improving PUE), providing empirical evidence for understanding its adaptation mechanism. Detailed Implementation

[0017] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0018] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0019] Example 1 The test materials in this embodiment were seeds (F01-F13) from 13 families of slash pine from the superior tree seed generation of Changle Forest Farm (Xishan Village, Jingshan Town, Yuhang District, Hangzhou City, China). The seeds were sown and cultivated in a greenhouse for 5 months (day and night temperature 20℃–12℃; relative humidity 50%–60%; natural light), and seedlings with a height of about 12cm were selected for the experiment.

[0020] The experiment adopted a single-factor design with phosphorus supply level as the only independent variable. Eighteen identical seedlings from each family were randomly divided into three groups (six seedlings in each group) and given three different phosphorus treatments: control (normal phosphorus: 136 mg / kg KH2PO4, NP), low phosphorus (1.36 mg / kg KH2PO4, LP) and phosphorus deficiency (0 mg / kg KH2PO4, -P).

[0021] All seedlings were planted in 1L plastic pots containing 1kg of soil (available phosphorus: 0.01mg / kg), with the addition of essential nutrients (mg). kg - ¹Soil): 506 KNO3, 80 NH4NO3, 241 MgSO4, 36.7 FeNaEDTA, 0.83 KI, 6.2H3BO3, 16.9 MnSO4 H2O, 8.6 ZnSO4 7H2O, 0.25 NaMoO4 2H₂O, 0.025 CuSO₄ 5H₂O, 0.025 CoCl₂ 6H2O and 945 Ca(NO3)2 7H2O.

[0022] The culture room environment was set at a 14-hour light / 10-hour dark cycle, with a temperature of 23-25℃ and humidity of 50%-60%. Watering was carried out regularly each week, and seedlings were randomly placed in the culture room. After two months of cultivation, relevant morphological and physiological indicators were measured.

[0023] The morphological parameters measured included plant height, ground diameter, total biomass (divided into aboveground and underground parts; after harvesting the seedlings whole, the soil around the roots was carefully washed, the surface moisture was absorbed with absorbent paper, and the fresh weight of the aboveground parts (stems and leaves) and underground parts (roots) was weighed separately. Then, each part of the sample was placed in a 105℃ oven for 30 minutes to blanch, and then dried at 80℃ to constant weight to measure the dry weight), root-shoot ratio (the ratio of the dry weight of the underground part to the dry weight of the aboveground part), and root morphological parameters (the root system was scanned using a root scanner (model EPSONPerfectionV800), and the total root length, root surface area, root volume, and average root diameter were measured using WinRHIZO root analysis system software).

[0024] Physiological indicators were measured, including leaf chlorophyll content (using a SPAD-502Plus portable chlorophyll meter, selecting fully expanded functional leaves at the top of seedlings, measuring three different sites on each leaf, and taking the average value), and leaf net photosynthetic rate (using a Li-6400XT portable photosynthesis measurement system, measured from 9:00 to 11:00 AM, with measurement conditions set as follows: photosynthetically active radiation 1000 μmol·m⁻¹). -2 ·s -1 CO2 concentration 400 μmol·mol -1 The leaf chamber temperature was 25℃, and the relative humidity was 50%-60%. Healthy functional leaves at the top were selected for measurement. The leaf soluble sugar content was measured (using the anthrone colorimetric method: 0.2g of fresh leaf sample was weighed, ground and extracted with 80% ethanol, centrifuged, and the supernatant was mixed with anthrone reagent and developed in a boiling water bath. After cooling, the absorbance was measured at a wavelength of 620nm, and the content was calculated according to the standard curve). The leaf free proline content was measured (using the acidic ninhydrin method: 0.5g of fresh leaf sample was weighed, ground and extracted with 3% sulfosalicylic acid, extracted in a boiling water bath, and the supernatant was mixed with acidic ninhydrin reagent and developed in a boiling water bath. After cooling, it was extracted with toluene, and the absorbance was measured at a wavelength of 520nm. The content was calculated according to the standard curve). In addition, the following measurements were taken: root acid phosphatase activity (using the disodium phenyl phosphate colorimetric method; 0.5 g of fresh roots were weighed, added to phosphate buffer (pH 5.0), ground into a homogenate, centrifuged, and the supernatant was used as the crude enzyme extract, mixed with the disodium phenyl phosphate substrate, reacted in a 37°C water bath, a colorimetric reagent was added, and the absorbance was measured at 400 nm; enzyme activity was expressed as milligrams of phenol released per gram of fresh root weight per hour) and total phosphorus content of the plant (dried aboveground and underground samples of the plant were pulverized separately, digested using the H2SO4-H2O2 digestion method, and then the absorbance was measured at 700 nm using the molybdenum antimony colorimetric method; total phosphorus content was calculated, and further, phosphorus accumulation (the product of total phosphorus content and the corresponding biomass) and phosphorus use efficiency (the ratio of biomass to total phosphorus content) were calculated). All indicators were measured in triplicate to ensure the accuracy and reliability of the data.

[0025] The results are shown in Tables 1 and 2.

[0026] Table 1. Comparison of the performance of phosphorus-efficient (F01) and phosphorus-inefficient (F03) families under low phosphorus conditions. Table 2. Trends in phosphorus efficiency-related indicators of family F01 under different phosphorus treatments. Based on the typical data and analysis in Tables 1 and 2 above, the following conclusions can be drawn: Slash pine exhibits a significant adaptive response under low phosphorus stress. Specifically, total root length and root surface area increase significantly by 16%-18%, and the root-to-shoot ratio rises, indicating that slash pine adapts to low phosphorus environments by strengthening root development. Acid phosphatase activity is significantly increased, exceeding 16%, thereby promoting the activation and absorption of organic phosphorus in the soil. Phosphorus use efficiency (PUE) is significantly improved, exceeding 50% under phosphorus-deficient conditions, demonstrating highly efficient phosphorus utilization.

[0027] Significant genetic differences in phosphorus efficiency exist among families. Phosphorus-efficient families (such as F01, F04, and F07) can maintain high growth, root development, and phosphorus metabolism activity even under low phosphorus conditions; while phosphorus-inefficient families (such as F03, F06, and F09) show significantly inhibited growth and limited improvement in phosphorus efficiency under low phosphorus stress.

[0028] Phosphorus-efficient families exhibit excellent overall tolerance to low phosphorus. The typical high-efficiency family F01 achieved a comprehensive evaluation value (D) of 0.86 under low phosphorus conditions, demonstrating strong potential for low phosphorus adaptation. These families can be preliminarily screened at the seedling stage based on root morphology and physiological indicators, providing a reliable phenotypic data foundation for subsequent genotype-phenotype association analysis.

[0029] This embodiment provides phenotypic evidence for subsequent molecular marker development and seedling identification. Phosphorus-efficient families exhibited consistent and stable performance in root development, acid phosphatase activity, and phosphorus use efficiency, demonstrating potential as candidate materials for gene mining and marker development.

[0030] Example 2 To determine and comprehensively evaluate the core phenotype of phosphorus efficiency in slash pine, the plant materials were consistent with those in Example 1, using seedlings from 13 slash pine families. Two phosphorus treatments were established: normal phosphorus (NP, 136 mg / kg KH₂PO₄) and low phosphorus (LP, 1.36 mg / kg KH₂PO₄). Six plants from each family were selected under each treatment, using a completely randomized design. Culture conditions were the same as in Example 1, and sampling was performed two months after cultivation. Phosphorus concentration was determined using methods including soil available phosphorus concentration (SPC) and plant tissue phosphorus concentration.

[0031] The procedure for determining soil available phosphorus concentration (SPC) is as follows: Weigh 1.0 g of air-dried and sieved soil sample into a centrifuge tube, add 10 mL of 0.03 M NH4F-0.025 M HCl extraction solution, and shake for 30 min. Then, take the supernatant, add ammonium molybdate-potassium antimony tartrate-ascorbic acid chromogenic reagent, and let stand at room temperature for 30 min. Measure the absorbance at 880 nm using a microplate reader, and calculate the soil available phosphorus concentration (SPC) based on the standard curve, in mg / kg.

[0032] The procedure for determining phosphorus concentration in plant tissues is as follows: Weigh 0.2g of dried samples from both the aboveground and underground parts, and completely digest them using the H2SO4-H2O2 digestion method. Adjust the volume of the digestion solution to 50mL. Take an appropriate amount of diluent and add molybdenum antimony anti-chromic reagent under controlled acidity conditions. Measure the absorbance at 700nm using an ELISA reader. Calculate the phosphorus concentration (APC) in the aboveground parts (APC) and the phosphorus concentration (BPC) in the underground parts (roots) according to the standard curve. The units are both mg / gDW.

[0033] Based on the measured phosphorus concentration data, the following core parameters were calculated: 1. Phosphorus accumulation (PA, mg / plant) ; ADW and BDW represent the dry weight (g) of the aboveground and underground parts, respectively.

[0034] 2. Bioaccumulation Factor (BCF) .

[0035] 3. Transfer coefficient .

[0036] 4. Phosphorus utilization efficiency (PUE, g / mg) .

[0037] A comprehensive evaluation was conducted using the Low Phosphorus Tolerance Factor (LPTF) combined with the fuzzy membership function method.

[0038] 1. Calculate the low phosphorus tolerance factor (LPTF) for each indicator. ; Where i represents the family lineage and j represents the indicators (including ADW, BDW, PA, BCF, TF, PUE).

[0039] 2. Calculate the membership function value ; Among them, X ij X is the LPTF value of the j-th indicator in the i-th family;jmin and X jmax The minimum and maximum values ​​of this indicator across all families. 3. Calculate the indicator weights (Wj) Principal component analysis was used to determine the weights based on the contribution rate of each indicator.

[0040] 4. Calculate the comprehensive evaluation value (D) for low phosphorus tolerance. .

[0041] The experimental results (typical results) are shown in Tables 3 and 4.

[0042] Table 3. Phosphorus concentration and phosphorus efficiency parameters of representative families (under low phosphorus treatment) Table 4 Low Phosphorus Tolerance Factor (LPTF) and Overall Evaluation Value (D) Based on the data in Tables 3 and 4, the core indicator phosphorus use efficiency (PUE) shows significant inter-family differences under low phosphorus conditions. High-efficiency families (e.g., F01, F04, F07) have an average PUE approximately 38% higher than low-efficiency families (e.g., F03, F06, F09). The low-phosphorus tolerance comprehensive evaluation value (D) can effectively identify the phosphorus efficiency type of a family. Families with a D value greater than 0.80 are classified as "phosphorus-efficient," sharing the common characteristics of maintaining high relative growth and significantly improved phosphorus use efficiency under low phosphorus stress. Example 3 Total RNA was extracted using the RNAprep Pure Plant Plus Kit (DP441, Tiangen, Beijing, China). The concentration and purity of the obtained RNA were determined using a Nanodrop-2000. First-strand cDNA synthesis was performed using the PrimeScript™ 1st Strand cDNA Synthesis Kit: 5 μg of RNA sample was taken, and the required RNA volume was calculated based on the RNA concentration, using 50 μmol·L⁻¹. -1 oligo(dT) 1 μl, 10 mmol·L -1 Add 1 μl of dNTPs and ddH2O to a final volume of 10 μl. Mix thoroughly and incubate at 65°C for 5 min. Immediately transfer to ice for 2 min. Add the following reagents sequentially to the above reaction solution. The reaction system is shown in Table 5.

[0043] Table 5 Reaction System Mix thoroughly, incubate at 50°C for 50 min, then at 85°C for 5 min. Add 1 μL of RNase H, incubate at 37°C for 20 min, and place on ice. Obtain the cDNA template and store at -20°C.

[0044] Forward primer PeSTOP1-F: 5′-ATGATGGCAGGTCAGGTTATGG-3′ (SEQ ID NO.1); Reverse primer PeSTOP1-R: 5′-TTAATTCCTCTACCTTGCTG-3′ (SEQ ID NO.2).

[0045] PCR amplification was performed using 2×PhantaMax Master Mix high-fidelity enzyme. The PCR reaction system (25 μl) and procedure are shown in Tables 6 and 7.

[0046] Table 6. Gene Cloning PCR Reaction System Table 7. Gene Cloning PCR Reaction Procedure After the PCR reaction, the amplification products were detected by 1% agarose gel electrophoresis. All family samples showed a single, clear target band, approximately 1950 bp in size, consistent with expectations. The target band in the correct position was excised and purified using a gel extraction kit (Xinjing, Hangzhou). The purified product was then... PeSTOP1 The gene PCR product was ligated into the pEASY®-Blunt Zero cloning vector, transformed into Trans1-T1 competent cells, plated on LB agar plates containing Kanamycin (50 μg / mL), and incubated overnight at 37°C. Single clones were randomly selected for colony PCR validation, and positive clones were sent to Shanghai Sangon Biotech for Sanger bidirectional sequencing. Sequencing results were compared with... PeSTOP1 The consistency of the gene reference sequence alignment was higher than 99.5%, and all samples were successfully obtained. PeSTOP1 The gene coding region sequence provides an accurate template for subsequent SNP identification.

[0047] The results of total RNA extraction quality testing are shown in Table 8.

[0048] Table 8 Results of Total RNA Extraction Quality Detection The A260 / A280 ratios of all samples were between 2.0 and 2.1, and the A260 / A230 ratios were all greater than 2.0, indicating that the RNA purity was high and the protein and salt contamination was minimal, meeting the requirements for reverse transcription.

[0049] cDNA synthesis efficiency was validated using extracted RNA as a template and PrimeScript was employed. TM The first-strand cDNA was synthesized using the 1st Strand cDNA Synthesis Kit. The cDNA synthesis efficiency was assessed using real-time quantitative PCR (qPCR) with the Actin gene as an internal control. The reaction volume (20 μL) is as follows: Table 9 Reaction System The qPCR program consists of 95℃ for 30 seconds; 40 cycles: 95℃ for 5 seconds, 60℃ for 30 seconds.

[0050] Table 10 shows the cDNA synthesis efficiency assessment (Ct value, Actin gene) of some families.

[0051] Table 10: Evaluation of cDNA synthesis efficiency in some families (Ct value, Actin gene) All samples had Ct values ​​between 18 and 19, indicating high cDNA synthesis efficiency and consistent template quality, making them suitable for subsequent gene cloning and expression analysis.

[0052] Primers designed based on the TsBLH3 gene were used to perform PCR amplification using synthesized cDNA as a template. The amplification product was detected by 1% agarose gel electrophoresis, and a target band of approximately 1500 bp was visible, consistent with the expected size.

[0053] Forward (SEQ ID NO.3): 5′-ATGGCCGAGCTGTTCGAC-3′; Reverse (SEQ ID NO.4): 5′-TCAGTCGACCTGCAGGAT-3′.

[0054] After gel extraction, purification, and ligation, the PCR products were ligated into the pEASY-Blunt vector, transformed into Trans1-T1 competent cells, plated on LB agar plates containing Kanamycin, and incubated overnight at 37°C. Single clones were randomly selected for colony PCR verification, and positive clones were sent for sequencing. Results showed high PCR amplification efficiency and good band specificity; the positive rate of clones was consistently above 70%, with 80% for F02-LP samples and 90% for F07-LP samples, indicating a stable and reliable cloning system that provided high-quality templates for subsequent gene sequence analysis and SNP site identification.

[0055] This embodiment successfully extracted high-quality total RNA from the root tip tissue of slash pine seedlings, synthesized a highly efficient cDNA template, and successfully cloned the target gene fragment via PCR amplification. The high quality of the RNA, the stable efficiency of cDNA synthesis, and the reliability of the PCR amplification and cloning system provide a reliable technical foundation for subsequent gene sequence analysis, SNP site identification, and molecular marker development.

[0056] Example 4 The positive clones obtained in Example 3 were sent to BGI Genomics for Sanger sequencing. The sequencing results were analyzed using VectorNTI software and compared with those of *Pinus slashii*. PeSTOP1 Gene reference sequence alignment identified two missense mutation SNP sites significantly associated with phosphorus efficiency, located at [locations to be inserted here]. PeSTOP1 The 620th and 1897th positions of the gene CDS sequence (Table 11).

[0057] Table 11 Two missense mutation SNP sites significantly associated with phosphorus efficiency Genotyping was performed on the two SNP loci mentioned above in 13 pine families (78 samples in total), and the frequency of each genotype was counted. The results are shown in Tables 12 and 13.

[0058] Table 12 Genotype distribution of SNP-1 loci Table 13 Genotype distribution of SNP-2 loci Association analysis between SNP genotype and phosphorus efficiency phenotype: The SNP genotype was associated with the phosphorus use efficiency (PUE) and low phosphorus tolerance comprehensive evaluation value (D) measured in Example 2. One-way ANOVA was used to compare phenotypic differences among different genotypes. The results are shown in Tables 14 and 15.

[0059] Table 14. Association Analysis between SNP-1 Genotype and Phosphorus Efficiency Indicators Table 15 Association Analysis between SNP-2 Genotype and Phosphorus Efficiency Indicators The association between double SNP combination genotypes and phosphorus efficiency was further analyzed. The relationship between SNP-1 and SNP-2 combination genotypes and phosphorus efficiency was further analyzed. The "high-efficiency haplotype" was defined as the combination of SNP-1-G allele and SNP-2-GC allele. The results are shown in Table 16.

[0060] Table 16 Information on the combination of SNP-1-G alleles and SNP-2-GC alleles There were highly significant differences in both PUE and D value between efficient and inefficient combinations (P<0.001).

[0061] In summary, this embodiment successfully identified... PeSTOP1 Two missense single nucleotide polymorphism (SNP) sites in the gene showed a significant correlation with phosphorus efficiency: SNP-1 (C→G) and SNP-2 (AG→GC). The GG genotype (SNP-1) and the GC / GC genotype (SNP-2) were significantly associated with high phosphorus use efficiency (PUE) and a high overall phosphorus tolerance score (D). The double SNP combination genotype (GG+GC / GC) can serve as a molecular marker haplotype for efficient phosphorus utilization in slash pine and can be used for seedling-assisted selection.

[0062] PeSTOP1 The genomic DNA sequence (SEQ ID NO.5) is as follows:

Claims

1. A molecular marker related to high phosphorus efficiency in Pinus elliottii, characterized in that, The molecular marker is a single nucleotide polymorphism (SNP) site in the coding region of a Pinus elliottii PeSTOP1 gene, the SNP site being SNP-1 and / or SNP-2. The SNP-1 is located at the 620th nucleotide of the coding region of the gene, wherein the reference allele is C and the variant allele is G. PeSTOP1 The SNP-1 is located at the 620th nucleotide of the coding region of the gene, wherein the reference allele is C and the variant allele is G. The SNP-2 is located at the 1897th nucleotide of the coding region of the gene, and the reference allele sequence is AG, and the variant allele sequence is GC. PeSTOP1 The SNP-2 is located at the 1897th nucleotide of the coding region of the gene, and the reference allele sequence is AG, and the variant allele sequence is GC.

2. Use of the molecular marker associated with phosphorus efficiency utilization in Pinus elliottii of claim 1 in identifying or assisting in identifying the phosphorus efficiency utilization ability of Pinus elliottii.

3. A reagent for detecting the molecular marker associated with phosphorus efficiency utilization in Pinus elliottii of claim 1.

4. Use of the reagent of claim 3 in preparing a product for assisting in screening or identifying a Pinus elliottii family or individual with phosphorus efficiency utilization.

5. A primer pair or a probe for detecting the molecular marker of phosphorus high efficiency utilization related to Pinus elliottii of claim 1, characterized in that, The primer pair or probe is capable of specifically amplifying or binding to a gene segment comprising the SNP-1 and / or SNP-2 site PeSTOP1 gene segment.

6. A kit for assisting in screening or identifying phosphorus efficient slash pines, comprising, comprising the primer pair or probe of claim 5.

7. A method for screening P-efficient slash pines using the P-efficient utilization related molecular marker of claim 1, wherein the method comprises the steps of: a) obtaining a sample of a slash pine; b) determining whether the sample contains the P-efficient utilization related molecular marker of claim 1; and c) selecting the sample as a P-efficient slash pine if the sample contains the P-efficient utilization related molecular marker of claim 1. comprising the following steps: (1) obtaining total RNA of a Pinus elliottii individual to be tested and reverse transcribing it into cDNA; (2) detecting the nucleotide type or allele at position 620 and / or position 1897 of the coding region of PeSTOP1 gene of the individual; (3) making a judgment based on the detection results: a) if the G allele at position 620 is homozygous (GG) and / or the GC allele at position 1897 is homozygous (GC / GC), it is judged that the individual has the potential for phosphorus efficiency utilization; b) if the G allele at position 620 and the GC allele at position 1897 are both present, it is judged that the individual has a higher potential for phosphorus efficiency utilization.

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