Molecular markers associated with soybean seed linolenic acid content and uses thereof

Abstract

The application discloses a molecular marker related to linolenic acid content of soybean kernels and application thereof, and belongs to the technical field of molecular marker assisted breeding. The nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1, and a T / G mutation exists at the 36th base of the sequence shown in SEQ ID NO. 1; the linolenic acid content of soybean kernels with the mutation base site G is significantly higher than that of soybean kernels with the mutation base site T. The molecular marker provided by the application can be used for rapidly identifying and screening soybean varieties with high linolenic acid content, and the result is accurate and reliable, and the operation is simple, so that the breeding process of new soybean varieties can be shortened, the work efficiency of breeding is improved, and a theoretical basis and technical support are provided for early identification and breeding of soybean with high linolenic acid content.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, and in particular to molecular markers related to the linolenic acid content in soybean seeds and their applications. Background Technology

[0002] Soybeans contain approximately 5%-10% alpha-linolenic acid (ALA), an essential polyunsaturated fatty acid that the human body cannot synthesize, making them a valuable source of nutritional and health benefits. With increasing awareness of healthy eating, the demand for soybeans rich in ALA continues to rise, exacerbating the supply-demand imbalance of high-ALA soybeans in China. Therefore, the rapid and effective breeding of high-ALA varieties is of paramount importance for increasing the ALA content of soybeans.

[0003] Studies have shown that soybean linolenic acid (LA) content is regulated by multiple genes and is easily influenced by the environment, making it a complex quantitative trait. Although many scholars have studied the overall fatty acid composition of soybean, reports on key genes controlling LA content are still limited, which significantly restricts the efficiency of molecular breeding for high-LA varieties. Genome-wide association studies (GWAS), as an effective gene mapping tool, can quickly and accurately identify genes controlling LA in soybean.

[0004] Traditional soybean breeding relies on single-plant selection from offspring, a method that is time-consuming, labor-intensive, susceptible to environmental interference, and inaccurate. Developing specific molecular markers to assist selection based on base differences in target genes is the optimal method for improving the selection efficiency of high-linolenic acid soybeans. Molecular markers offer advantages in crop breeding, including early selection, immunity to environmental influences, accuracy, speed, and efficiency, making them a valuable tool. Among these, Kompetitive Allele-Specific PCR (KASP) is a novel SNP genotyping method based on Amplification Refractory Mutation System (ARMS) and highly sensitive fluorescence detection. Its principle involves designing two forward primers and one universal reverse primer targeting allele SNP sites. Each forward primer has a specific sequence that can bind to different fluorescent markers. PCR amplification of DNA samples using forward primers with different fluorescent binding sequences and universal reverse primers can reveal allelic variations through different fluorescence signals (He CL, et al. SNP genotyping: the KASP assay. Methods Mol Biol, 2014, 1145, 75-86). While numerous QTLs and SNPs related to soybean linolenic acid content have been reported, patents for KASP markers targeting soybean linolenic acid content are scarce.

[0005] Therefore, developing KASP markers closely linked to soybean linolenic acid (LAA) content for early-stage (low-generation) selection can significantly reduce breeding workload and accelerate the breeding process, while also yielding substantial economic benefits. Thus, strengthening the mining and utilization of soybean LAA content genes and developing molecular markers for assisted breeding is crucial for breeding high-LAA soybeans. Summary of the Invention

[0006] The purpose of this invention is to provide molecular markers related to the linolenic acid (LA) content in soybean seeds and their applications, thereby addressing the problems existing in the prior art. This invention identifies an SNP locus, S11_614877, located on soybean chromosome 11 that controls LA content, and simultaneously develops the KASP molecular marker and KASP primer set. This enables rapid identification and screening of soybean varieties with high LA content, which is beneficial for shortening the breeding process of new soybean varieties and improving breeding efficiency.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a molecular marker related to the linolenic acid content of soybean seeds. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1. A T / G mutation exists at the 36th base of the sequence shown in SEQ ID NO.1. This mutation site is located at the 614,877th base of chromosome 11 of the soybean genome Glycine_max_Wm82.a2.v1. The linolenic acid content in soybean seeds with the G mutation site was significantly higher than that in soybean seeds with the T mutation site.

[0008] This invention also provides the application of the above-mentioned molecular markers as targets in any of the following: (1) Identification of the linolenic acid content in soybean seeds; (2) Screen soybean varieties with high linolenic acid content in their seeds; (3) Assist in the breeding of soybean varieties with high linolenic acid content in the grains.

[0009] The present invention also provides a KASP primer set for detecting the above-mentioned molecular markers, the KASP primer set comprising primers with nucleotide sequences as shown in SEQ ID NO.2-4.

[0010] This invention also provides the application of the above-described KASP primer set in any of the following: (1) Identification of the linolenic acid content in soybean seeds; (2) Screen soybean varieties with high linolenic acid content in their seeds; (3) Assist in the breeding of soybean varieties with high linolenic acid content in the grains; (4) Prepare products for identifying the linolenic acid content in soybean seeds; (5) Prepare products from soybean varieties with high linolenic acid content in the seeds; (6) Prepare products to assist in the breeding of soybean varieties with high linolenic acid content in the seeds.

[0011] Optionally, the product may include reagents, kits, or chips.

[0012] The present invention also provides a kit for detecting the above-mentioned molecular markers, the kit comprising the above-mentioned KASP primer set.

[0013] This invention also provides a method for identifying the linolenic acid content in soybean seeds, comprising the following steps: Using the genomic DNA of the soybean to be tested as a template, PCR amplification was performed using the above-mentioned KASP primer set or the above-mentioned kit, and the results were determined based on the fluorescence signal of the amplification product. When the amplification product carries a FAM fluorescence signal, the genotype of the soybean to be tested is TT; when the amplification product carries a HEX fluorescence signal, the genotype of the soybean to be tested is GG. The linolenic acid content in soybean seeds with genotype GG was significantly higher than that in soybean seeds with genotype TT.

[0014] Optionally, the PCR amplification reaction system is as follows: 2 μL template; 5 μL 2×KASP Master mix; 0.14 μL KASP Assay Mix; water to a final volume of 10 μL; the KASP Assay Mix is ​​prepared by mixing 10 µM primers shown in SEQ ID NO.2, 10 µM primers shown in SEQ ID NO.3, and 10 µM primers shown in SEQ ID NO.4 in a volume ratio of 2:2:5. The PCR amplification reaction program is as follows: activation at 94℃ for 15 min; denaturation at 94℃ for 20 sec, annealing at 61-55℃ for 60 sec, decreasing by 0.6℃ for each cycle, for 10 cycles; denaturation at 94℃ for 20 sec, annealing at 55℃ for 60 sec, for 26 cycles.

[0015] This invention also provides a method for assisting in the breeding of soybean varieties with high linolenic acid content in the seeds, comprising the following steps: Using the genomic DNA of the soybean to be tested as a template, PCR amplification was performed using the KASP primer set of claim 3 or the kit described in claim 6, and the results were determined based on the fluorescence signal of the amplification product. When the amplification product carries a FAM fluorescence signal, the genotype of the soybean to be tested is TT; when the amplification product carries a HEX fluorescence signal, the genotype of the soybean to be tested is GG; soybeans with the GG genotype are selected for breeding.

[0016] Optionally, the PCR amplification reaction system is as follows: 2 μL template; 5 μL 2×KASP Master mix; 0.14 μL KASP Assay Mix; water to a final volume of 10 μL; the KASP Assay Mix is ​​prepared by mixing 10 µM primers shown in SEQ ID NO.2, 10 µM primers shown in SEQ ID NO.3, and 10 µM primers shown in SEQ ID NO.4 in a volume ratio of 2:2:5. The PCR amplification reaction program is as follows: activation at 94℃ for 15 min; denaturation at 94℃ for 20 sec, annealing at 61-55℃ for 60 sec, decreasing by 0.6℃ for each cycle, for 10 cycles; denaturation at 94℃ for 20 sec, annealing at 55℃ for 60 sec, for 26 cycles.

[0017] The present invention discloses the following technical effects: This invention identifies an SNP locus, S11_614877, located on soybean chromosome 11 that controls soybean linolenic acid (LAA) content. Simultaneously, it develops a rapid and efficient KASP molecular marker and primer set for molecularly-assisted breeding of soybeans with high LAA content. The KASP molecular marker provided by this invention can directly and specifically distinguish and detect the G or T bases at SNP mutation sites, rapidly identifying and screening soybean varieties with high LAA content. The results are accurate and reliable, and the operation is simple, which helps to shorten the breeding process of new soybean varieties and improve breeding efficiency. Both in terms of detection throughput and accuracy, it is superior to traditional markers such as SSR, CAPS, and SCAR. This invention provides a theoretical basis and technical support for the early identification and breeding of soybeans with high LAA content. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The Manhattan and QQ-plots of the GWAS results for soybean linolenic acid are shown; where A and B represent the Manhattan and QQ-plots of the GWAS results for soybean linolenic acid in 2023 and 2024, respectively. The gray dashed line in the figure represents the significance threshold, -log(p-value)≥5.93. Figure 2 The results of genotyping of different soybean varieties using the KASP primers of Example 2 are shown. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0026] Example 1: Identification of SNP sites significantly associated with soybean linolenic acid content 1. 270 representative accessions were selected from 1084 soybean germplasm resources (deposited at the Institute of Economic Crops, Jiangsu Academy of Agricultural Sciences) (see Table 1, published in the literature "Zhang, W., Xu, W., Zhang, H. et al. Comparative selective signature analysis and high-resolution GWAS reveal a new candidate gene controlling seed weight in soybean. Theor Appl Genet (2021). https: / / doi.org / 10.1007 / s00122-021-03774-6"), including 10 wild species, 49 local species and 211 cultivated species, to form a microcore germplasm resource. 10× resequencing was performed, and after filtering, a high-density SNP molecular marker map covering the entire genome was obtained.

[0027] Table 1 Representative soybean varieties 2. The linolenic acid (LA) content in soybean seeds under two different environmental conditions (2023 and 2024) was determined using gas chromatography (GC). The dried laboratory sample was thoroughly mixed, pulverized, and all seeds were passed through a 0.25 mm sieve and placed in a sample vial. 0.3 g of the sample, accurate to 0.0001 g, was weighed and placed in a 10 mL test tube. 3 mL of fat extraction solution was added, shaken to mix, and allowed to stand for 1 h. 2 mL of 0.5 mol / L sodium methoxide was added for methyl esterification, and the mixture was allowed to stand for 2 h. 5 mL of distilled water was added, and 2 mL of the supernatant was transferred to a brown sample vial. Using a gas chromatograph, a combination of methyl esterification extraction and GC analysis was employed. The relative LA content (%) was calculated using the peak area normalization method, based on the ratio of the target fatty acid signal peak area to the total area of ​​all fatty acid signal peaks.

[0028] 3. Genomic DNA was extracted from 270 young soybean leaves using the CTAB method and whole-genome resequencing was performed.

[0029] 4. Genome-wide association analysis (GWAS) was performed on soybean linolenic acid phenotypic data using the GAPIT algorithm package in R software. GWAS analysis detected 5 SNP loci significantly associated with soybean linolenic acid content, all located on chromosome 11. Figure 1 The SNP site S11_614877, which is significantly associated with soybean linolenic acid, was detected in both environments. It is located at 614,877 bp on chromosome 11 of the soybean genome Glycine_max_Wm82.a2.v1.

[0030] The S11_614877 site exhibits a T / G polymorphism at the 36th base of the nucleotide sequence shown in SEQ ID NO.1, as follows: TAACATGTATTATATTATATTTATTTTACTCTATTKAAAAATATTGAAGAATTATTTACTTTTATATATAAATTTATGACTTTCCTAATAGGTTTTAGA, SEQ ID NO. 1; K represents T or G.

[0031] Example 2: Development of KASP-labeled specific primers A KASP molecular marker was developed for the SNP site S11_614877 identified in Example 1. Using the Primer-BLAST function of NCBI (https: / / www.ncbi.nlm.nih.gov / ), a KASP primer set was designed, including upstream primer F1 (SEQ ID NO.2), upstream primer F2 (SEQ ID NO.3), and downstream primer R (SEQ ID NO.4). F1 and F2 contain FAM and HEX fluorescent linker sequences (underlined), respectively, as shown below: F1: 5'- GAAGGTGACCAAGTTCATGCT TAACATGTATTATATTATATTTATTTTACTCTA TTT-3'; F2: 5'- GAAGGTCGGAGTCAACGGATT AACATGTATTATATTATATTTATTTTACTCTAT TG-3'; R: 5'-TCTAAAACCTATTAGCGAAAGTCAT-3'.

[0032] Example 3: Application of KASP primers in the breeding of high-linolenic acid soybean varieties Fifty soybean varieties were randomly selected from Table 1, and genomic DNA was extracted from the samples to be tested. Using the genomic DNA as a template, PCR amplification was performed using the KASP primers from Example 2 to obtain the PCR amplification products. PCR amplification was performed using an ABI 7500 real-time quantitative PCR instrument. After PCR, the instrument can perform genotyping based on the fluorescence signal.

[0033] The total volume of the PCR amplification system was 10 μL: 2 μL of soybean sample DNA template (25 ng / μL); 5 μL of 2×KASP Master mix; 0.14 μL of KASP Assay Mix; and water to a final volume of 10 μL. The KASP Assay Mix was prepared by mixing primers shown in SEQ ID NO.2 (10 µM), SEQ ID NO.3 (10 µM), and SEQ ID NO.4 (10 µM) in a volume ratio of 2:2:5.

[0034] The reaction conditions were: activation at 94℃ for 15 min; denaturation at 94℃ for 20 sec, annealing at 61-55℃ for 60 sec, with a decrease of 0.6℃ per cycle, for 10 cycles; and denaturation at 94℃ for 20 sec, annealing at 55℃ for 60 sec, for 26 cycles.

[0035] After the reaction is complete, the ABI 7500 real-time quantitative PCR instrument can directly read the fluorescence data of the PCR reaction products, and the results are as follows: Figure 2Among them, the blue dots near the Y-axis represent soybean varieties carrying the G allele and having the genotype GG (10 varieties); the red dots near the X-axis represent soybean varieties carrying the T allele and having the genotype TT (39 varieties); the green dots represent soybean varieties carrying both the G and T alleles and having the genotype TG (1 variety); and the dots near the origin of the XY axes represent the blank control (water). The average linolenic acid content of different genotype soybean varieties is shown in Table 2.

[0036] Figure 2 As shown in Table 2, the KASP primers can clearly distinguish between the two soybean genotypes. The linolenic acid content of soybeans with the GG genotype is significantly higher than that of soybeans with the TT genotype, which is consistent with the expected results.

[0037] Table 2. Results of average linolenic acid content determination in soybean varieties of different genotypes The above results indicate that the KASP primers of this invention can rapidly and accurately detect the linolenic acid content phenotype of soybeans and can be used for auxiliary selection in soybean quality breeding.

[0038] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A molecular marker associated with the linolenic acid content of soybean seeds, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

1. A T / G mutation exists at the 36th base of the sequence shown in SEQ ID NO.

1. This mutation site is located at the 614,877th base of chromosome 11 of the soybean genome Glycine_max_Wm82.a2.v1. The linolenic acid content in soybean seeds with the G mutation site was significantly higher than that in soybean seeds with the T mutation site.

2. The use of the molecular marker of claim 1 as a target in any of the following: (1) Identification of the linolenic acid content in soybean seeds; (2) Screen soybean varieties with high linolenic acid content in the grains; (3) Assist in the breeding of soybean varieties with high linolenic acid content in the grains.

3. A KASP primer set for detecting the molecular marker of claim 1, characterized in that, The KASP primer set includes primers with nucleotide sequences as shown in SEQ ID NO.2-4.

4. The use of the KASP primer set according to claim 3 in any of the following: (1) Identification of the linolenic acid content in soybean seeds; (2) Screen soybean varieties with high linolenic acid content in the grains; (3) Assist in the breeding of soybean varieties with high linolenic acid content in the grains; (4) Prepare products for identifying the linolenic acid content of soybean seeds; (5) Prepare products from soybean varieties with high linolenic acid content in the seeds; (6) Prepare products for the auxiliary breeding of soybean varieties with high linolenic acid content in the seeds.

5. The application according to claim 4, characterized in that, The products include reagents, reagent kits, or chips.

6. A kit for detecting the molecular marker of claim 1, characterized in that, The kit contains the KASP primer set as described in claim 3.

7. A method for identifying the linolenic acid content in soybean seeds, characterized in that, Includes the following steps: Using the genomic DNA of the soybean to be tested as a template, PCR amplification was performed using the KASP primer set of claim 3 or the kit described in claim 6, and the results were determined based on the fluorescence signal of the amplification product. When the amplification product carries a FAM fluorescence signal, the genotype of the soybean to be tested is TT; when the amplification product carries a HEX fluorescence signal, the genotype of the soybean to be tested is GG. The linolenic acid content in soybean seeds with genotype GG was significantly higher than that in soybean seeds with genotype TT.

8. The method according to claim 7, characterized in that, The PCR amplification reaction system was as follows: 2 μL template; 5 μL 2×KASP Master mix; 0.14 μL KASP Assay Mix; water to a final volume of 10 μL; the KASP Assay Mix was prepared by mixing primers shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 at a volume ratio of 2:2:

5. The PCR amplification reaction program is as follows: activation at 94℃ for 15 min; denaturation at 94℃ for 20 sec, annealing at 61-55℃ for 60 sec, decreasing by 0.6℃ for each cycle, for 10 cycles; denaturation at 94℃ for 20 sec, annealing at 55℃ for 60 sec, for 26 cycles.

9. A method for assisting in the breeding of soybean varieties with high linolenic acid content in the seeds, characterized in that, Includes the following steps: Using the genomic DNA of the soybean to be tested as a template, PCR amplification was performed using the KASP primer set of claim 3 or the kit described in claim 6, and the results were determined based on the fluorescence signal of the amplification product. When the amplification product carries a FAM fluorescence signal, the genotype of the soybean to be tested is TT; when the amplification product carries a HEX fluorescence signal, the genotype of the soybean to be tested is GG; soybeans with the GG genotype are selected for breeding.

10. The method according to claim 9, characterized in that, The PCR amplification reaction system was as follows: 2 μL template; 5 μL 2×KASP Master mix; 0.14 μL KASP Assay Mix; water to a final volume of 10 μL; the KASP Assay Mix was prepared by mixing primers shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 at a volume ratio of 2:2:

5. The PCR amplification reaction program is as follows: activation at 94℃ for 15 min; denaturation at 94℃ for 20 sec, annealing at 61-55℃ for 60 sec, decreasing by 0.6℃ for each cycle, for 10 cycles; denaturation at 94℃ for 20 sec, annealing at 55℃ for 60 sec, for 26 cycles.

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