Use of a SNP molecular marker for identifying the phosphorus concentration trait of the above-ground part of onobrychis viciaefolia

Abstract

The application discloses a kind of for identifying the application of SNP molecular marker of column flower ground part phosphorus concentration character, belong to plant molecular marker assisted breeding field.The SNP site SGM.chr3:3078952 is located at the 3078952 bp of column flower chr3 chromosome, and G / A base mutation exists at the 401th position of the sequence shown in SEQ ID NO.1.The application provides specific primer pair for detecting the site and kit comprising the primer pair.The genotype of the site of the material to be tested can be judged by detecting the height of ground part phosphorus concentration: GG genotype is low ground part phosphorus concentration, AA or GA genotype is high ground part phosphorus concentration.The interpretation rate of the SNP marker to the character is 11.04%, and early identification and high-throughput screening of column flower low-phosphorus-tolerant germplasm in acidic low-phosphorus soil environment can be realized, and the screening efficiency of germplasm resources is improved.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular marker-assisted breeding and molecular biology, and in particular to the application of an SNP molecular marker for identifying phosphorus concentration traits in the upper part of Stylosanthes meadow. Background Technology

[0002] Phosphorus is an essential macronutrient for plant growth and development, participating in several key physiological processes such as nucleic acid synthesis, energy metabolism, and photosynthesis. In acidic soils (especially strongly acidic soils with a pH < 4.5), available phosphorus is readily fixed by iron and aluminum oxides, resulting in a phosphorus concentration in the soil solution far below the levels required for normal plant growth. Aboveground phosphorus concentration, expressed as phosphorus mass per unit dry weight (mg P / g DW), is an intensity trait independent of plant biomass. It directly reflects the phosphorus nutritional status and cellular physiological activity within plant tissues. Its level directly regulates the efficiency of core physiological processes such as photosynthetic phosphorylation and key enzyme activity, making it a core parameter for evaluating the intrinsic phosphorus utilization efficiency of plants. Therefore, screening and cultivating plant materials that can maintain a high aboveground phosphorus concentration under acidic, low-phosphorus conditions is of great significance for improving the utilization efficiency of acidic soils and tapping the productive potential of marginal land.

[0003] However, aboveground phosphorus concentration, as a quantitative trait controlled by multiple genes, has a complex genetic mechanism and is easily affected by environmental factors such as soil pH, available phosphorus levels, and climatic conditions. Traditional phenotypic identification requires planting trials under specific acidic soil conditions, involving steps such as sample collection, organ isolation, drying, digestion, and phosphorus concentration determination. This process is time-consuming, labor-intensive, and susceptible to environmental fluctuations, making it difficult to meet the needs of rapid screening of large-scale germplasm resources.

[0004] Guyana penguin beans ( Stylosanthes guianensis Stylosanthes kirilowii, commonly known as stylosanthes, is a perennial forage grass belonging to the legume family. Native to tropical America, it has been widely introduced into tropical and subtropical acidic soil regions worldwide. Through long-term evolution, this species has developed a unique low-phosphorus tolerance adaptation mechanism, enabling it to maintain relatively stable growth and phosphorus nutrition under conditions of extreme phosphorus scarcity. Its aboveground phosphorus concentration exhibits rich natural variation, making it an ideal material for low-phosphorus tolerance breeding and improvement. Therefore, identifying genetic loci related to phosphorus concentration in the aboveground parts of Stylosanthes kirilowii is of significant value for breeding superior forage varieties adapted to acidic, low-phosphorus soil environments.

[0005] Currently, reports on molecular markers for phosphorus concentration traits in the aboveground parts of *Stylos stylossum* are relatively limited, especially lacking stable SNP molecular markers that can distinguish between materials with high and low aboveground phosphorus concentrations. This restricts the efficient evaluation of *Stylos stylossum* germplasm resources and marker-assisted selection breeding. Therefore, developing SNP molecular markers stably associated with phosphorus concentration traits in the aboveground parts of *Stylos stylossum* and establishing a rapid genotype-based screening method are of great significance for the early identification of superior low-phosphorus-tolerant *Stylos stylossum* germplasm and for efficient phosphorus breeding. Summary of the Invention

[0006] This invention provides an application of SNP molecular markers for identifying phosphorus concentration characteristics in the upper part of Stylosanthes meadows, in order to overcome the above-mentioned problems.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: The first aspect of this invention provides an application of SNP molecular marker primer pair related to phosphorus concentration in the aboveground parts of Stylosanthes stylosanthes in identifying phosphorus concentration traits in the aboveground parts of Stylosanthes stylosanthes and screening for Stylosanthes stylosanthes germplasm with low or high aboveground phosphorus concentrations. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein a G / A base mutation exists at position 401 of the sequence shown in SEQ ID NO.1. The SNP site of the SNP molecular marker is named SGM.chr3:3078952, located at the 3078952 bp position on chromosome chr3 of Stylosanthes stylosanthes. The nucleotide sequences of the SNP molecular marker primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.3; When the genotype of the SNP molecular marker locus is GG, it is determined to be a low-phosphorus-concentration Stylosanthes germplasm; when the genotype of the SNP molecular marker locus is AA or GA, it is determined to be a high-phosphorus-concentration Stylosanthes germplasm.

[0008] A second aspect of the present invention provides a kit containing the SNP molecular marker primer pair for the identification of phosphorus concentration in the aboveground parts of *Stylosanthes stylosanthes* and the screening of *Stylosanthes stylosanthes* germplasm with low or high aboveground phosphorus concentrations. When the genotype of the SNP molecular marker locus is GG, it is determined to be *Stylosanthes stylosanthes stylosanthes* germplasm with low aboveground phosphorus concentration; when the genotype of the SNP molecular marker locus is AA or GA, it is determined to be *Stylosanthes stylosanthes stylosanthes stylosanthes* germplasm with high aboveground phosphorus concentration.

[0009] A third aspect of the present invention provides a method for identifying phosphorus concentration characteristics in the upper part of *Stylosanthes stylosum* meadow, comprising the following steps: S1. Extract genomic DNA from the *Stylosanthes* material to be tested; S2. The SNP molecular marker primers were used to perform PCR amplification on the genomic DNA of the test plant, and the amplification products were sequenced to detect the genotype of the SNP molecular marker sites in the test plant material. S3. Determine the aboveground phosphorus concentration trait of the tested Stylosanthes material based on genotype: if the genotype is GG, it is determined to be a Stylosanthes germplasm with low aboveground phosphorus concentration; if the genotype is AA or GA, it is determined to be a Stylosanthes germplasm with high aboveground phosphorus concentration.

[0010] Furthermore, the PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; 72℃ extension for 7 min.

[0011] The beneficial effects of this invention are: This invention developed the SNP molecular marker locus SGM.chr3:3078952 in *Stylos stylosus*, which explains 11.04% of the phosphorus concentration in the aboveground parts and plays a crucial role in the genetic regulation of this trait. Based on specific primers designed for this locus, genotypes can be rapidly and accurately identified through PCR amplification and sequencing or direct resequencing, enabling early screening of phosphorus concentration in the aboveground parts of *Stylos stylosus*. The detection is unaffected by environmental factors or growth stages, and is simple, low-cost, and quick. Compared to traditional phenotypic identification methods, which require seedling cultivation, have long cycles, are susceptible to environmental interference, and suffer from poor accuracy, this invention significantly improves breeding efficiency and provides a reliable technical tool for marker-assisted breeding of phosphorus concentration in the aboveground parts of *Stylos stylosus*, possessing significant scientific and industrial application value. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is a frequency distribution diagram of phosphorus concentration characteristics in the aboveground parts of the Styrax chinensis population in Example 1 of the present invention; Figure 2 This is a Manhattan plot of the genome-wide association analysis in Example 1 of this invention. The horizontal axis represents chromosome location, and the vertical axis represents... P negative logarithm, i.e. -log 10 ( P ); Figure 3This is a box plot of aboveground phosphorus concentration for different haplotypes of materials with peak SNP loci (SGM.chr3:3078952) in Example 1 of the present invention. The horizontal axis represents the genotype (hap1: GG genotype; hap2: AA or GA genotype), and the vertical axis represents the aboveground phosphorus concentration (mgP / gDW).

[0014] Figure 4 This is a DNA sequence alignment diagram of a specific region of the *Stylosanthes* cultivar or breeding material in Example 4 of the present invention. The red-marked bases in the diagram are the SGM.chr3:3078952 sites, and the total sequence range is from the 137th to the 620th base of SEQ ID NO.1. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1: Construction of a genome-wide association population of Stylosanthes kirilowii and development of SNP molecular markers (1) Material collection and phenotypic identification Twenty-three natural accessions of *Stylos sinensis* were collected for genome-wide association analysis (GWAS) population construction and phenotypic identification. The materials were grown in the germplasm resource nursery of the Institute of Tropical Crops Germplasm Resources, Chinese Academy of Tropical Agricultural Sciences, located in Danzhou, Hainan Province, in strongly acidic lateritic soil (pH = 4.2). Mature upper leaves and stems were collected after the plants reached the peak functional leaf stage. The aboveground phosphorus concentration (mg P / g DW) of the 253 *Stylos sinensis* accessions was determined using the national standard GB / T 6437-2018, "Determination of Total Phosphorus in Feed - Spectrophotometric Method". The aboveground phosphorus concentrations of the 253 *Stylos sinensis* accessions (average of three biological replicates) are shown in Table 1.

[0017] Table 1. Average values ​​of phosphorus concentration in the aboveground parts of 253 *Stylosanthes* specimens.

[0018] The mean shoot phosphorus concentration of 253 Stylosanthes germplasm accessions is 1.71 mg P / g DW. Accessions with shoot phosphorus concentration higher than the mean are defined as high shoot phosphorus concentration accessions, and there are 106 such Stylosanthes germplasm accessions; accessions with shoot phosphorus concentration lower than the mean are defined as low shoot phosphorus concentration accessions, and there are 147 such Stylosanthes germplasm accessions. Based on the above data, a schematic diagram of the phenotypic distribution of the associated population is drawn, as shown in Figure 1 . The shoot phosphorus concentration of the 253 Stylosanthes germplasm accessions varies in the range of 0.85 to 3.58 (mg P / g DW). The trait generally presents a unimodal skewed continuous distribution, which conforms to the genetic characteristics of quantitative traits, and it is speculated that there may be major gene regulatory loci for this trait.

[0019] (2) Genomic DNA extraction and resequencing Samples are taken in the Stylosanthes germplasm resource nursery. Approximately 2 g of young leaves are taken from each of the above 253 Stylosanthes germplasm accessions, quick-frozen in liquid nitrogen, and then sent to BGI Genomics. The construction of the resequencing library is completed using a commercial DNA library preparation kit supporting the DNBSEQ platform, and resequencing is performed on the DNBSEQ-T7 platform. Quality control and filtering are carried out on the off-machine sequencing data to obtain clean data. Using the Stylosanthes cultivar S1979 as the reference genome (the genomic data has been published at https: / / github.com / LiuChun-Lab / Sguianensis_T2T), a genome index file is constructed. The BWA-MEM algorithm of the BWA software is used to align the quality-controlled sequencing data (clean data) to the reference genome, obtain the position information of the sequencing fragments on the chromosomes, and output the alignment file in BAM format. The SAMtools software is used to sort the BAM alignment file according to the chromosomal coordinates, and the PicardTools software is used to mark and remove duplicate sequencing fragments introduced by PCR amplification. For each Stylosanthes germplasm accession, the HaplotypeCaller module of the GATK software is used to perform genome-wide variation detection on the processed BAM alignment file to generate an individual GVCF file. Subsequently, the CombineGVCFs module of GATK is used to integrate the variation information of the GVCF files of all individuals, and then the GenotypeGVCFs module is used for population joint genotyping, to output a population variation VCF file containing the variation information of all samples.

[0020] Quality control was performed on the above population variation information. First, hard filtering was performed using the VariantFiltration module of GATK software. Candidate variant sites were marked as low-quality variant sites if they met any of the following conditions: Quality Depth Ratio (QD) less than 2.0, Fisher Chain Bias (FS) greater than 60.0, Alignment Quality Value (MQ) less than 40.0, Alignment Quality Rank Sum (MQRankSum) less than -12.5, or the Rank Sum of the variant site's position in the sequencing fragment less than -8.0. Variants that passed the hard filtering were retained and further filtered using BCFtools software based on allele count, genotype loss rate, and minor allele frequency. The screening criteria were: the variant site contained exactly two alleles, the genotype loss rate was less than 30%, and the minor allele frequency (MAF) was greater than 0.05. After this filtering, a high-quality biallelic SNP dataset (high-quality population SNP dataset) was obtained for subsequent population genetic analysis.

[0021] (3) Genome-wide association analysis The VCF files corresponding to the high-quality population SNP dataset (6,118,683 SNP loci) obtained in the above steps were converted using VCFtools software to output input files compatible with rMVP software. Subsequently, a population kinship matrix was constructed using rMVP software based on the converted files, and a genome-wide association analysis (GWAS) was performed using the FarmCPU model. The Bonferroni correction method was used to calculate the significance threshold for the association analysis. P <1 / Number of SNP sites = 1.63 × 10 -7 As a criterion for determining a significant association between a SNP and a trait. The Manhattan plot of the genome-wide association analysis results is shown below. Figure 2 As shown in the figure, the vertical axis is log 10 ( P The higher the value, the stronger the association with the corresponding SNP. P The lower the value; the red dashed line is the significance threshold line after Bonferroni correction, and the sites above the dashed line are SNPs that are significantly associated with aboveground phosphorus concentration; the site with the highest ordinate is the most significant SNP among all SNPs. P The peak SNP with the lowest value was identified in this study as SGM.chr3:3078952. All population materials were grouped according to the different allelic genotypes corresponding to this peak SNP, and analysis of variance was performed on the aboveground phosphorus concentration phenotype. The proportion of between-group variance to total variance was defined as the phenotypic contribution rate of this peak SNP to the target trait of aboveground phosphorus concentration.

[0022] GWAS analysis identified a major QTL region controlling aboveground phosphorus concentration in *Stylos stylosus*, located 400 bp upstream and downstream of chromosome 3078952 on chromosome chr3 (801 bp in length). The peak SNP within this region was named SGM.chr3:3078952, and its genomic nucleotide sequence is shown in SEQ ID NO.1, with a G / A mutation at 401 bp. This SNP explained 11.04% of the phenotypic variation in aboveground phosphorus concentration in *Stylos stylosus*. The population was divided into two groups based on the genotype at this SNP: hap1 (GG genotype) with a mean aboveground phosphorus concentration of 1.66 mg P / g DW, and hap2 (AA or GA genotype) with a mean aboveground phosphorus concentration of 2.20 mg P / g DW. Independent samples t-test results are shown below. Figure 3 As shown, the overall phosphorus concentration in the upper part of the *Stylosanthes* meadow in group hap1 was significantly lower than that in group hap2. P <0.01). Therefore, the nucleotide sequence shown in SEQ ID NO.1 was identified as an SNP molecular marker for identifying the phosphorus concentration trait in the upper part of Stylosanthes meadow.

[0023] Example 2: Specific primer design and primer effectiveness verification for SNP molecular markers (1) Primer design Based on the nucleotide sequence containing the SGM.chr3:3078952 site shown in SEQ ID NO.1, specific PCR amplification primers were designed using the NCBI primer design tool. The primer amplification fragments covered the 401 bp mutation site. Upstream primer: 5'-ACATTAAGCCATTTTTGGTTTTGCT-3' (SEQ ID NO.2); Downstream primer: 5'-GCACACGCGTCAATCTGAG-3' (SEQ ID NO.3); (2) Genomic DNA extraction and PCR amplification of the test material DNA Extraction: Approximately 100 mg of young leaves from each of the tested *Stylosanthes cusia* materials were ground into powder using liquid nitrogen. The powder was then added to 500 μL of 2×CTAB extraction buffer (containing 100 mmol / L Tris-HCl pH 8.0, 20 mmol / L EDTA, 1.4 mol / L NaCl, 2% CTAB, and freshly prepared 40 mmol / L mercaptoethanol) preheated at 65°C. The mixture was incubated in a water bath at 65°C for 30–60 min. After cooling to room temperature, an equal volume of chloroform-isoamyl alcohol (24:1, v / v) was added for extraction. The mixture was centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected, and 2 / 3 volume of pre-chilled 100% isopropanol was added. The mixture was incubated at -20°C for at least 30 min to precipitate the DNA. The precipitate was centrifuged at 12000 rpm for 10 min at 4°C, and washed twice with 70% ethanol to obtain genomic DNA. The precipitate was dried at room temperature and then extracted with TE buffer (containing 10 mmol / L Tris-HCl pH 7.4 and 1% EDTA). Dissolve the DNA in 1 mmol / L EDTA and adjust the DNA concentration to 50 ng / μL as a template for PCR amplification.

[0024] PCR amplification: PCR amplification was performed using the extracted genomic DNA as a template. The total PCR system was 10 μL: 1 μL template DNA (50 ng / μL), 5 μL 2×PCR Master Mix (containing Taq DNA polymerase, dNTPs, MgCl2, and PCR reaction buffer), 0.5 μL 10 μmol / L upstream primer, 0.5 μL 10 μmol / L downstream primer, and 3 μL nuclease-free water. The amplification program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and 72℃ extension for 7 min. The amplified products were detected by 1.5% agarose gel electrophoresis. Once a single, clear target band appeared, the samples were sent to a sequencing company for Sanger sequencing.

[0025] (3) Sequencing of PCR products and interpretation of genotypes The PCR amplification products were subjected to Sanger sequencing. The sequence peak diagram obtained from the sequencing was compared with the sequence of SEQ ID NO.1. The base at position 401bp was read to determine whether the sample was of genotype GG, AA, or GA.

[0026] (4) Validation of primer and marker effectiveness Ten *Stylosanthes* materials with known aboveground phosphorus concentration phenotypes were selected from the 253 natural populations of *Stylosanthes* in Example 1. Five of these were classified as low aboveground phosphorus concentration materials, and five as high aboveground phosphorus concentration materials. The classification of low and high aboveground phosphorus concentration materials was based on the aboveground phosphorus concentration distribution of the 253 materials; materials with aboveground phosphorus concentrations below the population mean were classified as low aboveground phosphorus concentration materials, and those above the population mean were classified as high aboveground phosphorus concentration materials. Genomic DNA templates from the 10 materials were amplified and detected using the primers and PCR conditions described above. The detection results are shown in Table 2.

[0027] Table 2. Genotypes and aboveground phosphorus concentrations at the SGM.chr3:3078952 locus in 10 *Stylosanthes* accessions.

[0028] Table 2 shows that the genotypes of all five materials with low phosphorus concentration in the aboveground parts were GG, while the genotypes of the five materials with high phosphorus concentration in the aboveground parts were AA or GA. The molecular marker genotype results were completely consistent with the actual phenotypes, thus verifying the accuracy and effectiveness of the primers and SNP molecular markers.

[0029] Example 3: Preparation of a kit for detecting SNP sites of phosphorus concentration in the upper part of Stylosanthes meadow This embodiment provides a detection kit (molecular marker name: SGM.chr3:3078952) for detecting SNP molecular marker genotypes related to phosphorus concentration in the upper part of *Stylosanthes stylosum*. This kit is used in conjunction with Sanger sequencing to determine the genotype of *Stylosanthes stylosum* germplasm.

[0030] (1) The kit composition is shown in Table 3 below.

[0031] Table 3. Reagent Kit Composition

[0032] (2) Kit preparation method A. Prepare DNA extraction buffer reagents according to the standard CTAB formula; after the reagents pass quality testing, dispense them. B. Prepare 2×PCR Master Mix, aliquot it after the reagents pass quality testing, and store it at -20℃. C. The primers SEQ ID NO.2 and SEQ ID NO.3 were synthesized by a synthetic company. The primers were purified by HPLC with a purity of ≥90%. The primers were dissolved in ddH2O to prepare a 100 μmol / L stock solution and stored at -20℃ in the dark. Before use, the solution was diluted with ddH2O to a working concentration of 10 μmol / L. D. Assemble and package all components in Table 3 and the instructions for use according to the kit specifications to obtain the finished kit.

[0033] (3) Instructions for use of the reagent kit Genomic DNA was extracted from the *Stylosanthes spp.* and the DNA concentration was adjusted to 50 ng / μL. Using the primers and 2×PCR Master Mix provided in the kit, and supplementing the total reaction volume with nuclease-free water provided in the kit, PCR amplification was performed using the extracted and adjusted genomic DNA as a template, following the amplification system and procedure of Example 2. After Sanger sequencing of the PCR amplification products, the genotype was determined according to the genotype determination criteria described in Example 2.

[0034] Genotype determination: GG is a candidate material with low aboveground phosphorus concentration; AA and GA are candidate materials with high aboveground phosphorus concentration.

[0035] Example 4: Verification of aboveground phosphorus concentration in *Stylosanthes cultivars* or breeding materials based on SNP molecular markers The steps of the method for identifying phosphorus concentration in aboveground parts using the molecular markers of the present invention are as follows: (1) DNA extraction Take 100 mg of young leaves of *Stylosanthes stylosum* to be tested; use the DNA extraction reagent (CTAB method) provided with the kit in Example 3 to extract genomic DNA from the leaf sample; adjust the concentration of the extracted DNA to 50 ng / μL.

[0036] (2) PCR amplification and sequencing Using the primers SEQ ID NO.2 and SEQ ID NO.3 provided in the kit of Example 3, and with the adjusted concentration of genomic DNA as a template, PCR amplification was performed according to the amplification system and procedure of Example 2. Subsequently, the PCR amplification product was subjected to Sanger sequencing to read the 401st bp of the sequence shown in SEQ ID NO.1, obtaining the SGM.chr3:3078952 genotype.

[0037] (3) Genotype determination and evaluation of phosphorus concentration in aboveground parts When the SNP genotype of the material to be tested is GG, the material is determined to be a variety with low aboveground phosphorus concentration; when the SNP genotype of the material to be tested is AA or GA, the material is determined to be a variety with high aboveground phosphorus concentration.

[0038] (4) Practical application verification Six cultivated varieties or breeding materials of *Stylos sinensis* that did not overlap with the 253 natural populations of *Stylos sinensis* used in Example 1 for genome-wide association analysis were selected (the six materials were planted in the germplasm resource nursery of the Institute of Tropical Crops Germplasm Resources, Chinese Academy of Tropical Agricultural Sciences, located in Danzhou, Hainan). The genomic DNA samples of the six materials were genotyped at the SGM.chr3:3078952 locus using the identification methods in steps (1)-(3) above. The results are shown in [reference]. Figure 4 At the same planting and sampling time as the 253 natural population materials of Stylosanthes in Example 1, the leaves and stems of these 6 Stylosanthes to be tested were collected and the phosphorus concentration of the aboveground parts was measured. The results are shown in Table 4.

[0039] Table 4. Screening and validation of aboveground phosphorus concentration traits in 6 *Stylosanthes* specimens.

[0040] The results of the identification are as follows Figure 4 As shown in Table 4, four materials were predicted to have low aboveground phosphorus concentration, and two materials were predicted to have high aboveground phosphorus concentration. Using the average aboveground phosphorus concentration of 1.71 mg P / g DW for the 253 *Stylosanthes* germplasm populations in Example 1 as the standard, materials with measured values ​​below this average were identified as having low aboveground phosphorus concentration, and those above this average were identified as having high aboveground phosphorus concentration. Field trial results showed that the four materials predicted to have low aboveground phosphorus concentration had aboveground phosphorus concentrations of 1.20 mg P / g DW–1.57 mg P / g DW, all below 1.71 mg P / g DW; the two materials predicted to have high aboveground phosphorus concentrations had aboveground phosphorus concentrations of 1.87 mg P / g DW–2.03 mg P / g DW, both above 1.71 mg P / g DW. The trait results predicted based on genotype perfectly matched the trait determination results from the field trials. The entire detection process of this method only takes 3-5 days, which greatly shortens the breeding and screening cycle and confirms that this SNP molecular marker can be used for rapid and high-throughput screening of Stylosanthes germplasm resources.

[0041] In summary, this application successfully identified the major SNP locus for phosphorus concentration in the aboveground parts of *Stylos stylossum* through genome-wide association analysis. A complete molecular identification technology chain was established through specific primer development, validation of the effectiveness of known phenotypic materials, construction of a detection kit system, and blind sample validation of unknown germplasm. This SNP locus showed stable association with phosphorus concentration in the aboveground parts of *Stylos stylossum*, and the genotype determination results highly matched the actual phenotype. The entire detection process was time-efficient, standardized, and reproducible, providing stable and reliable technical support for early screening of *Stylos stylossum* germplasm with varying aboveground phosphorus concentrations and for marker-assisted breeding of superior varieties.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a SNP molecular marker primer pair related to phosphorus concentration in the aboveground parts of *Stylosanthes stylosanthes* grassland in identifying phosphorus concentration traits in the aboveground parts of *Stylosanthes stylosanthes* grassland and screening *Stylosanthes stylosanthes* germplasm with low or high aboveground phosphorus concentrations, characterized in that... The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, wherein a G / A base mutation exists at position 401 of the sequence shown in SEQ ID NO.1; The SNP site of the SNP molecular marker is named SGM.chr3:3078952, located at the 3078952 bp position on chromosome chr3 of Stylosanthes stylosanthes. The nucleotide sequences of the SNP molecular marker primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.3; When the genotype of the SNP molecular marker locus is GG, it is determined to be a low-phosphorus-concentration Stylosanthes germplasm; when the genotype of the SNP molecular marker locus is AA or GA, it is determined to be a high-phosphorus-concentration Stylosanthes germplasm.

2. The application of a kit containing the SNP molecular marker primer pair described in claim 1 in identifying the aboveground phosphorus concentration trait of *Stylosanthes stylosanthes* and screening *Stylosanthes stylosanthes* germplasm with low or high aboveground phosphorus concentration, wherein when the genotype of the SNP molecular marker locus is GG, it is determined to be *Stylosanthes stylosanthes stylosanthes* germplasm with low aboveground phosphorus concentration; when the genotype of the SNP molecular marker locus is AA or GA, it is determined to be *Stylosanthes s ...

3. A method for identifying phosphorus concentration characteristics in the upper part of *Stylosanthes stylosum* meadow, characterized in that, Includes the following steps: S1. Extract genomic DNA from the *Stylosanthes* material to be tested; S2. The SNP molecular marker primers described in claim 1 are used to perform PCR amplification on the genomic DNA of the test plant, and the amplification products are sequenced to detect the genotype of the SNP molecular marker sites described in claim 1 in the test plant material. S3. Determine the aboveground phosphorus concentration trait of the tested Stylosanthes material based on genotype: if the genotype is GG, it is determined to be a Stylosanthes germplasm with low aboveground phosphorus concentration; if the genotype is AA or GA, it is determined to be a Stylosanthes germplasm with high aboveground phosphorus concentration.

4. The method according to claim 3, characterized in that, The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ extension for 7 min.

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