Molecular marker InDel20 for identifying soybean oil content, primer and application thereof
By developing a 321 bp InDel20 molecular marker on soybean chromosome 20 and designing specific primers, the problems of complexity and inefficiency in soybean oil content determination in existing technologies have been solved, enabling rapid and accurate oil identification and efficient breeding.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTH CHINA AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-01-28
- Publication Date
- 2026-05-29
Smart Images
Figure CN121592807B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of plant molecular biology and crop genetics and breeding, and more specifically, to a molecular marker InDel20 for identifying soybean oil content, primers, and their applications. Background Technology
[0002] Soybeans Glycine max (Linn.) Merr.) is an important food and oilseed crop worldwide, and the oil content of its grains directly affects its edible quality, industrial utilization, and economic value. Oil content has always been one of the key targets in soybean quality improvement and breeding. However, currently used oil content determination methods, such as near-infrared spectroscopy (NIRS) and Soxhlet extraction, although capable of accurately assessing oil levels, are time-consuming, costly, and complex to operate, making them unsuitable for rapid screening of large-scale materials.
[0003] With the continuous development of marker-assisted selection (MAS) technology, crop breeding has gradually shifted from phenotypic screening to genotypic screening. By establishing molecular markers closely linked to target traits, precise identification of target genotypes can be achieved in the early stages of breeding, thereby significantly improving breeding efficiency. In soybean, molecular markers based on single nucleotide polymorphisms (SNPs) and insertion / deletion (InDel) variations have been used to locate and screen genes related to oil content. Using genome-wide association analysis (GWAS) and quantitative trait locus (QTL) mapping technology, researchers have identified a series of genetic regions closely related to oil content traits. For example, Chinese patent applications CN117248061A, CN117230240A, and CN117965778A have disclosed InDel sites, molecular markers, and primers related to soybean seed oil content, respectively. The accuracy of identifying soybean seed oil content can reach up to 72.28%–79.22%, but further improvements are still needed. At the same time, there is a need to provide more different InDel molecular markers for selection. Therefore, there is an urgent need to develop an InDel molecular marker with higher detection accuracy related to soybean oil content for rapid and accurate identification of soybean oil content. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of existing molecular markers related to soybean oil content, and to provide a molecular marker InDel20 for identifying soybean oil content.
[0005] A second objective of the present invention is to provide primers for detecting the molecular marker InDel20.
[0006] A third objective of this invention is to provide the application of the molecular marker InDel20 and its primers.
[0007] The above-mentioned objective of this invention is achieved through the following technical solution:
[0008] A molecular marker InDel20 for identifying soybean oil content, wherein the molecular marker InDel20 is a 321 bp insertion / deletion variant, and its nucleotide sequence is shown in SEQ ID No. 1; wherein soybean material containing the insertion fragment is high-oil-content soybean, and soybean material not containing the insertion fragment is low-oil-content soybean.
[0009] This invention, through comparative analysis of genomic resequencing data from high-oil and low-oil soybean materials, identified a specific insertion / deletion (InDel) 321 bp variant site within a genomic region significantly associated with grain oil content. This 321 bp insertion / deletion variant is located at... qOil-20 The relevant QTL region is specifically located at Chr20:31728602-31728922 on soybean chromosome 20. Based on the upstream and downstream sequences of this site, specific primer pairs were designed, and the target fragment containing this InDel variant was obtained by PCR amplification. Compared with traditional oil content determination methods relying on near-infrared spectroscopy and chemical extraction, the InDel20 molecular marker detection process of this invention is simple, requiring only DNA extraction, PCR amplification, and electrophoretic analysis. It is unaffected by plant growth stage or environmental conditions, making it suitable for high-throughput detection of large-scale samples.
[0010] The present invention also provides a primer for detecting the molecular marker InDel20, the primer comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer being as shown in SEQ ID No. 2: 5'-ACTGCATCAACCAAGCCTTATGC-3', and the nucleotide sequence of the downstream primer being as shown in SEQ ID No. 3: 5'-TGTACGTTTCTAACTCACTTAACTTATTGG-3'.
[0011] The present invention also provides the application of the above-mentioned molecular marker InDel20 or primers in the preparation of products for identifying soybean oil content.
[0012] The present invention also provides a product for identifying the content of soybean oil, the product containing the aforementioned primers.
[0013] Furthermore, the product is a reagent kit.
[0014] The present invention also provides the application of the above-mentioned molecular marker InDel20 or primers or products in the identification of soybean oil content.
[0015] The present invention also provides the application of the above-mentioned molecular marker InDel20 or primers or products in the cultivation of soybeans with high oil content.
[0016] The present invention also provides a method for identifying the soybean oil content, the method comprising the following steps:
[0017] S1. Extract genomic DNA from the soybean material to be tested;
[0018] S2. Using the extracted genomic DNA as a template, perform PCR amplification using the primers shown in SEQ ID No. 2 to SEQ ID No. 3 above;
[0019] S3. Detect the difference in PCR amplification product length. If the PCR amplification product length is 635 bp, the genotype of the soybean being tested is determined to be L; if the amplification product length is 956 bp, its genotype is determined to be H. The oil content of soybean material corresponding to genotype H is higher than that of soybean material corresponding to genotype L. Soybean material with genotype L is low-oil soybean material, and soybean material with genotype H is high-oil soybean material. That is, soybean material with a shorter 635 bp amplification fragment is low-oil soybean material, and soybean material with a 956 bp fragment is high-oil soybean material.
[0020] Further, the PCR amplification reaction system described in step S2 consists of 1 µL of DNA template, 1.5 µL each of forward and reverse primers, 21 µL of ddH2O, and 25 µL of PCR Taq Mix, for a total of 50 µL.
[0021] Further, the PCR amplification reaction program in step S2 is as follows: 94℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ final extension for 2 min, and the final product is stored at 4℃.
[0022] Furthermore, the difference in length of the PCR amplification products was detected using agarose gel electrophoresis.
[0023] Preferably, the mass concentration of the agarose gel used in the agarose gel electrophoresis is 3%.
[0024] Furthermore, the application in the breeding of soybeans with high oil content involves using the molecular marker InDel20 or primers to identify the genotypes of soybean hybrid parents, segregating populations, and / or germplasm resources. Based on the amplified banding or allele differences, high oil content genotype materials are efficiently identified and enriched, thereby achieving the directed aggregation of superior alleles related to the high oil content trait of soybeans. This significantly improves the selection efficiency of breeding new high oil content soybean varieties and shortens the breeding cycle.
[0025] Furthermore, the application is applicable to soybean materials with different genetic backgrounds, including local varieties, cultivars, improved varieties, and transgenic / gene-edited soybean materials.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] This invention provides a molecular marker, InDel20, for identifying soybean oil content. The InDel20 marker is a 321 bp insertion / deletion variant located on soybean chromosome 20 (Chr20:31728602-31728922). Soybean materials containing this insertion fragment are high-oil-content soybeans, while those without are low-oil-content soybeans. The oil content type of the material can be determined by detecting the difference in amplification product length using corresponding detection primers: smaller amplified bands correspond to low-oil-content soybeans, and larger amplified bands correspond to high-oil-content soybeans. The InDel20 marker provided by this invention has been validated in multiple soybean materials with known oil content. The amplified band pattern shows high consistency with the actual grain oil phenotype, with an identification accuracy of up to 90.61%, indicating that the marker has good stability and repeatability and can be used to distinguish between high-oil and low-oil materials. The molecular marker InDel20 of this invention can be used for early marker-assisted selection of high oil content traits in soybeans. By using this marker to pre-select genotypes of large population materials in the early stage of breeding, the screening efficiency of high oil content germplasm can be significantly improved, the aggregation of superior alleles can be accelerated, and the breeding cycle of new high oil soybean varieties can be shortened. It has broad prospects for promotion and application. Attached Figure Description
[0028] Figure 1 LOD plot for QTL mapping of seed oil content in a population of Guizao 1 × Brazil 13 recombinant inbred line using ICIM method.
[0029] Figure 2 Bar chart showing the difference in oil content between Guizao 1 and Brazil 13, and frequency distribution of seed oil content in recombinant inbred line populations.
[0030] Figure 3 This is a schematic diagram of the electrophoresis results of Guizao No. 1 and Brazilian 13 InDel labeled.
[0031] Figure 4 This is a schematic diagram of the electrophoresis results for materials numbered 1 to 47.
[0032] Figure 5 This is a schematic diagram of the electrophoresis results for materials numbered 48 to 95.
[0033] Figure 6 This is a schematic diagram of the electrophoresis results for materials numbered 96 to 143.
[0034] Figure 7 This is a schematic diagram of the electrophoresis results for materials numbered 144 to 181.
[0035] Figure 8 Scatter plots and frequency distribution statistics of differences in oil content in 181 soybean resources. Detailed Implementation
[0036] Unless otherwise specified, the experimental operations involved in the embodiments of this invention adopt standard procedures generally accepted in the art. For specific details, please refer to the technical methods and experimental conditions reported in relevant literature, or directly follow the product instructions of the reagents and instruments used. Unless otherwise specified, the experimental materials, chemical reagents and consumables used in each embodiment are all commercially available products that can be purchased through conventional commercial channels.
[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can modify or make equivalent substitutions to the technical solutions of this invention within the scope of the claims, and all such modifications or substitutions should be covered within the protection scope of this invention.
[0038] Example 1: Determination of soybean seed oil content by Soxhlet extraction
[0039] 1. Instrument preheating and preparation
[0040] Before starting the experiment, turn on the fume hood, start the low-humidity coolant circulation pump and connect the instrument power supply. Turn the key to the locked position to preheat the instrument, and set the temperature to 80℃.
[0041] 2. Sample processing
[0042] Mature, dry, and mold-free soybean seeds were crushed and filtered through a 60-mesh sieve to remove larger particles. Then, 1.0000 g of sample (accurate to 0.0001 g) was accurately weighed, placed in a pre-dried and constant-weighted filter paper tube, sealed, and kept for later use.
[0043] 3. Extraction Steps
[0044] Place the sample filter paper tube into the Soxhlet extractor and connect the condenser to the extraction bottle;
[0045] Add 100 mL of petroleum ether to the extraction flask;
[0046] The apparatus is used for continuous reflux extraction in a 65±2 ℃ water bath for 6–8 hours, allowing the solvent to continuously circulate and dissolve the lipids in the sample.
[0047] After extraction, the extraction flask was removed and the petroleum ether residue was evaporated at 105°C.
[0048] Place the extraction bottle along with the oil residue into a 105 ℃ drying oven and dry at a constant temperature for 1 hour. After cooling to room temperature, weigh the contents.
[0049] Repeat drying and weighing until the difference between two weighings does not exceed 1 mg.
[0050] Method for calculating the oil content of soybean seeds:
[0051] Oil content = (M2 - M1) / M0 × 100%;
[0052] in:
[0053] M0: Sample mass (g);
[0054] M1; constant weight of empty extraction bottle (g).
[0055] M2: Total weight (g) of the extraction flask and residue after extraction and drying.
[0056] Example 2: QTL positioning of total oil content in soybeans
[0057] Based on a recombinant inbred line population constructed using Guizao 1 (a low-oil-content soybean variety) and Brazil 13 (a high-oil-content soybean variety) as parents, a total of 248 families were constructed. The parental materials showed significant differences in oil content, and the oil content of the offspring conformed to a normal distribution (e.g., ...). Figure 1 A high-density genetic map was constructed using the above materials, and QTLs were identified using the ICIM composite interval mapping method in QTL ICiMapping 4.0 software. At the 5% significance level, a LOD threshold ≥ 5.0 was used as the basis for QTL existence. The QTL naming method was "q + trait + chromosome," for example, a QTL locus related to oil content detected on chromosome 20 was named... qOil-20 .
[0058] QTL mapping analysis was performed on the recombinant inbred line population to analyze genetic loci associated with soybean seed oil content. The results are shown in Table 1. Two QTL loci significantly associated with soybean oil content were detected in this recombinant inbred line population. One of these loci is located on chromosome 5. qOil-5 The highest LOD value is 15.19 (e.g., Figure 2 The phenotypic explanation rate was 17.54%, and major genes controlling soybean seed oil content have been identified in the candidate regions. GmMFT This demonstrates the accuracy of this population's localization of soybean seed oil content. The QTL locus located on chromosome 20 (named...) qOil-20 It also exhibits a very strong genetic effect: its LOD value is 13.15 (e.g., Figure 2 The phenotypic variation explained 14.96% of the variation. This result indicates that... qOil-20 The site may contain, except qOil-5 The novel major genes regulating soybean oil content outside the original region can be used as candidate regions for further gene screening and functional verification.
[0059] Table 1. QTL mapping results of grain oil content in recombinant inbred line population
[0060]
[0061] Example 3: Establishment and Validation of InDel Molecular Markers for Identifying High-Oil Traits in Soybeans
[0062] Resequencing was performed on two parental soybean materials, Guizao 1 and Brazil 13. Sequence alignment was performed within candidate genes or QTL segments related to oil content traits to screen for sites with stable insertion / deletion variations. The InDel site is located on a segment of soybean chromosome 20, where Brazil 13 material carries a 321 bp insertion (its nucleotide sequence is shown in SEQ ID No. 1), while Guizao 1 material does not have an insertion at this site. Based on the conserved sequences flanking the InDel site, paired specific primers were designed online using the NCBI website. The upstream primer sequence is shown in SEQ ID No. 2 in Table 2, and the downstream primer sequence is shown in SEQ ID No. 3 in Table 2.
[0063] Table 2. Nucleotide sequences of InDel molecular marker primers
[0064]
[0065] The parental materials Guizao 1 and Brazil 13 were identified by PCR using the above primers and detected by agarose gel electrophoresis. The PCR reaction system is shown in Table 3.
[0066] Table 3 Molecular marker PCR reaction system
[0067]
[0068] The reaction procedure was as follows: pre-denaturation at 94℃ for 3 min; denaturation at 98℃ for 10 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, for a total of 35 cycles; final extension at 72℃ for 2 min, and the final product was stored at 4℃. The amplified product was then separated by 3% agarose gel electrophoresis.
[0069] Agarose gel electrophoresis results are as follows Figure 3As shown, when the length of the PCR amplification product is 635 bp, the genotype of the soybean to be tested is determined to be L; when the length of the amplification product is 956 bp, its genotype is determined to be H (L banding is Guizao 1, H banding is Brazil 13).
[0070] 181 soybean materials with known stable oil content and reliable measurement data were selected as test materials, including 98 high-oil-content materials and 83 low-oil-content materials. PCR was performed using the aforementioned molecular marker primers according to the PCR reaction system and reaction conditions described in Table 3. The soybean materials were then classified as high- or low-oil-content by agarose gel electrophoresis. The electrophoresis results are shown below. Figures 4-7 As shown, the results indicated that 89 samples exhibited H-band patterns and 92 samples exhibited L-band patterns. After comparison with actual data, the accuracy rate of this molecular marker identification was calculated to be 90.61%. Based on existing oil content determination results and the correct molecular marker banding patterns, 85 soybean samples with high oil content (H-band pattern) were successfully screened. The soybean samples with different oil contents followed a normal distribution, demonstrating the randomness and reliability of sample selection (e.g., ...). Figure 8 The average seed oil content was 20.80%, while the average seed oil content of the 79 L-band type low-oil soybean materials was 19.16%, and the two were statistically significant (e.g., ...). Figure 8 The specific details are shown in Table 4.
[0071] Table 4. Indel molecular marker typing results of 181 resource materials
[0072]
[0073] As shown in Table 4, the two markers exhibit a high degree of consistency. Statistical analysis reveals that the InDel marker is accurate in identifying high-oil-content materials, indicating that it possesses good stability, repeatability, and application value, and can serve as a screening criterion for determining the oil content of soybean materials.
[0074] Those skilled in the art can make appropriate adjustments to the annealing temperature, number of cycles, and extension time according to different instruments and reagents to obtain amplification products with clear bands and good specificity.
[0075] This invention has provided a detailed description of the technical solution for molecular markers of soybean oil content with specific embodiments. However, those skilled in the art should understand that this invention is not limited to the above embodiments. Any modifications, substitutions, combinations, or optimizations made regarding soybean material type, molecular marker genotyping conditions, oil content determination methods, and data analysis procedures, as long as they do not deviate from the core concept and essential technical features of this invention, are all within the scope of protection of this invention. Steps or conditions not described in detail in this specification can be implemented by referring to conventional methods known in the art. All equivalent changes or improvements made to specific embodiments based on the technical concept of this invention should be included within the scope of protection defined by the appended claims and their equivalent technical solutions.
Claims
1. The application of a primer for the molecular marker InDel20 in distinguishing high / low oil content soybean materials or in the preparation of products that distinguish high / low oil content soybean materials, characterized in that, The primers include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.
3. The distinction is made by amplifying the genomic DNA of the soybean material to be tested using the above primers. If the length of the PCR amplification product is 635 bp, the genotype of the soybean material to be tested is determined to be L; if the length of the amplification product is 956 bp, its genotype is determined to be H. Among them, soybean material with genotype L is low-oil soybean material, and soybean material with genotype H is high-oil soybean material.
2. The application of a primer for the molecular marker InDel20 in the cultivation of soybeans with high oil content, characterized in that, The primers include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.
3. The application involves using the primers of the molecular marker InDel20 to identify the genotypes of soybean hybrid parents, segregating populations, and / or germplasm resources. High-oil-content genotype materials are identified and enriched based on the amplified band pattern or allelic genotype differences. If the length of the PCR amplification product is 635 bp, the genotype of the soybean to be tested is determined to be L; if the length of the amplification product is 956 bp, its genotype is determined to be H. Among them, soybean materials with genotype L are low-oil-content soybean materials, and soybean materials with genotype H are high-oil-content soybean materials.
3. A method for distinguishing high / low oil content soybean materials, characterized in that, Includes the following steps: S1. Extract genomic DNA from the soybean material to be tested; S2. Using the extracted genomic DNA as a template, perform PCR amplification using the primers described in SEQ ID No. 2 to SEQ ID No. 3; S3. Detect the difference in length of PCR amplification products. If the length of the PCR amplification product is 635 bp, the genotype of the soybean to be tested is determined to be L; if the length of the amplification product is 956 bp, the genotype is determined to be H. Among them, soybean materials with genotype L are low-oil soybean materials, and soybean materials with genotype H are high-oil soybean materials.
4. The method according to claim 3, characterized in that, The PCR amplification reaction system described in step S2 consists of 1 µL of DNA template, 1.5 µL each of forward and reverse primers, 21 µL of ddH2O, and 25 µL of PCR Taq Mix, for a total of 50 µL.
5. The method according to claim 3, characterized in that, The PCR amplification reaction program in step S2 is as follows: 94℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ final extension for 2 min, and the final product is stored at 4℃.