Labeling for detecting soybean protein content and its applications
By using dCAPS and KASP markers, and utilizing Indel variant sites and quantitative real-time PCR, the problems of low accuracy and environmental dependence in soybean protein content detection have been solved, enabling efficient and low-cost early screening and breeding of high-protein soybeans.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NORTHEAST INST OF GEOGRAPHY & AGRIECOLOGY C A S
- Filing Date
- 2026-04-14
- Publication Date
- 2026-06-02
AI Technical Summary
Existing methods for detecting soybean protein content suffer from problems such as low accuracy, high cost, susceptibility to environmental influences, and inability to perform early screening. They also lack molecular markers with high stability and specificity, which affects the breeding efficiency of high-protein soybeans.
Using dCAPS and KASP markers, specific primers were designed for rapid detection of soybean protein content via Indel mutation sites and quantitative real-time PCR, respectively. dCAPS markers were genotyped using TaqI digestion and agarose gel electrophoresis, while KASP markers were genotyped using a fluorescent reporter group and high-throughput PCR.
It enables efficient, accurate, and low-cost early screening of high-protein soybeans, eliminating environmental influences, improving detection and breeding efficiency, and adapting to the detection needs of different soybean breeding materials.
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Figure CN122128458A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural molecular biology, specifically relating to a labeling method for detecting soybean protein content and its application. Background Technology
[0002] Soybeans are a dual-purpose grain crop, used for both food and feed, and are a core source of plant protein and edible oil. Their industrial supply capacity directly impacts national food security and protein feed security. Currently, my country faces a significant supply-demand gap in soybean production, with a persistently high dependence on imports. This makes it a typical bottleneck industry in agriculture. The vast majority of imported soybeans are used for oil extraction, with soybean meal, a byproduct, serving as a core feed protein source supporting domestic livestock and aquaculture. Domestically produced soybeans, however, play a crucial role in supplying edible protein, and their high protein content is their core quality and market competitiveness. The molecular mechanisms underlying soybean protein traits are not fully understood, leading to insufficient exploration and utilization of high-quality, high-protein germplasm resources. Furthermore, protein content is greatly influenced by the environment, and compared to conventional soybeans, high-protein soybeans have longer breeding cycles and lower production efficiency.
[0003] Traditional methods for identifying soybean protein content typically employ near-infrared spectroscopy or chemical methods. However, these methods can only be performed after soybean maturity. While chemical methods are precise, they are time-consuming, costly, and cumbersome. Furthermore, the results are easily affected by factors such as planting location, climate, and cultivation management, making it difficult to quickly and accurately select soybean plants with high and stable protein content. Molecular marker-assisted breeding offers advantages such as rapid detection, accurate results, and independence from environmental and growth cycle limitations, making it a key technology in molecular breeding. dCAPS (derived enzyme digestion amplification polymorphic sequence) markers and KASP (competitive allele-specific polymerase chain reaction) markers offer advantages such as high site specificity, good result stability, simple operation, low detection cost, and high throughput. They have been widely used in the molecular identification of quality, yield, and resistance traits in various crops, providing a feasible technical pathway for efficient breeding of high-protein soybeans.
[0004] Currently, there is a lack of specific dCAPS and KASP markers for the high-protein trait in soybeans. Existing molecular markers suffer from drawbacks such as weak linkage to protein content traits and insufficient stability, thus failing to develop effective molecular identification tools for high-protein soybeans. This hinders the efficiency and progress of breeding new high-protein soybean varieties. Therefore, developing a molecular marker with strong stability, high specificity, high throughput, and high detection efficiency has significant theoretical guiding value and industrial upgrading implications for the efficient breeding of new high-protein soybean varieties. Summary of the Invention
[0005] To address the problems of weak linkage and insufficient stability of some existing molecular markers with protein content traits, this invention provides markers for detecting soybean protein content and their applications. The markers for detecting soybean protein content described in this invention are dCAPS and KASP markers.
[0006] The Indel variant site of the dCAPS-tagged base used in this invention for detecting soybean protein content is located at 3877327 on chromosome 15, i.e., Indel_3877327.
[0007] The primer sequence for the dCAPS marker used in this invention to detect soybean protein content is as follows:
[0008] Forward primer F: 5'-AGGTTCCAAATGTGGGGGGGGT-3', sequence as shown in SEQ ID NO:1.
[0009] The reverse primer R is 5'-ATCATCCACTTCCTCTGCGATCG-3', and its sequence is shown in SEQ ID NO:2;
[0010] The corresponding endonuclease is TaqI.
[0011] Furthermore, the method for detecting soybean protein content using dCAPS labeling is performed according to the following steps:
[0012] I. Extraction of soybean genomic DNA;
[0013] 2. Using soybean genomic DNA as a template, amplification is performed using the primers described in claim 2 to obtain PCR amplification products;
[0014] 3. The PCR amplification products are digested with the restriction endonuclease TaqI, and the digested products are detected by agarose gel electrophoresis. If the electrophoresis result shows a single band of 157bp, with the sequence shown in SEQ ID NO.3, the soybean to be identified is a low-protein soybean; if the electrophoresis result shows a single band of 179bp, with the sequence shown in SEQ ID NO.4, the soybean to be identified is a high-protein soybean.
[0015] Furthermore, the enzyme digestion conditions in step three are 65℃ water bath digestion for 1 hour.
[0016] Furthermore, the PCR reaction program in step three is as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s, annealing at 59℃ for 15 s, extension at 72℃ for 10 s, for a total of 30 cycles; final extension at 72℃ for 5 min, and storage at 4℃. This program can effectively achieve efficient amplification of the target fragment and reduce the generation of nonspecific bands.
[0017] The KASP marker used in this invention for detecting soybean protein content has an Indel mutation site located at 3877327 on chromosome 15.
[0018] The primer sequence for the KASP marker used in this invention to detect soybean protein content is as follows:
[0019] Upstream primer F1:
[0020] 5'-GAAGGTGACCAAGTTCATGCTGGGCCTGCTGAAGTCTTCAA-3', the sequence is shown in SEQ ID NO.5.
[0021] Upstream primer F2:
[0022] 5'-GAAGGTCGGAGTCAACGGATTGGGCCTGCTGAAGTCTTCCC-3', the sequence is shown in SEQ ID NO.6;
[0023] Downstream primer R: 5'-TCCTTGGCATCCTTCTCTTGAT-3', sequence as shown in SEQ ID NO.7.
[0024] Furthermore, upstream primers F1 and F2 are linked to different fluorescent reporter groups, including FAM, HEX, FITC, RED, TET, JOE, or R110.
[0025] Furthermore, the method for detecting soybean protein content using KASP markers is performed according to the following steps:
[0026] I. Extraction of soybean genomic DNA;
[0027] 2. Using soybean genomic DNA as a template, amplification is performed using the primers described in claim 7, and the amplification primers are used to amplify and read the fluorescence signal in a real-time PCR instrument.
[0028] Third, the signal was read in the PCR instrument to obtain a clear genotype cluster. The CC genotype is high-protein soybean, and the -- genotype is low-protein soybean, thus realizing the detection of soybean protein content.
[0029] Furthermore, in step one, the CTAB method was used to extract genomic DNA from the leaves of soybean samples.
[0030] Furthermore, in step two, the PCR reaction was performed using a Douglas Scientific Array Tape system; primers F1, F2, and R, each at 10 μM, were combined in a volume ratio of 1:1:2.5 to form a Primer Mix; the PCR system consisted of 50 ng of DNA, 5 μL of KASP PCR Mix Plus, 0.76 μL of Primer mxi, and ultrapure water to a final volume of 10 μL; the PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 61℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 10 cycles; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 28 cycles; data was read at 30℃ for 60 s.
[0031] The application of the dCAPS or KASP markers described in this invention in the breeding of high-protein crops.
[0032] The beneficial effects of this invention are as follows:
[0033] 1. Stable and reliable loci with high detection accuracy: The Indel variant loci used in this invention are significant and stable loci obtained through GWAS analysis of 1206 diverse soybean materials. They have a high phenotypic explanatory power and can stably distinguish between high-protein and low-protein genotypes under different genetic backgrounds. Compared with traditional phenotypic identification, they have good genetic stability and detection reliability, and are suitable for the detection needs of different soybean breeding materials.
[0034] 2. Highly efficient and rapid identification, suitable for early screening: The dCAPS marker of this invention has the advantages of simple operation, high precision, strong specificity and low cost; the KASP marker does not require enzyme digestion and electrophoresis, and can be quickly amplified and genotyped by fluorescence quantitative PCR; at the same time, the two markers can be detected in the early stage of soybean growth or even at the seed stage, eliminating the influence of environmental factors, growth stage and subjective judgment of operators, greatly shortening the detection cycle, improving identification efficiency, and solving the pain point of traditional high protein identification relying on mature plants and long cycle.
[0035] 3. High throughput and strong adaptability, reducing breeding costs: The high-throughput detection characteristics of the KASP marker of this invention are adapted to the real-time PCR platform, which can realize the simultaneous detection of batch soybean samples. Compared with traditional molecular marker detection, the detection efficiency is significantly improved. Compared with dCAPS marker, it is more suitable for large-scale soybean breeding screening, solving the shortcomings of traditional markers that are difficult to adapt to batch sample detection.
[0036] 4. Outstanding application value, assisting targeted breeding: This invention provides two high-protein molecular marker schemes, dCAPS and KASP, which are adapted to different scenarios. It can realize low-cost and high-precision identification of high-protein soybeans in a small amount of soybean material, and can also carry out high-throughput and high-efficiency batch detection and identification of high-protein soybeans in a large population. Molecular marker-assisted breeding is highly flexible and provides efficient and diverse technical tools for targeted breeding of high-protein soybeans.
[0037] This invention overcomes the shortcomings of existing technologies that rely on mature soybean phenotyping for identification of high-protein traits, which has a long cycle, is greatly affected by growth environment and cultivation conditions, and cannot achieve early screening. It provides a method and application for high-protein molecular markers and detection, enabling rapid, low-cost, and accurate detection of high-protein traits, and providing an efficient technical tool for targeted breeding of high-protein crops. Attached Figure Description
[0038] Figure 1 Manhattan and corresponding QQ plots are shown for GWAS results of protein content;
[0039] Figure 2 A schematic diagram of the genotyping electrophoresis results of dCAPS markers on different soybean germplasm resources;
[0040] Figure 3 Genotyping results for KASP molecular markers. Detailed Implementation
[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and 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.
[0042] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.
[0043] Example 1: Development and Verification of dCAPS Tags
[0044] 1. Experimental Materials: 1206 soybean accessions were selected, covering various genetic backgrounds. GWAS analysis of protein content was conducted based on previously collected protein content data and whole-genome sequencing data. Figure 1 The Manhattan and corresponding QQ plots are shown for the GWAS results of soybean protein content. The results indicate that a significant Indel marker, Indel_3877327, was identified at base 3877327 on soybean chromosome 15, with the polymorphic base InDel CC / --.
[0045] 2. dCAPS marker design: Targeting the characteristics of the significant site marker Indel_3877327, specific primer pairs were designed using PrimerPremier 5.0 software (forward primer F: 5'-AGGTTCCAAATGTGGGGGGGT-3', sequence as shown in SEQ ID NO:1; reverse primer R: 5'-ATCATCCACTTCCTCTGCGATCG-3', sequence as shown in SEQ ID NO:2). Through mismatch design, this site forms a recognition site for the restriction endonuclease TaqI at TCAA, while high-protein soybeans do not carry this restriction site, thus achieving accurate and efficient differentiation between high-protein and low-protein soybeans.
[0046] 3. Label verification: PCR amplification and TaqI restriction enzyme digestion experiments were performed on 100 soybean samples using the designed primers. Figure 2 This is a schematic diagram of the genotyping electrophoresis results of dCAPS markers for different soybean germplasm resources; in the figure: M is the DNA molecular weight standard (Marker); among the two control templates, 1 is low-protein soybean DNA and 2 is high-protein soybean DNA; the arrows in the gel indicate the high-protein soybean band type, which is a single band of 179bp, and the rest are low-protein soybean band types, which are single bands of 157bp.
[0047] Example 2: Validation method of dCAPS markers related to soybean protein content
[0048] 1. Experimental materials: 100 samples of unknown soybean material were selected as the samples to be identified.
[0049] 2. Genomic DNA extraction: Genomic DNA was extracted from the leaves of 100 soybean samples using the CTAB method. The specific steps are as follows:
[0050] (1) Take 0.1g of fresh soybean leaves, place them in a mortar, add liquid nitrogen and grind them into powder;
[0051] (2) Transfer the powder into a centrifuge tube, add 600 μL of CTAB extraction buffer preheated to 65°C, mix gently, and incubate in a 65°C water bath for 30 min, inverting and mixing once every 10 min during the process.
[0052] (3) Add an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1), gently invert and mix for 10 min, then centrifuge at 12000 r / min for 10 min;
[0053] (4) Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, gently invert to mix, and let stand at -20℃ for 30 min.
[0054] (5) Centrifuge at 12000r / min for 10min, discard the supernatant, wash the precipitate twice with 75% ethanol, air dry, add 50μL LTE buffer to dissolve the DNA, and store at 4℃ for later use.
[0055] 3. PCR Amplification: Using the extracted genomic DNA as a template, PCR amplification was performed using forward primer F and reverse primer R. PCR reaction system (20 μL): 10 μL 2×Magic Green Taq SuperMix, 0.8 μL forward primer (10 μmol / L), 0.8 μL reverse primer (10 μmol / L), 1 μL template DNA, 7.4 μL ddH2O. PCR reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 59℃ annealing for 15 s, 72℃ extension for 10 s, for a total of 30 cycles; final extension at 72℃ for 5 min, stored at 4℃.
[0056] 4. Enzyme digestion: Take 5 μL of PCR amplification product, add 1 μL of 10× enzyme digestion buffer, 0.3 μL of TaqI restriction enzyme (10 U / μL) and 3.7 μL of ddH2O, mix gently, and digest in a water bath at 65℃ for 1 h.
[0057] 5. Electrophoresis Detection: Prepare a 2.0% agarose gel. Mix the enzyme digestion product with the loading buffer and add it to the gel wells. Use 1×TAE buffer as the electrophoresis buffer, and perform electrophoresis at 100V for 5 min, followed by 80V for 45 min. After electrophoresis, place the gel in a gel imaging system to observe and photograph the results. Determine the soybean protein content based on the electrophoretic bands: a single 179-bp band (SEQ ID NO.4) indicates high-protein soybeans; a single 157-bp band (SEQ ID NO.3) indicates low-protein soybeans.
[0058] 6. Result Validation: The molecular marker identification results were compared with the soybean protein content detection results to verify the accuracy of the method. The results showed that the molecular marker identification results of 100 soybean samples were consistent with the soybean protein content detection results (Table 1: Information on 100 cultivated soybean materials used for genotyping, protein content, and genotyping results), with an accuracy rate of 86%, proving that the dCAPS marker of the present invention can be effectively used for the rapid identification of high-protein soybeans.
[0059] Table 1. Information on 100 cultivated soybean accessions used for genotyping, protein content, and genotyping results.
[0060]
[0061] Example 3: KASP markers associated with soybean protein content
[0062] 1. Obtaining KASP molecular markers
[0063] (1) Primer design: Based on the genetic variation information in Example 1, 48 core germplasm resources that had undergone protein phenotypic identification were selected as the main experimental materials. The approximately 100bp sequence before and after the key variation of Indel_3877327 was obtained from GWAS, and KASP primer markers were designed using an online primer design website (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The marker has three primers (specific primer: upstream primer F1: 5'-GAAGGTGACCAAGTTCATGCTGGGCCTGCTGAAGTCTTCAA-3', specific primer: upstream primer F2: 5'-GAAGGTCGGAGTCAACGGATTGGGCCTGCTGAAGTCTTCCC-3', and universal primer: downstream primer R: 5'-TCCTTGGCATCCTTCTCTTGAT-3'), with the 5' ends of the two specific primers connected to FAM and VIC fluorescent sequences, respectively. After the design was completed, a genome-wide copy number analysis was performed on the primer sequences to ensure that a single copy was obtained, and the primer was assigned the tag ID KASPpro327.
[0064] (2) Genotype detection: Molecular markers designed using the above-mentioned gene KASP reaction principle can be used to detect high and low protein content in soybean materials in a high-throughput manner. When the genotype obtained by KASPpro327 molecular marker detection is CC, it indicates that the soybean material has a high protein content (average protein content ≥44%), and when the detected genotype is --, it indicates that the soybean material has a low protein content (average protein content is about 41%).
[0065] 2. KSAP marker genotyping verification method
[0066] (1) DNA extraction. Genomic DNA was extracted from 48 soybean samples using the CTAB method.
[0067] (2) Validation of KASP markers. PCR reactions were performed using the Douglas Scientific Array Tape system. Primers were mixed with 10 μM Primer Mix (volume ratio 1:1:2.5) for F1, F2, and R. The PCR system consisted of 50 ng DNA, 5 μL KASP PCR Mix Plus, 0.76 μL Primer mxi, and ultrapure water to a final volume of 10 μL. PCR reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 61℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 10 cycles; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 28 cycles; data reading at 30℃ for 60 s.
[0068] (3) Genotyping. In the genotyping data, the genotypes of the samples were mainly divided into three clusters. Among them, the X cluster indicates that the sample contains the homozygous X allele at this KASP marker locus (…). The Y cluster indicates that the sample contains the homozygous Y allele at this KASP marker site (marked in red in the lower right corner of the genotype). The blank control is marked in black in the genotype.
[0069] Table 2. Information on 48 cultivated soybean materials used for genotyping, protein content, and genotyping results. The genotyping results for the 48 samples are as follows: Figure 3 As shown ( Figure 3 The genotyping results of the KASP molecular marker are shown in the figure. The marker genotyping is excellent, the fluorescence signals are compact, and all sites are single copies, with a detection efficiency of 100%. The genotyping results are relatively consistent with the phenotype, reaching 91%. Therefore, the genotyping quality of the KASPpro327 molecular marker is fully capable of accurately detecting high and low soybean protein content.
[0070] Table 2 Information on 48 cultivated soybean accessions used for genotyping, protein content, and genotyping results.
[0071]
Claims
1. A dCAPS marker for detecting soybean protein content, characterized in that... The Indel variant site of the dCAPS-tagged base is located at position 3877327 on chromosome 15, i.e., Indel_3877327.
2. A dCAPS-labeled primer for detecting soybean protein content, characterized in that, The primer sequence is as follows: Forward primer F: 5'-AGGTTCCAAATGTGGGGGGGGT-3', sequence as shown in SEQ ID NO:
1. The reverse primer R is 5'-ATCATCCACTTCCTCTGCGATCG-3', with the sequence shown in SEQ ID NO:
2.
3. The method for detecting soybean protein content using dCAPS labeling according to claim 1, characterized in that, The detection method is performed according to the following steps: I. Extraction of soybean genomic DNA; 2. Using soybean genomic DNA as a template, amplification is performed using the primers described in claim 2 to obtain PCR amplification products; 3. The PCR amplification products are digested with the restriction endonuclease TaqI, and the digested products are detected by agarose gel electrophoresis. If the electrophoresis result shows a single band of 157bp, with the sequence shown in SEQ ID NO.3, the soybean to be identified is a low-protein soybean; if the electrophoresis result shows a single band of 179bp, with the sequence shown in SEQ ID NO.4, the soybean to be identified is a high-protein soybean.
4. The method for detecting soybean protein content using dCAPS labeling according to claim 3, characterized in that, In step three, the enzyme digestion conditions are: 65℃ water bath for 1 hour.
5. The method for detecting soybean protein content using dCAPS labeling according to claim 3, characterized in that, The PCR reaction program in step three is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 59℃ annealing for 15 s, 72℃ extension for 10 s, for a total of 30 cycles; 72℃ final extension for 5 min, and storage at 4℃.
6. A KASP marker for detecting soybean protein content, characterized in that, The base Indel variant site of the KASP molecular marker for soybean protein content is located at 3877327 on chromosome 15.
7. A KASP-labeled primer for detecting soybean protein content, characterized in that, The primer sequence is as follows: Upstream primer F1: 5'-GAAGGTGACCAAGTTCATGCTGGGCCTGCTGAAGTCTTCAA-3', the sequence is shown in SEQ ID NO.
5. Upstream primer F2: 5'-GAAGGTCGGAGTCAACGGATTGGGCCTGCTGAAGTCTTCCC-3', the sequence is shown in SEQ ID NO.6; Downstream primer R: 5'-TCCTTGGCATCCTTCTCTTGAT-3', sequence as shown in SEQ ID NO.
7.
8. The primer according to claim 7, characterized in that... The upstream primers F1 and F2 are linked to different fluorescent reporter groups, including FAM, HEX, FITC, RED, TET, JOE, or R110.
9. The method for detecting soybean protein content using KASP labeling according to claim 7, characterized in that, The detection method is performed according to the following steps: I. Extraction of soybean genomic DNA; 2. Using soybean genomic DNA as a template, amplification is performed using the primers described in claim 7, and the amplification primers are used to amplify and read the fluorescence signal in a real-time PCR instrument. Third, the signal was read in the PCR instrument to obtain a clear genotype cluster. The CC genotype is high-protein soybean, and the -- genotype is low-protein soybean, thus realizing the detection of soybean protein content.
10. The application of the dCAPS marker of claim 1 or the KASP marker of claim 6 in high-protein crop breeding.