A molecular marker InDel05 related to soybean oil content, detection primers and their applications

Abstract

This invention discloses a molecular marker InDel05 related to soybean oil content, detection primers, and their applications. The molecular marker InDel05 is located at the physical location 41854660-41854682 on soybean chromosome 5, and its nucleotide sequence is shown in SEQ ID No. 1. Based on this, detection primers for the molecular marker InDel05 were designed. The length difference of the amplification products is detected by PCR amplification. Soybean materials without the inserted fragment in the PCR amplification product are high-oil-content soybeans, while those with the inserted fragment are low-oil-content soybeans, thus achieving the identification of soybean oil content phenotypes with an accuracy of up to 87.5%. The molecular marker InDel05 and detection primers provided by this invention can be used for rapid, accurate, and efficient screening of high-oil-content soybean germplasm, significantly improving the efficiency of marker-assisted selection breeding and accelerating the breeding process of high-oil-content soybean varieties.

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 InDel05 related to soybean oil content, detection primers, and their applications. Background Technology

[0002] Soybeans Glycine max Soybean is an important oilseed and protein crop worldwide, and its oil content directly affects its edible, industrial, and economic value. In soybean breeding, increasing oil content has always been a crucial goal for quality improvement. However, traditional oil content determination methods, such as near-infrared spectroscopy (NIRS) and Soxhlet extraction, while accurate, are typically time-consuming, expensive, and complex, and are insufficient for the rapid screening needs of large-scale populations.

[0003] In recent years, marker-assisted selection (MAS) has been widely used in crop breeding. By developing molecular markers closely linked to target traits, target genotypes can be rapidly identified in the early stages of breeding, thereby significantly improving breeding efficiency. In soybeans, some progress has been made in locating and screening for oil content-related traits using molecular markers such as single nucleotide polymorphisms (SNPs) and insertions / deletions (InDels). For example, Chinese patent applications CN117248061A, CN117230240A, and CN117965778A have disclosed InDel sites, molecular markers, and primers related to soybean seed oil content, respectively. Although their accuracy in identifying soybean seed oil content can reach up to 72.28%–79.22%, their detection accuracy still needs further improvement. At the same time, more molecular markers related to soybean oil content (InDel05) are needed for selection. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects and deficiencies in the prior art and to provide a molecular marker InDel05 related to soybean oil content.

[0005] A second objective of this invention is to provide a detection primer for the molecular marker InDel05.

[0006] A third objective of this invention is to provide the application of the molecular marker InDel05 and the detection primers.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] A molecular marker InDel05 related to soybean oil content, wherein the molecular marker InDel05 is a 23 bp insertion variation, which is not present in high-oil soybean materials and is present in low-oil soybean materials, and its nucleotide sequence is shown in SEQ ID No. 1.

[0009] This invention systematically analyzed genetic loci related to soybean oil content through QTL mapping analysis, detecting five QTL loci significantly associated with soybean oil content, including a QTL locus located on chromosome 5 (named...). q-Oil- 5 It exhibits a very strong genetic effect, further targeting q-Oil-5 Based on the relevant QTL intervals and resequencing data from both parents (Huachun 2 and Wayao soybean), InDel sites with a difference greater than 15 bp were screened. A significant genetic variation was detected at the physical location 41854660-41854682 (23 bp in length) on soybean chromosome 5 (Chr05). This InDel variation is a 23 bp insertion located at Chr05:41854660_41854682. This 23 bp InDel variation exists in different soybean materials, present in low-oil-content soybeans but absent in high-oil-content soybeans. Based on the upstream and downstream sequences of this site, specific primer pairs were designed, and the target fragment containing this InDel variation was obtained by PCR amplification. Compared with traditional oil determination methods that rely on near-infrared spectroscopy and chemical extraction, the detection process of the molecular marker InDel05 of this invention is simple, requiring only DNA extraction, PCR amplification and electrophoretic analysis. It is not affected by the plant growth stage or environmental conditions and is suitable for high-throughput detection of large-scale samples.

[0010] The present invention also provides a detection primer for the molecular marker InDel05, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID No.2: 5'-CCAGGCAATTGGACTTGGGA-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID No.3: 5'-GGCACCACTCATGTCTTGGA-3'.

[0011] The present invention also provides a kit containing the above-mentioned detection primers.

[0012] The present invention also provides the application of the above-mentioned molecular marker InDel05, detection primers or kits in the identification of soybean oil.

[0013] This invention also provides the application of the above-mentioned molecular marker InDel05, detection primers or kits in the cultivation of soybeans with high oil content.

[0014] The present invention also provides a method for identifying the content of soybean oil, the method comprising the following steps:

[0015] S1. Extract genomic DNA from the soybean material to be tested;

[0016] 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;

[0017] S3. The difference in PCR amplification product length was detected. When the amplification product length was 241 bp, the genotype of the soybean being tested was determined to be A; when the amplification product length was 264 bp, its genotype was determined to be B. Soybean materials corresponding to genotype A had a higher oil content than soybean materials corresponding to genotype B. Soybean materials with genotype A were high-oil soybean materials, and soybean materials with genotype B were low-oil soybean materials. In other words, soybean materials with a shorter 241 bp amplification fragment were high-oil soybean materials, and soybean materials with a longer 264 bp amplification fragment were low-oil soybean materials.

[0018] Further, the PCR amplification reaction system described in step S2 consists of 1 µL of DNA template, 2 µL each of forward and reverse primers, 20 µL of ddH2O, and 25 µL of PCR Taq Mix, for a total of 50 µL.

[0019] Further, the PCR amplification reaction program in step S2 is as follows: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 2 min, and storage at 4℃.

[0020] Furthermore, the difference in length of the PCR amplification products was detected using agarose gel electrophoresis.

[0021] Preferably, the mass concentration of the agarose gel used in the agarose gel electrophoresis is 5%.

[0022] Furthermore, the application involves using the molecular marker InDel05, the detection primers, or the kit to identify the genotypes of soybean parents, segregating populations, or breeding progeny, in order to rapidly screen high-oil-content soybean germplasm and accelerate the breeding of new varieties.

[0023] Furthermore, the application is applicable to soybean materials with different genetic backgrounds, including local varieties, cultivars, improved varieties, or transgenic / gene-edited materials.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention provides a molecular marker, InDel05, related to soybean oil content. InDel05 is a 23 bp insertion variant located at physical position 41854660-41854682 on soybean chromosome 5. High-oil-content materials do not contain this insertion fragment, while low-oil-content materials do. By detecting the difference in the length of the amplified products using corresponding detection primers via electrophoresis, the oil content type of the material can be determined: smaller amplified bands correspond to high-oil-content soybean materials, and larger amplified fragments correspond to low-oil-content soybean materials. This achieves the identification of soybean oil content phenotypes with an accuracy of up to 87.5%. The molecular marker InDel05 and detection primers provided by this invention can be used for rapid, accurate, and efficient screening of high-oil-content soybean germplasm, significantly improving the efficiency of marker-assisted selection breeding and accelerating the breeding process of high-oil-content soybean varieties. Attached Figure Description

[0026] Figure 1 ICIM method QTL mapping LOD plot for seed oil content of Huachun 2 × Wayao soybean RIL population.

[0027] Figure 2 Bar chart showing the difference in oil content between Huachun 2 and Wayao soybeans, and frequency distribution of seed oil content in the RIL population.

[0028] Figure 3 A schematic diagram of the electrophoresis results of Huachun No. 2 and Wayao soybeans labeled with InDel.

[0029] Figure 4 This is a schematic diagram of the electrophoresis results for materials numbered 1 to 48.

[0030] Figure 5 This is a schematic diagram of the electrophoresis results for materials numbered 49 to 96.

[0031] Figure 6 This is a schematic diagram of the electrophoresis results for materials numbered 97 to 144.

[0032] Figure 7 Box plots and frequency distribution diagrams showing the differences in oil content among 144 soybean resource materials. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] Example 1: Extraction of total oil from soybeans

[0036] Before oil extraction, the sample must be pulverized and dried to prevent moisture and ether from jointly dissolving sugars and causing measurement errors. This experiment used a Danish Forsøe-Søren Kibsen extractor (characterized by high precision, ease of operation, and high extraction efficiency). The specific measurement method is as follows:

[0037] 1. Instrument preheating and preparation

[0038] 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, select program #1 and set the temperature to 80℃.

[0039] 2. Sample preparation and weighing

[0040] Accurately weigh approximately 1.0000 g of sample (accurate to ±0.0002 g), take a filter paper tube, place a small amount of defatted cotton at the bottom, fill it with the weighed soybean powder, and then cover the top with a layer of defatted cotton to prevent the sample from leaking out.

[0041] 3. Filter paper cartridges are loaded into the extraction system.

[0042] Arrange the prepared filter paper tubes in order, and use the sleeve clamp to insert the 6 filter paper tubes into the extraction condenser tube one by one. Pull down the left operating lever to the lowest position to pick up the filter paper tubes. After the sleeve is firmly attracted by the magnet, push the left operating lever to the top and remove the sleeve clamp.

[0043] 4. Preparation and loading of extraction cups

[0044] Weigh the aluminum extraction cups to constant weight (denoted as M2) beforehand. Then add 80 mL of petroleum ether to each of the six extraction cups and place them on the six-position extraction cup rack in the corresponding order. Place the rack and extraction cups together into the main unit of the instrument, cover with the outer glass cover and press the rack tightly.

[0045] 5. Start the extraction process.

[0046] Adjust the control levers on both sides of the instrument to the lowest position, press the start button and the timer button, and the instrument will begin to execute the first step of the program (extraction stage), with a set time of 4 hours.

[0047] 6. Rinse Stage Operation

[0048] After 4 hours, the instrument will emit a beep. Push the left control lever to the middle position (to lift the extraction paper tube), press the timer button, and the instrument will enter the second step of the program (rinsing stage), with a set time of 2 hours.

[0049] 7. Petroleum ether recovery

[0050] Two hours later, the prompt sound will sound again. Push the left control lever to the top and press the timer button to start the instrument's third step (petroleum ether recovery). Open the recovery valve to allow the remaining petroleum ether to flow out along the pipeline and collect it in a waste liquid bucket or other container (the recovered petroleum ether can be reused). The program will automatically stop after about 10 minutes. Manually drain the remaining petroleum ether from the pipeline and close the valve.

[0051] 8. Weighing of extraction cups and maintenance of instruments

[0052] Open the instrument compartment door, remove the extraction cup and immediately place it in a desiccator to cool. Weigh the extraction cup and oil mixture using a 1 / 2 quartile electronic balance (record as M3). Clean and maintain the instrument according to the instruction manual after use.

[0053] 9. Formula for calculating oil content in a sample:

[0054] W(EE)=(M3-M2) / M1×100%;

[0055] In the formula:

[0056] W(EE): Oil content of the sample;

[0057] M1: Sample mass (g);

[0058] M2: Constant weight of empty aluminum immersion cup (g);

[0059] M3: Total constant weight (g) of aluminum extraction cup and oil after extraction.

[0060] To ensure the reliability of the test results, the oil content determination of each sample must be performed independently and repeatedly. During the operation, the weighing process must be strictly controlled for accuracy, and latex or nylon gloves must be worn throughout the process to avoid sample contamination.

[0061] Regarding safe operation, when using ether, open flame heating is strictly prohibited, and the operating environment must be well ventilated (e.g., by turning on the fume hood). During the extraction stage, the temperature must be closely monitored to prevent the risk of explosion due to overheating of the ether.

[0062] Example 2: QTL positioning of total oil content in soybeans

[0063] Based on Huachun No. 2 (high oil) and Wayao soybean (low oil) as parent varieties ( Figure 1A high-density genetic map was constructed using 196 RIL populations as the material. QTL localization was performed using the CIM composite interval mapping method in WinQTLCart 2.0 software. At the 5% significance level, a LOD threshold ≥ 3.0 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 5 was named... q-Oil-5 .

[0064] This embodiment systematically analyzed genetic loci related to soybean oil content using QTL mapping analysis. The results are shown in Table 1. In the tested population, a total of 5 QTL loci significantly associated with soybean oil content were detected, and the phenotypic data conformed to a normal distribution. Figure 1 The LOD values ​​of these QTL loci ranged from 3.76 to 41.65, and the phenotypic variation explained (PVE) ranged from 3.20% to 52.32%, indicating that different QTLs have significantly different genetic regulatory effects on the oil content phenotype. Of particular note is the QTL locus located on chromosome 5 (named...). q-Oil-5 It exhibits a very strong genetic effect: its LOD value is as high as 41.65 ( Figure 2 Furthermore, the phenotypic variation explained 52.32%, the highest among all detected QTL sites. This result suggests that... q-Oil-5 The locus may contain major genes that regulate soybean oil content, and their genetic contribution to oil accumulation is particularly prominent. It can be used as a key target region for candidate gene discovery and functional verification in the future.

[0065] Table 1. QTL Location Results of Seed Oil Content in RIL Population

[0066]

[0067] Example 3: Development and Application of Indel Tags

[0068] For in-depth analysis q-Oil-5 To investigate the molecular genetic basis of locus regulation of soybean oil content, this embodiment further integrates expression profile data, genotype information, homologous gene function annotations, and parental genome resequencing data. A strategy combining molecular biology experiments and comparative genomics analysis is employed to perform a detailed genetic analysis of this locus.

[0069] Analysis revealed a significant genetic variation in the genome sequence alignment of the parents (Huachun 2 and Wayao soybean) at the 41854660-41854682 region (23 bp in length) on soybean chromosome 5 (Chr05): compared to Huachun 2, Wayao soybean exhibited a 23 bp insertional structural variation in this region (its nucleotide sequence is shown in SEQ ID No. 1). Based on the conserved sequences flanking this 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.

[0070] Table 2 InDel molecular marker nucleotide sequences

[0071]

[0072] The above primers were used to identify the parent (Huachun 2 and Wayao soybean) by PCR and detected by 5% agarose gel electrophoresis. The PCR reaction system was as follows: DNA template 1 μL, forward primer 2 μL, reverse primer 1 μL, dd H2O 20 μL, TaqMix 25 μL, total 50 μL. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; 72℃ final extension for 2 min. After the reaction, the sample was stored at 4℃.

[0073] The results of agarose gel electrophoresis are shown below. Figure 3 As stated, when the length of the amplification product is 241 bp, the genotype of the soybean to be tested is determined to be A; when the length of the amplification product is 264 bp, its genotype is determined to be B (A band type is Huachun 2, B band type is Wayao soybean).

[0074] Using 144 soybean resource materials with known stable oil content and reliable measurement data as subjects, genotyping was performed on them using the above-mentioned molecular marker primers. The electrophoresis detection results are as follows: Figures 4-6 As shown in Table 3, the statistical results are as follows: There were 69 samples of material with band type A and 75 samples of material with band type B. Further analysis of the oil content phenotypic data revealed that the average oil content of material with band type A was 20.20%, while the average oil content of material with band type B was 19.48%. Statistical analysis showed a significant difference in oil content between the two groups (P<0.005), and the average oil content of material with band type A was significantly higher than that of material with band type B. Figure 7 The accuracy rate is as high as 87.5%.

[0075] Table 3 Indel marker typing results of 144 resource materials

[0076]

[0077] The above results indicate that A-band and B-band molecular markers can specifically correspond to soybean materials with high and low oil content, respectively. These molecular markers can effectively distinguish the differences in soybean oil content, providing key theoretical basis and technical support for molecular marker-assisted breeding of soybean oil content.

[0078] This invention has described in detail the technical solution for molecular markers of soybean oil content. For those skilled in the art, without departing from the core concept and scope of protection of this invention, and without conducting additional unnecessary experiments, this invention can be implemented in a wide range based on equivalent soybean material types, molecular marker genotyping conditions, and oil content determination parameters. Although specific embodiments are listed in this invention, it should be understood that the presented embodiments are only part and not all. Other embodiments derived from the technical principles of this invention, as long as they do not depart from the technical essence of this invention, are all within the scope of protection of this invention. For the optimization of molecular marker detection procedures, statistical methods for oil content phenotypic data, etc., not specifically described in this invention, conventional technical means known in the art can be used for implementation. The scope of protection of this invention is not limited to the specific forms disclosed above. Any modifications, equivalent substitutions, or improvements to the technical solutions of this invention, as long as they do not substantially deviate from the technical concept of this invention, should be included within the scope of protection defined by the appended claims and their equivalents.

Claims

1. The application of a primer pair containing the molecular marker InDel05, associated with the detection of soybean oil content, in the identification of soybean oil content, characterized in that, The molecular marker InDel05 is a 23 bp insertion variant, and the insertion sequence of the molecular marker InDel05 is shown in SEQ ID No.

1. High-oil soybean materials do not contain the molecular marker InDel05, while low-oil soybean materials do contain the molecular marker InDel05. The nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID No. 2, and the nucleotide sequence of the downstream primer of the primer pair is shown in SEQ ID No.

3.

2. The application of a kit for detecting the molecular marker InDel05 related to soybean oil content in the identification of soybean oil content, characterized in that, The molecular marker InDel05 is a 23 bp insertion variant, and the insertion sequence of the molecular marker InDel05 is shown in SEQ ID No. 1; high-oil soybean materials do not contain the molecular marker InDel05, while low-oil soybean materials do contain the molecular marker InDel05; the kit contains the primer pair described in claim 1.

3. The application of a primer pair for detecting the molecular marker InDel05 related to soybean oil content in screening soybean varieties with high oil content, characterized in that, The application involves using the primer pair to identify the genotypes of soybean parents, segregating populations, or breeding progeny, in order to rapidly screen high-oil-content soybean germplasm and accelerate the breeding of new varieties. When the length of the amplified product is 241 bp, the genotype of the soybean to be tested is determined to be A; when the length of the amplified product is 264 bp, its genotype is determined to be B. Among them, soybean materials with genotype A are high-oil-content soybean varieties, and soybean materials with genotype B are low-oil-content soybean varieties. The nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID No. 2, and the nucleotide sequence of the downstream primer of the primer pair is shown in SEQ ID No.

3.

4. A method for identifying the content of soybean oil, 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 a PCR amplification reaction using the primer pair described in claim 1; S3. Detect the difference in length of PCR amplification products. When the length of the amplification product is 241 bp, the genotype of the soybean to be tested is determined to be A; when the length of the amplification product is 264 bp, its genotype is determined to be B. Among them, soybean materials with genotype A are high-oil soybean materials, and soybean materials with genotype B are low-oil soybean materials.

5. The method for identifying soybean oil content according to claim 4, characterized in that, The PCR amplification reaction system described in step S2 consists of 1 µL of DNA template, 2 µL each of forward and reverse primers, 20 µL of ddH2O, and 25 µL of PCR Taq Mix, for a total of 50 µL.

6. The method for identifying soybean oil content according to claim 4, characterized in that, The PCR amplification reaction program in step S2 is as follows: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 2 min, and then stored at 4℃.

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