Application of SNP molecular marker for identifying the trait of leaf crude protein content of Stipa krylovii Roshev.

Abstract

This invention discloses the application of a SNP molecular marker for identifying the crude protein content trait in *Stylosanthes cuspidatum* leaves, belonging to the field of plant molecular marker-assisted breeding technology. The SNP locus, named SGM.chr9:3360969, is located at 3360969 bp on chromosome chr9 of *Stylosanthes cuspidatum*, with a G / A base mutation at position 401 of the corresponding sequence. This invention also provides a specific primer pair for amplifying this SNP marker and a detection kit containing this primer pair. By detecting the genotype of the test material at this SNP locus, its leaf crude protein content can be quickly determined: individuals with genotype GG exhibit high leaf crude protein content, while individuals with genotypes AA or GA exhibit low leaf crude protein content. This marker and detection method are unaffected by the environment, are simple to operate, and have a short cycle time, which can significantly improve the screening efficiency of high-quality germplasm resources and accelerate the breeding process.

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 the crude protein content trait in leaves of Stylosanthes kirilowii. Background Technology

[0002] Aluminum ion toxicity and available phosphorus deficiency in acidic soils are two major obstacles limiting plant growth and crop production, especially in strongly acidic soils (pH < 4.5). Most crops experience limited growth and reduced nitrogen use efficiency in strongly acidic, low-available-phosphorus soils, leading to decreased crude protein concentration and lower yields, thus hindering the full realization of the production potential of such marginal lands. Therefore, screening and cultivating plant materials adapted to acidic soils with high nitrogen absorption and protein accumulation capabilities is of great significance for improving the utilization efficiency of acidic soils and tapping the production potential of marginal lands.

[0003] Guyana penguin beans ( Stylosanthes guianensis Stylosanthes kirilowii, commonly known as stylosanthes, is a perennial forage and green manure crop belonging to the legume family. It possesses strong tolerance to acidic, aluminum-rich, and low-phosphorus soils, allowing it to grow and be cultivated in acidic soils in tropical and subtropical regions. Stylosanthes kirilowii not only has high forage and ecological value but can also be used for soil degradation improvement, soil fertility enhancement, and soil and water conservation. The abundant genetic resources of acid-tolerant, aluminum-tolerant, and low-phosphorus-tolerant traits in Stylosanthes kirilowii are of significant value for breeding superior forage varieties adapted to acidic, low-phosphorus soil environments and for identifying genetic loci related to forage quality, such as crude protein content in plant leaves.

[0004] Crude protein content in plant leaves is an important indicator reflecting a plant's nitrogen assimilation, translocation, and accumulation capacity. It is also a crucial trait for evaluating the forage nutritional quality of *Stylosanthes spp.* and its adaptability to acidic, low-phosphorus soils. *Stylosanthes spp.* with higher crude protein content in their leaves typically exhibit greater nutrient utilization efficiency and superior forage value, thus becoming a key target trait for *Stylosanthes spp.* breeding improvement. Traditional methods for identifying crude protein content in *Stylosanthes spp.* leaves usually require planting trials under specific soil conditions, involving steps such as plant sample collection, digestion, and nitrogen content determination. This approach suffers from drawbacks such as long identification cycles, high workload, and susceptibility to environmental influences.

[0005] Currently, reports on molecular markers for the crude protein content trait in *Stylosanthes stylosus* leaves are relatively limited, especially lacking SNP molecular markers that can distinguish between materials with high and low leaf crude protein content. Therefore, developing molecular markers stably associated with the crude protein content trait in *Stylosanthes stylosus* leaves is of great significance for the rapid screening of *Stylosanthes stylosus* germplasm resources, the identification of superior materials, and molecular marker-assisted breeding. Summary of the Invention

[0006] This invention provides an application of SNP molecular markers for identifying the crude protein content trait in *Stylosanthes cusia* leaves, 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 crude protein content in leaves of Stylosanthes stylosanthes in identifying crude protein content traits in leaves of Stylosanthes stylosanthes and screening Stylosanthes stylosanthes germplasm with low or high crude protein content in leaves. 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.chr9: 3360969, located at the 3360969 bp position on chromosome chr9 of *Stylosanthes chinensis*. The nucleotide sequences of the primer pairs for the SNP molecular markers 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 Stylosanthes germplasm with high leaf crude protein content; when the genotype of the SNP molecular marker locus is AA or GA, it is determined to be a Stylosanthes germplasm with low leaf crude protein content.

[0008] The second aspect of this invention provides a kit containing the SNP molecular marker primer pair for the identification of crude protein content in leaves of *Stylosanthes cuspidatum* and the screening of *Stylosanthes cuspidatum* germplasm with low or high crude protein content in leaves. When the genotype of the SNP molecular marker locus is GG, it is determined to be *Stylosanthes cuspidatum* germplasm with high crude protein content in leaves; when the genotype of the SNP molecular marker locus is AA or GA, it is determined to be *Stylosanthes cuspidatum* germplasm with low crude protein content in leaves.

[0009] A third aspect of the present invention provides a method for identifying the crude protein content trait of *Stylosanthes cuspidatum* leaves, 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 crude protein content of the leaves of the tested Stylosanthes material based on genotype: if the genotype is GG, it is determined to be a Stylosanthes germplasm with high crude protein content in leaves; if the genotype is AA or GA, it is determined to be a Stylosanthes germplasm with low crude protein content in leaves.

[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.chr9:3360969 in *Stylosanthes stylosus*, which explains 6.85% of the crude protein content in leaves and plays a crucial role in the genetic regulation of this trait. Based on specific primers designed for this locus, PCR amplification and sequencing can rapidly and accurately identify genotypes, enabling early screening of crude protein content in *Stylosanthes stylosus* leaves. 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 *Stylosanthes stylosus* with high leaf crude protein content, 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 the crude protein content trait in the leaves 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 3 Box plots of leaf crude protein content of different genotypes at the peak SNP locus (SGM.chr9:3360969) in Example 1 of this invention. The horizontal axis represents the genotype (hap1: GG genotype; hap2: AA or GA genotype), and the vertical axis represents the leaf crude protein content (%). Figure 4 This is a DNA sequence alignment diagram of a specific region in the *Stylosanthes* cultivar or breeding material in Example 4 of the present invention. The bases marked in red in the diagram are the SGM.chr9:3360969 sites, and the total sequence range is from the 224th to the 607th base of SEQ ID NO.1. Detailed Implementation

[0014] 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.

[0015] 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 chinensis* 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 were collected after the plants reached the peak functional leaf stage. The crude protein content (%) of the leaves of the 253 *Stylos chinensis* accessions was determined using the Kjeldahl method, a standard specified in GB / T 6432-2018, "Determination of Crude Protein in Feed". The crude protein content of the leaves of the 253 *Stylos chinensis* accessions (average of three biological replicates) is shown in Table 1.

[0016] Table 1. Average values ​​of crude protein content in the leaves of 253 *Stylosanthes cusia* samples.

[0017] The average crude protein content of leaves in the 253 *Stylosanthes* germplasms was 16.86%. Germplasms with a crude protein content greater than the average were defined as having high leaf crude protein content, and 129 of these germplasms had high leaf crude protein content. Germplasms with a crude protein content lower than the average were defined as having low leaf crude protein content, and 124 of these germplasms had low leaf crude protein content. Based on the data in Table 1, a schematic diagram of the phenotypic distribution of the associated populations was drawn, as shown below. Figure 1 As shown, the crude protein content of leaves in 253 Stylosanthes germplasm samples ranged from 7.76% to 23.44%, and the trait showed a continuous distribution with a single-peak skewness, consistent with the genetic characteristics of quantitative traits. It is speculated that there may be significant associated loci for this trait.

[0018] (2) Genomic DNA extraction and resequencing Samples are collected in the stylosanthes germplasm resource nursery, wherein about 2g of young leaves are respectively taken from each of the 253 stylosanthes germplasm materials described above, 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. The off-machine sequencing data is subjected to quality control and filtration 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 quality-controlled sequencing data (clean data) is aligned to the reference genome via the BWA-MEM algorithm of BWA software, to obtain the position information of sequencing fragments on the chromosomes, and an alignment file in BAM format is output. The BAM alignment file is sorted by chromosome coordinates using SAMtools software, and Picard Tools software is used to mark and remove duplicate sequencing fragments introduced by PCR amplification. For each stylosanthes germplasm, the HaplotypeCaller module of GATK software is used to perform genome-wide variation detection on the processed BAM alignment file, so as 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.

[0019] Quality control is performed on the above population variation information. First, the VariantFiltration module of GATK software is used for hard filtering. When a candidate variation site meets any of the following conditions, the candidate variation site is marked as a low-quality variation site: the quality depth ratio QD is less than 2.0, the Fisher strand bias value FS is greater than 60.0, the mapping quality value MQ is less than 40.0, the mapping quality rank sum test value MQRankSum is less than -12.5, or the rank sum test value ReadPosRankSum of the position of the variation site in the sequencing fragment is less than -8.0. The variation sites that pass the above hard filtering are retained, and further filtered for the number of alleles, genotype deletion rate and minor allele frequency using BCFtools software, with the screening conditions as follows: the variation site contains exactly two alleles, the site genotype deletion rate is less than 30%, and the minor allele frequency MAF is greater than 0.05. After the above filtering, a high-quality biallelic SNP dataset (high-quality population SNP dataset) for subsequent population genetic analysis is obtained.

[0020] (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 significance threshold after Bonferroni correction was used... P <1 / Total number of SNP sites = 1.63 × 10 -7 This determines whether there is a significant association between SNP sites and target traits. The Manhattan plot of 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 greater 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 leaf crude protein content; 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.chr9:3360969. All population materials were grouped according to the different alleles corresponding to this peak SNP, and an analysis of variance was conducted on the leaf crude protein content phenotype. The proportion of the variance between groups to the total variance was defined as the phenotypic contribution rate of this peak SNP to the target trait of leaf crude protein content.

[0021] GWAS analysis identified a QTL interval that was significantly associated with the crude protein content of *Stylosanthes cuspidatum* leaves (the association was significant across the entire genome). P <1.63×10 -7 The SNP located 400 bp upstream and downstream of chromosome 3360969 on chromosome chr9 of *Stylosanthes spp.*, with a length of 801 bp, was named SGM.chr9:3360969. The nucleotide sequence of the genomic segment containing this SNP is shown in SEQ ID NO.1, with a G / A base mutation at 401 bp. This SNP explained 6.85% of the phenotypic variation in crude protein content of *Stylosanthes spp.* leaves. The population was divided into two groups based on the genotype of this SNP: group hap1 (GG genotype) had a mean crude protein content of 17.24% in leaves, and group hap2 (AA or GA genotype) had a mean crude protein content of 15.79% in leaves. The independent samples t-test results are shown below. Figure 3 As shown, the crude protein content of *Stylosanthes pubescens* leaves in group hap1 was significantly higher than that in group hap2. P<0.001). Therefore, the nucleotide sequence shown in SEQ ID NO.1 was identified as an SNP molecular marker for identifying the crude protein content trait in Stylosanthes leaves.

[0022] Example 2: Specific primer design and primer effectiveness verification for SNP molecular markers (1) Primer design Based on the nucleotide sequence containing the SGM.chr9:3360969 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'-CGAAGGAGTGAGGCGAACAT-3' (SEQ ID NO.2); Downstream primer: 5'-AAGCAAGCTCCCAGGGAATG-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.

[0023] 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.

[0024] (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, GA or AA.

[0025] (4) Validation of primer and marker effectiveness Ten *Stylosanthes* materials with known leaf crude protein content phenotypes were selected from the 253 natural populations of *Stylosanthes* in Example 1. Five of these were high-leaf crude protein content germplasm, and five were low-leaf crude protein content germplasm. The classification of low-leaf crude protein content and high-leaf crude protein content materials was based on the leaf crude protein content distribution of the 253 materials; materials with leaf crude protein content below the population mean were classified as low-leaf crude protein content materials, and those above the population mean were classified as high-leaf crude protein content 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.

[0026] Table 2. Genotypes and crude protein content of leaves from 10 *Stylosanthes stylosus* SGM.chr9:3360969 locus.

[0027] Table 2 shows that the genotypes of all five materials with high leaf crude protein content were GG, while the genotypes of the five materials with low leaf crude protein content were AA or GA. The molecular marker genotype results were in complete agreement with the actual phenotypes, verifying the accuracy and effectiveness of the primers and SNP molecular markers.

[0028] Example 3: Preparation of a kit for detecting SNP sites of crude protein content in *Stylosanthes cuspidatum* leaves This embodiment provides a detection kit (molecular marker name: SGM.chr9:3360969) for detecting SNP molecular marker genotypes related to crude protein content in *Stylosanthes spp.* leaves. This kit is used in conjunction with Sanger sequencing to determine the genotype of *Stylosanthes spp.* germplasm.

[0029] (1) The kit composition is shown in Table 3 below.

[0030] Table 3. Reagent Kit Composition

[0031] (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.

[0032] (3) Instructions for use of the reagent kit Genomic DNA was extracted from the *Stylosanthes stylosum* species to be tested, 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.

[0033] Genotype determination: GG is a candidate material with high leaf crude protein content; AA and GA are candidate materials with low leaf crude protein content.

[0034] Example 4: Verification of crude protein content in leaves of *Stylosanthes cultivars* or breeding materials based on SNP molecular markers The method for identifying leaf crude protein content based on the molecular markers of this invention comprises the following steps: (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.

[0035] (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.chr9:3360969 genotype.

[0036] (3) Genotype determination and evaluation of crude protein content in leaves When the SNP genotype of the material to be tested is GG, the material is determined to be a variety with high leaf crude protein content; when the SNP genotype of the material to be tested is AA or GA, the material is determined to be a variety with low leaf crude protein content.

[0037] (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.chr9:3360969 locus using the identification methods in steps (1)-(3) above. The results are shown in [reference]. Figure 4 Meanwhile, at the same planting and sampling time as the 253 samples of Stylosanthes natural population in Example 1, the leaves of these 6 samples of Stylosanthes were collected and the crude protein content of the leaves was measured. The results are shown in Table 4.

[0038] Table 4. Screening and validation results of crude protein content in leaves of 6 *Stylosanthes* samples.

[0039] The results of the identification are as follows Figure 4As shown in Table 4, three materials were predicted to have low leaf crude protein content, and three materials were predicted to have high leaf crude protein content. Using the average leaf crude protein content of 16.86% for the 253 *Stylosanthes* germplasm populations in Example 1 as the standard, materials with measured values ​​below this average were classified as having low leaf crude protein content, and those above were classified as having high leaf crude protein content. Field trial results showed that the three materials predicted to have low leaf crude protein content had leaf crude protein content ranging from 13.31% to 14.54%, all below 16.86%; the three materials predicted to have high leaf crude protein content had leaf crude protein content ranging from 18.81% to 22.06%, all above 16.86%. The trait predictions based on genotype perfectly matched the trait determinations from the field trials. This method's entire detection process takes only 3-5 days, significantly shortening the breeding and screening cycle and confirming that this SNP molecular marker can be used for rapid, high-throughput screening of *Stylosanthes* germplasm resources.

[0040] In summary, this application identified a SNP locus significantly associated with crude protein content in *Stylosanthes cuspidata* leaves 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 a stable association with the crude protein content trait in *Stylosanthes cuspidata* leaves, 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 the early screening of *Stylosanthes cuspidata* germplasm with varying leaf crude protein content and for marker-assisted breeding of superior varieties.

[0041] 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 crude protein content in *Stylosanthes cuspidatum* leaves in identifying the crude protein content trait in *Stylosanthes cuspidatum* leaves and screening *Stylosanthes cuspidatum* germplasm with high or low crude protein content in leaves, 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.chr9:3360969, located at the 3360969th bp position on chromosome chr9 of Stylosanthes stylosum. 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 Stylosanthes germplasm with high leaf crude protein content; when the genotype of the SNP molecular marker locus is AA or GA, it is determined to be a Stylosanthes germplasm with low leaf crude protein content.

2. The application of a kit containing the SNP molecular marker primer pair described in claim 1 in identifying the crude protein content trait of *Stylosanthes cuspidatum* leaves and screening *Stylosanthes cuspidatum* germplasm with high or low leaf crude protein content, characterized in that... When the genotype of the SNP molecular marker locus is GG, it is determined to be a Stylosanthes germplasm with high leaf crude protein content; when the genotype of the SNP molecular marker locus is AA or GA, it is determined to be a Stylosanthes germplasm with low leaf crude protein content.

3. A method for identifying the crude protein content trait in *Stylosanthes cuspidatum* leaves, 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 crude protein content of the leaves of the tested Stylosanthes material based on genotype: if the genotype is GG, it is determined to be a Stylosanthes germplasm with high crude protein content in leaves; if the genotype is AA or GA, it is determined to be a Stylosanthes germplasm with low crude protein content in leaves.

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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