Development and application of functional molecular markers of soybean lipoxygenase gene

Abstract

The present invention discloses the development of a functional molecular marker of soybean lipoxygenase gene and its application. The present invention relates to the field of molecular biotechnology, and specifically to the development of a functional molecular marker of soybean lipoxygenase gene and its application. The present invention detects the polymorphism or genotype of SNP in the soybean genome to be tested, and identifies or assists in identifying the soybean fishy odor trait according to the genotype. SNP-S0003 is the 1112th nucleotide of SEQ ID No.1 in the sequence list, which is A or T; SNP-S0037 is the 2955th nucleotide of SEQ ID No.2 in the sequence list, which is T or C; SNP-S0051 is the 4505th nucleotide of SEQ ID No.3 in the sequence list, which is A or C. The substance for detecting the SNP polymorphism and genotype can be combined with other substances to prepare a product for identifying soybeans without fishy odor.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and mainly relates to the development and application of functional molecular markers of soybean lipoxygenase genes. Background Art

[0002] Soybean (Glycine max [L.] Merr.) is a globally important crop for human dietary protein, vegetable oil, and animal feed. However, lipoxygenases (LOXs) present in mature soybean seeds can catalyze the oxidation of unsaturated fatty acids such as linoleic acid and linolenic acid to generate conjugated unsaturated fatty acid hydroperoxides and convert them into volatile compounds with an unpleasant bean aroma. The soy flavor of soybean seed products limits human consumption of soybeans. In the food industry, the bean aroma in soy products (oil, soy milk, tofu, etc.) is removed by heat treatment, microwave treatment, organic solvent extraction, etc., which increases the production and processing costs of soybeans. The breeding of lipoxygenase-free soybean varieties is a promising method to eliminate the fishy smell in soy products without incurring cost losses.

[0003] Studies have shown that lipoxygenase is the main factor causing beany smell, so detecting the type of lipoxygenase deficiency in soybean varieties is the fundamental way to solve the problem of beany smell. Mature seeds mainly contain three lipoxygenases, LOX1, LOX2 and LOX3, and the genes are Glyma.13g347600 (GmLox1), Glyma.13g347500 (GmLox2), and Glyma.15g026300 (GmLox3). There are many traditional methods for screening lipoxygenase deficiency, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), enzymology (IEF-PAGE) technology, colorimetric assay, etc., but they are relatively time-consuming and laborious, and the steps are also cumbersome. The low detection throughput makes it difficult to meet the needs of commercial breeding, and even breeders use manual tasting to identify the lack of odor.

[0004] To solve this problem, a safe, rapid, cost-effective and high-throughput detection technology is urgently needed to develop molecular markers for soybean lipoxygenase-deficient soybean lines to improve the detection of lipoxygenase-deficient soybean lines, so as to accelerate the breeding process of new soybean varieties, improve breeding efficiency, and solve problems such as the difficulty of phenotypic identification. Summary of the invention

[0005] The problem to be solved by the present invention is how to identify or assist in identifying the trait of soybean lipoxygenase deficiency.

[0006] In order to solve the above technical problems, the present invention provides a KASP molecular marker of soybean lipoxygenase gene and its application, and verifies the practicability of the marker, providing a reliable molecular marker for identifying or assisting in the cultivation of odorless soybean varieties.

[0007] The present invention first provides an application, which is the use of a substance for detecting the polymorphism or genotype of three, two or one of the three SNPs, SNP-S0003, SNP-S0037 and SNP-S0051, in identifying or assisting in identifying the soybean lipoxygenase deficiency trait or in preparing a product for identifying or assisting in identifying the soybean lipoxygenase deficiency trait.

[0008] The SNP-S0003 is a SNP in the soybean genome, which is the 1112th nucleotide of SEQ ID No. 1 in the sequence list, and is A or T;

[0009] The SNP-S0037 is a SNP in the soybean genome, which is the 2955th nucleotide of SEQ ID No. 2 in the sequence list, and is T or C;

[0010] The SNP-S0051 is a SNP in the soybean genome, which is the 4505th nucleotide of SEQ ID No. 3 in the sequence list, and is A or C.

[0011] In the above application, the application is the use of a substance for detecting the polymorphism or genotype of three, two or one of the three SNPs, SNP-S0003, SNP-S0037 and SNP-S0051, in soybean breeding or preparation of soybean breeding products; the SNP-S0003 is a SNP in the soybean genome, which is the 1112th nucleotide of SEQ ID No.1 in the sequence list, which is A or T; the SNP-S0037 is a SNP in the soybean genome, which is the 2955th nucleotide of SEQ ID No.2 in the sequence list, which is T or C; the SNP-S0051 is a SNP in the soybean genome, which is the 4505th nucleotide of SEQ ID No.3 in the sequence list, which is A or C.

[0012] The present invention also provides a product, which contains the above-mentioned substance for detecting the polymorphism or genotype of the three SNPs SNP-S0003, SNP-S0037, and SNP-S0051 in the soybean genome, and is any one of the following G1)-G3):

[0013] G1) A product for detecting single nucleotide polymorphisms or genotypes associated with soybean lipoxygenase deficiency traits;

[0014] G2) Products for identifying or assisting in identifying soybean lipoxygenase deficiency traits;

[0015] G3) Products for soybean breeding.

[0016] The invention also provides a method for identifying or assisting in identifying the soybean lipoxygenase deficiency trait.

[0017] The method for identifying or assisting in identifying the soybean lipoxygenase deficiency trait provided by the present invention comprises detecting the genotypes of three, two or one of the three SNPs SNP-S0003, SNP-S0037 and SNP-S0051 in the soybean to be tested, and identifying or assisting in identifying the soybean odor trait according to the genotype of the soybean to be tested:

[0018] The soybean with the genotype of SNP-S0003 being AA is or is a candidate for being a soybean lacking lipoxygenase LOX1, and the soybean with the genotypes of SNP-S0003 being TT and AT are both soybeans containing lipoxygenase LOX1; the genotype of SNP-S0003 being AA is a homozygous type in which the nucleotide at position 1112 of SEQ ID No.1 in the soybean genome is A, the genotype of SNP-S0003 being TT is a homozygous type in which the nucleotide at position 1112 of SEQ ID No.1 in the soybean genome is T, and the genotype of SNP-S0003 being AT is a heterozygous type in which the nucleotide at position 1112 of SEQ ID No.1 in the soybean genome is A and T respectively;

[0019] The soybean with the genotype of SNP-S0037 being TT is a soybean lacking lipoxygenase LOX2, and the soybean with the genotypes of SNP-S0037 being CC and TC are both soybeans containing lipoxygenase LOX2; the genotype of SNP-S0037 being TT is a homozygous type in which the nucleotide at position 2955 of SEQ ID No.2 in the soybean genome is T, the genotype of SNP-S0037 being CC is a homozygous type in which the nucleotide at position 2955 of SEQ ID No.2 in the soybean genome is C, and the genotype of SNP-S0037 being TC is a heterozygous type in which the nucleotide at position 2955 of SEQ ID No.2 in the soybean genome is T and C respectively;

[0020] The soybeans whose SNP-S0051 genotype is AA are soybeans lacking lipoxygenase LOX3, and the soybeans whose SNP-S0051 genotypes are CC and AC are both soybeans containing lipoxygenase LOX3; the SNP-S0051 genotype is AA, which is a homozygous type in which the 4505th nucleotide of SEQ ID No.3 in the soybean genome is A, the SNP-S0051 genotype is CC, which is a homozygous type in which the 4505th nucleotide of SEQ ID No.3 in the soybean genome is C, and the SNP-S0051 genotype is AC, which is a heterozygous type in which the 4505th nucleotide of SEQ ID No.3 in the soybean genome is A and C respectively.

[0021] The present invention also provides application of the method for identifying or assisting in identifying the soybean lipoxygenase deficiency trait in soybean breeding.

[0022] In the above application or method, the purpose of soybean breeding includes cultivating soybeans with lipoxygenase deficiency, that is, soybeans without fishy smell.

[0023] In the above application, product or method, the substance may be the following D1), D2) or D3):

[0024] D1) containing PCR primers for amplifying soybean genomic DNA fragments including SNP-S0003, SNP-S0037 and / or SNP-S0051;

[0025] D2) a PCR reagent containing the PCR primers described in D1);

[0026] D3) A kit containing the PCR primers described in D1) or the PCR reagents described in D2).

[0027] In the above application, product or method, the PCR primers contain SNP-S0003 primer set, SNP-S0037 primer set and / or SNP-S0051 primer set:

[0028] The SNP-S0003 primer set consists of SNP-S0003-A, SNP-S0003-B and SNP-S0003-C;

[0029] Primer SNP-S0003-A is a single-stranded DNA molecule whose nucleotide sequence is sequence 4 in the sequence list or a single-stranded DNA whose nucleotide sequence is the 22nd to 49th position of sequence 4 in the sequence list;

[0030] Primer SNP-S0003-B is a single-stranded DNA molecule whose nucleotide sequence is sequence 5 in the sequence list or a single-stranded DNA whose nucleotide sequence is the 22nd to 49th position of sequence 5 in the sequence list;

[0031] Primer SNP-S0003-C is a single-stranded DNA molecule whose nucleotide sequence is sequence 6 in the sequence list;

[0032] The SNP-S0037 primer set consists of two upstream primers SNP-S0037-A and SNP-S0037-B and one downstream primer SNP-S0037-C;

[0033] Primer SNP-S0037-A is a single-stranded DNA molecule whose nucleotide sequence is sequence 7 in the sequence list or a single-stranded DNA whose nucleotide sequence is the 22nd to 47th position of sequence 7 in the sequence list;

[0034] Primer SNP-S0037-B is a single-stranded DNA molecule whose nucleotide sequence is sequence 8 in the sequence list or a single-stranded DNA whose nucleotide sequence is the 22nd to 47th position of sequence 8 in the sequence list;

[0035] Primer SNP-S0037-C is a single-stranded DNA molecule whose nucleotide sequence is sequence 9 in the sequence list;

[0036] The SNP-S0051 primer set consists of two upstream primers SNP-S0051-A and SNP-S0051-B and one downstream primer SNP-S0051-C;

[0037] Primer SNP-S0051-A is a single-stranded DNA molecule whose nucleotide sequence is sequence 10 in the sequence list or a single-stranded DNA whose nucleotide sequence is the 22nd to 43rd position of sequence 10 in the sequence list;

[0038] Primer SNP-S0051-B is a single-stranded DNA molecule whose nucleotide sequence is sequence 11 in the sequence list or a single-stranded DNA whose nucleotide sequence is the 22nd to 43rd position of sequence 11 in the sequence list;

[0039] The primer SNP-S0051-C is a single-stranded DNA molecule whose nucleotide sequence is sequence 12 in the sequence list.

[0040] The present invention also provides a soybean breeding method, comprising detecting the genotypes of the SNP-S0003, SNP-S0037 and / or SNP-S0051 in the soybean genome, selecting soybeans whose genotype of SNP-S0003 is AA, whose genotype of SNP-S0037 is TT and / or whose genotype of SNP-S0051 is AA as parents for breeding, wherein the genotype of SNP-S0003 is AA, which is a homozygous type in which the nucleotide at position 1112 of SEQ ID No.1 in the soybean genome is A, the genotype of SNP-S0037 is TT, which is a homozygous type in which the nucleotide at position 2955 of SEQ ID No.2 in the soybean genome is T, and the genotype of SNP-S0051 is AA, which is a homozygous type in which the nucleotide at position 4505 of SEQ ID No.3 in the soybean genome is A.

[0041] The method includes crossing the recipient parent as the recurrent parent.

[0042] The present invention discloses functional molecular markers related to the loss of soybean lipoxygenase LOX1, LOX2 and LOX3 and their applications. The polymorphism or genotype of SNP in the soybean genome to be tested is detected, and the soybean fishy odor trait is identified or auxiliary identified according to the genotype, SNP-S0003 is the 1112th nucleotide of SEQ ID No.1 in the sequence list, which is A or T; SNP-S0037 is the 2955th nucleotide of SEQ ID No.2 in the sequence list, which is T or C; SNP-S0051 is the 4505th nucleotide of SEQ ID No.3 in the sequence list, which is A or C. The substance for detecting the SNP polymorphism and genotype can be combined with other substances to prepare a product for identifying soybeans without fishy odor. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The graph shows the genotyping results of mutants and wild types using five primer pairs: S0003, S0004, S0051, S0052 and S0037.

[0044] Figure 2 This is the SDS-PAGE image of soybean seed protein extract.

[0045] Figure 3 The figure shows the genotyping results of mutants and wild types using three primer pairs: S0003, S0037 and S0051.

[0046] Figure 4 This is a diagram showing the genotype results of backcrossing improved prospect testing materials for lipoxygenase deletion.

[0047] Figure 5 Improved background detection technology route for backcrossing of lipoxygenase deletion mutants.

[0048] Figure 6 The figure is the SDS-PAGE image of seed protein extracts of improved soybean lines. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0051] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0052] Example 1 Development of SNP Molecular Markers for Soybean Lipoxygenase Gene

[0053] Test Materials:

[0054] Parental materials: Mutant types: Suiwuxindou No. 2 (abbreviated as WX1), Suiwuxindou No. 3 (abbreviated as WX2), Dongfudou No. 1 (abbreviated as WX3).

[0055] Wild type: Beidou 40, Hefeng 50.

[0056] Population materials: The test materials were 2541 individual plants of the F4 generation in the field.

[0057] 1 Obtaining the full-length genes of LOX1 and LOX2

[0058] The full-length gene sequences of LOX1, LOX2 and LOX3 were found at NCBI.

[0059] https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=soybean+lipoxygenase-1

[0060] https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=soybean+lipoxygenase-2

[0061] https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=soybean+lipoxygenase-3

[0062] 2. Primer design

[0063] Sequencing primers were designed according to the sequences of LOX1, LOX2 and LOX3 for PCR amplification. The design software was Primer3. The Tm value of the primers was between 55 and 65°C. The primers were synthesized by Invitrogen.

[0064] 3. Tissue DNA Extraction

[0065] The SDS method was used to extract DNA from soybean seeds and leaves. The specific steps are as follows:

[0066] 1) Take the verified sample plate out of the -80℃ freezer, press the rubber cover with a plate press, and then oscillate at 1100 rpm for 90 seconds using a high-throughput tissue grinder;

[0067] 2) Centrifuge at 4000rpm at 4℃ for about 3min, add 300μL BGⅠ solution to each well using the automatic liquid dispensing system, and be careful to avoid contamination between wells when opening the gel cap. After covering the gel cap, oscillate at 1100rpm for 40s;

[0068] 3) Centrifuge at 4000 rpm at 4°C for about 2 minutes, add 150 μL of BGII solution to each well, cover with a rubber cap, and shake at 1100 rpm for 20 seconds (shake at 37°C for 15 minutes);

[0069] 4) Centrifuge at 4000 rpm and 4°C for 15 min, take 300 μL of supernatant from each well into a new 96-well plate, then add 200 μL of isopropanol to each well, cover with a plastic cap and shake by hand for 1 min;

[0070] 5) Centrifuge at 4000 rpm and 4°C for 15 min, discard the supernatant, add 600 μL 75% ethanol solution, cover with a plastic cap and shake by hand for 1 min;

[0071] 6) Centrifuge at 4000 rpm and 4°C for 15 min, discard the supernatant, and let stand to dry;

[0072] 7) After the 96-well plate is dried, add 80 μL of 65°C preheated Low TE to each well, cover with the rubber cover, mix on a vortexer for 15-20 minutes, and store in a 4°C refrigerator.

[0073] 4 High-fidelity PCR amplification

[0074] Use TransTaq DNA Polymerase High Fidelity from Quantachrome:

[0075] Table 1 PCR reaction system

[0076] Component Volume ddH2O 7μL 10 Buffer I / II 1μL primer 1μL dNTP 0.6μL TransTaq 0.1μL template 1μL Total 10.7μL

[0077] Table 2 PCR reaction conditions

[0078] Step 1 94℃ 5min Step 2 94℃ 30s Step 3 60℃ 30s Step 4 72℃ 1min Step 5 Step 2 to Step 4 for 35 cycles Step 6 72℃ 10min Step7 4℃ ∞

[0079] 5. Cloning of PCR products

[0080] The TransGen pEasy-T1 Cloning Kit was used.

[0081] Gently mix 1-4 μL PCR product (up to 4 μL) with 1 μL pEasy-T1 Cloning Vector and react at room temperature (22℃-37℃) for 5 minutes (for PCR fragments larger than 1 kb or PCR gel recovery products, incubate for 10-15 minutes). After the reaction, place on ice.

[0082] Take 50 μL of the freshly melted Trans-T1 competent cells, add 5 μL of the incubated PCR ligation product, flick to mix, and place on ice for 20-30 minutes;

[0083] The mixture was placed in a 42°C water bath for 30 seconds and then quickly placed on ice for 2 minutes.

[0084] Add 500 / 750ul LB liquid culture medium (without antibiotics) to the centrifuge tube and resume culture at 37°C, 200rpm for 1 hour. (Dry the plate 20 minutes in advance)

[0085] Centrifuge at 4,000 rpm for 5 minutes, discard 600 μL of supernatant, gently pipette to mix the precipitate, take 200 μL of bacterial solution and spread it on LB plate containing IPTG, X-gal and kanamycin, quickly spread it until it is dry, and culture at 37°C overnight (18:00-6:00 the next day).

[0086] Use a white pipette tip to pick up the white clones and put them into 20 μL of TE1.0 solution or 10 μL of sterile water (use a PCR plate and mark the number of the single clone picked on the plate), pipette evenly, boil at 100°C for 10 minutes in a PCR instrument, centrifuge at 3700 rpm for 10 minutes, and take 1 μL of the supernatant as a template for the PCR reaction.

[0087] The PCR product amplified with primer M13 (annealing temperature 55°C, extension time 10 min at 72°C, 30 cycles) was used to detect positive clones by electrophoresis, and the corresponding white clone bacteria were expanded and cultured with 5 ml of LB medium containing kanamycin at 37°C and 200 rpm overnight.

[0088] Take 2 mL of bacterial solution and send it to the company for sequencing (bidirectional sequencing), and add an appropriate amount of glycerol to the remaining bacterial solution and store it at -80℃.

[0089] 6 Sequencing, sequence assembly and gene structure prediction

[0090] Each primer of each mutant selected 12 clones for colony PCR. Based on the colony PCR results, 2-3 clones with the same size as the target fragment were selected and sent to Invitrogen for sequencing. Finally, a total of 133 clones were sequenced and bidirectionally tested. The sequencing results were spliced ​​and sorted using Cexpress software. The full-length genes were obtained as follows: L1 full length 5705bp, L2 full length 6035bp, L3 full length 5383bp.

[0091] That is, the mutants Suiwuxingdou No. 2 (WX1) and Suiwuxingdou No. 3 (WX2) LOX1 (SEQ ID No. 1 in the sequence list), the full-length LOX2 gene (SEQ ID No. 2 in the sequence list), and the full-length LOX3 gene (SEQ ID No. 3 in the sequence list) of the Dongfudou No. 1 were obtained.

[0092] The sequences of LOX1, LOX2, and LOX3 were compared with the Williams82 sequence, and the gene structure was predicted on www.softberry.com. The results are as follows: 35 variant sites were selected as development markers, and the variant sites were distributed in the promoter region, exon region, and intron. A total of 96 pairs of KASP primers were developed. After screening by variant sites, primer specificity, and amplification product size, 61 pairs of primers were selected, numbered S0001-S0061, including 33 pairs of LOX1, 15 pairs of LOX2, and 13 pairs of LOX3. Pre-fluorescent sequences FAM and HEX were added, respectively, and sent to Invitrogen to synthesize KASP primers. It was verified that 5 pairs of primers among the 61 pairs of primers had good typing results, including 2 pairs in LOX1, namely S0003 and S0004, 1 pair in LOX2, namely S0037, and 2 pairs in LOX3, namely S0051 and S0052. All 5 pairs of primers can be used for typing mutants and wild types in this experiment (see Figure 1 ). Finally, S0003, S0037, and S0051 markers were selected as specific markers. The S0003 molecular marker was named SNP-S0003, the S0037 molecular marker was named SNP-S0037, and the S0051 molecular marker was named SNP-S0051.

[0093] SNP-S0003 corresponds to position 43769559 on chromosome 13 of the reference sequence williams82 (version v2.1), and its nucleotide is A or T, corresponding to position 1112 of SEQ ID No.1 in the sequence list. The w in SEQ ID No.1 in the sequence list represents A or T;

[0094] SNP-S0037 corresponds to position 43763685 on chromosome 13 of the reference sequence williams82 (version v2.1), and its nucleotide is T or C, corresponding to position 2955 of SEQ ID No.2 in the sequence list. The y in SEQ ID No.2 in the sequence list represents T or C;

[0095] SNP-S0051 corresponds to position 2123903 on chromosome 15 of the reference sequence williams82 (version v2.1), and its nucleotide is A or C, corresponding to position 4505 of SEQ ID No. 3 in the sequence listing. m in SEQ ID No. 3 in the sequence listing represents A or C.

[0096] 7. Design of genome-specific KASP primers

[0097] The following three genome-specific KASP primers were designed: a primer set for identifying the polymorphism of the SNP-S0003 site (referred to as the SNP-S0003 primer set), a primer set for identifying the polymorphism of the SNP-S0037 site (referred to as the SNP-S0037 primer set), and a primer set for identifying the polymorphism of the SNP-S0051 site (referred to as the SNP-S0051 primer set).

[0098] The SNP-S0003 primer set consists of SNP-S0003-A, SNP-S0003-B, and SNP-S0003-C.

[0099] Primer SNP-S0003-A:

[0100] 5'-gaaggtcggagtcaacggattCGGTACGTACGTAACATAGTATTATTAT-3' (SEQ ID No. 4, the lowercase letters are the specific fluorescent tag sequence HEX);

[0101] Primer SNP-S0003-B:

[0102] 5'-gaaggtgaccaagttcatgctCGGTACGTACGTAACATAGTATTATTAA-3' (SEQ ID No. 5, the lowercase letter part is the specific fluorescent tag sequence FAM);

[0103] Primer SNP-S0003-C:

[0104] 5'-CCAAGAACGTATCCTTTCCAAC-3' (SEQ ID No. 6).

[0105] The primer set for identifying the polymorphism of the SNP-S0003 site is designed based on the sequence SEQ ID No.1.

[0106] The fragments in which the SNP-S0003 site is T in the single-stranded DNA molecular amplification sequence table SEQ ID No.1 shown in the above sequences SEQ ID No.4 and SEQ ID No.6 can read the fluorescence signal of the fluorescent group bound to the HEX sequence in the template using an enzyme reader or a fluorescence quantitative PCR instrument; the fragments in which the SNP-S0003 site is A in the single-stranded DNA molecular amplification sequence table SEQ ID No.1 shown in the above sequences SEQ ID No.5 and SEQ ID No.6 can read the fluorescence signal of the fluorescent group bound to the FAM sequence in the template using an enzyme reader or a fluorescence quantitative PCR instrument.

[0107] The SNP-S0037 primer set consists of SNP-S0037-A, SNP-S0037-B, and SNP-S0037-C.

[0108] Primer SNP-S0037-A:

[0109] 5'-gaaggtcggagtcaacggattCGTACCATATATTCACAAACAAATGC-3' (SEQ ID No. 7, the lowercase letters are the specific fluorescent tag sequence HEX);

[0110] Primer SNP-S0037-B:

[0111] 5'-gaaggtgaccaagttcatgctCGTACCATATATTCACAAACAAATGT-3' (SEQ ID No. 8, the lowercase letter part is the specific fluorescent tag sequence FAM);

[0112] Primer SNP-S0037-C:

[0113] 5'-CATCGGTCATCCATGCAG-3' (SEQ ID No. 9).

[0114] The primer set for identifying the polymorphism of the SNP-S0037 site is designed based on the sequence SEQ ID No.2.

[0115] The fragments in which the SNP-S0037 site is C in the single-stranded DNA molecular amplification sequence table SEQ ID No.2 shown in the above sequences SEQ ID No.7 and SEQ ID No.9 can read the fluorescence signal of the fluorescent group bound to the HEX sequence in the template using an enzyme reader or a fluorescence quantitative PCR instrument; the fragments in which the SNP-S0037 site is T in the single-stranded DNA molecular amplification sequence table SEQ ID No.2 shown in the above sequences SEQ ID No.8 and SEQ ID No.9 can read the fluorescence signal of the fluorescent group bound to the FAM sequence in the template using an enzyme reader or a fluorescence quantitative PCR instrument.

[0116] The SNP-S0051 primer set consists of SNP-S0051-A, SNP-S0051-B, and SNP-S0051-C.

[0117] Primer SNP-S0051-A:

[0118] 5'-gaaggtcggagtcaacggattGACCTTGACCAATAATTCCCCC-3' (SEQ ID No. 10, the lowercase letters are the specific fluorescent tag sequence HEX);

[0119] Primer SNP-S0051-B:

[0120] 5'-gaaggtgaccaagttcatgctGACCTTGACCAATAATTCCCCA-3' (SEQ ID No. 11, the lowercase letter part is the specific fluorescent tag sequence FAM);

[0121] Primer SNP-S0051-C:

[0122] 5'-GGTGTTGATGCGCAAGAATG-3' (SEQ ID No. 12).

[0123] The primer set for identifying the polymorphism of the SNP-S0051 site is designed based on the sequence SEQ ID No.3.

[0124] The fragments in which the SNP-S0051 site is C in the single-stranded DNA molecular amplification sequence table SEQ ID No.3 shown in the above sequences SEQ ID No.10 and SEQ ID No.12 can read the fluorescence signal of the fluorescent group bound to the HEX sequence in the template using an enzyme reader or a fluorescence quantitative PCR instrument; the fragments in which the SNP-S0051 site is A in the single-stranded DNA molecular amplification sequence table SEQ ID No.3 shown in the above sequences SEQ ID No.11 and SEQ ID No.12 can read the fluorescence signal of the fluorescent group bound to the FAM sequence in the template using an enzyme reader or a fluorescence quantitative PCR instrument.

[0125] 8 Establishment of molecular marker detection method

[0126] The mutant materials Suiwuxingdou No. 2 (WX1), Suiwuxingdou No. 3 (WX2), Dongfudou No. 1 (WX3), and wild-type materials Beidou 40 and Hefeng 50 were used for marker verification. The sample DNA concentration was homogenized, and the primers were mixed in proportion, and the SNP molecular markers were verified using the QuatStudio 12k Flex platform.

[0127] 1) Suiwuxingdou 2 (WX1), Suiwuxingdou 3 (WX2), Dongfudou 1 (WX3), Beidou 40, and Hefeng 50 were used as test varieties, and the genome-specific KASP primers in step 7 were used to establish a method for detecting soybean lipoxygenase deficiency. Genomic DNA was extracted from soybean seeds using the conventional SDS method. KASP reaction test was performed as follows:

[0128] 2) Prepare primer mixture:

[0129] Three primer mixtures were prepared, namely, SNP-S0003 primer mixture, SNP-S0037 primer mixture and SNP-S0051 primer mixture.

[0130] First, primers SNP-S0003A, SNP-S0003B, and SNP-S0003C were diluted with ddH2O to 100 mmol / L -1 , respectively obtain primer SNP-S0003A solution, primer SNP-S0003B solution and primer SNP-S0003C solution. Take 12 μL of primer A solution, 12 μL of primer B solution, 30 μL of primer C solution, add 46 μL of ddH2O, and obtain SNP-S0003 primer mixture.

[0131] First, primers SNP-S0037A, SNP-S0037B, and SNP-S0037C were diluted with ddH2O to 100 mmol / L -1 , respectively obtain primer SNP-S0037A solution, primer SNP-S0037B solution and primer SNP-S0037C solution. Take 12 μL of primer A solution, 12 μL of primer B solution, 30 μL of primer C solution, add 46 μL of ddH2O, and obtain SNP-S0037 primer mixture.

[0132] First, primers SNP-S0051A, SNP-S0051B, and SNP-S0051C were diluted with ddH2O to 100 mmol / L -1 , respectively obtain primer SNP-S0051A solution, primer SNP-S0051B solution and primer SNP-S0051C solution. Take 12 μL of primer A solution, 12 μL of primer B solution and 30 μL of primer C solution, add 46 μL of ddH2O to obtain SNP-S0051 primer mixture.

[0133] 4) Select LGC's QuatStudio 12k Flex platform for testing

[0134] 5μL PCR QuatStudio 12k Flex platform detection reaction system includes: 2.5μL of genomic DNA 50ng / μL, 0.072μL of primer mixture, and 2.5μL of LGC 2×KASP Mix (Std Rox). According to the QuatStudio 12kFlex platform instrument operation manual, write the sample table, run the program, and read the data.

[0135] Note: The above are recommended detection methods. Other detection methods that can achieve the same detection purpose can also be applied to the molecular marker-assisted breeding process of the above markers.

[0136] Amplification program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 20 s, annealing at 65°C-57°C for 60 s, 10 cycles, decreasing 0.8°C each cycle; denaturation at 94°C for 20 s, annealing at 57°C for 60 s, set 35 cycles.

[0137] In the experiment, no template DNA was added to the reaction system, and ddH2O with an equal volume to the DNA was added as a blank control (NTC). Two or more blank controls were set for each primer.

[0138] The scanned data is analyzed, and then the genotype of the SNP-S0003 site in the soybean genome to be tested is determined as follows: if the fluorescence signal data of the amplified product of the soybean to be tested is close to the X-axis (HEX signal) after analysis by the genotyping genotyping software, the genotype of the SNP-S0003 in the soybean genome to be tested is TT (i.e., the 1112th nucleotide of SEQ ID No.1 in the soybean genome is a homozygous type of TT); if the fluorescence signal data of the amplified product of the soybean to be tested is close to the Y-axis (FAM signal) after analysis by the genotyping software, the genotype of the SNP-S0003 in the soybean genome to be tested is AA (i.e., the 1112th nucleotide of SEQ ID No.1 in the soybean genome is a homozygous type of AA); if the fluorescence signal data of the amplified product of the soybean to be tested is located between the X-axis and the Y-axis (HEX and FAM signals) after analysis by the genotyping software, the genotype of the SNP-S0003 in the soybean genome to be tested is AT, i.e., the 1112th nucleotide of SEQ ID No.1 in the soybean genome is a homozygous type of AA. The 1112th nucleotide of No.1 is a heterozygous type of A and T). The black sample in the lower left corner is a blank control.

[0139] The scanned data is analyzed, and then the genotype of the SNP-S0037 site in the soybean genome to be tested is determined as follows: if the fluorescence signal data of the amplified product of the soybean to be tested is close to the X-axis (HEX signal) after analysis by the genotyping genotyping software, the genotype of the SNP-S0037 in the soybean genome to be tested is CC (i.e., the 2955th nucleotide of SEQ ID No. 2 in the soybean genome is a homozygous type of CC); if the fluorescence signal data of the amplified product of the soybean to be tested is close to the Y-axis (FAM signal) after analysis by the genotyping software, the genotype of the SNP-S0037 in the soybean genome to be tested is TT (i.e., the 2955th nucleotide of SEQ ID No. 2 in the soybean genome is a homozygous type of TT); if the fluorescence signal data of the amplified product of the soybean to be tested is located between the X-axis and the Y-axis (HEX and FAM signals) after analysis by the genotyping software, the genotype of the SNP-S0037 in the soybean genome to be tested is TC, i.e., the 2955th nucleotide of SEQ ID No. 2 in the soybean genome is a homozygous type of TT. The 2955th nucleotide of No.2 is a heterozygous type of T and C. The sample shown in black in the lower left corner is a blank control.

[0140] The scanned data is analyzed, and then the genotype of the SNP-S0051 site in the soybean genome to be tested is determined as follows: if the fluorescence signal data of the amplified product of the soybean to be tested is close to the X-axis (HEX signal) after analysis by the genotyping genotyping software, the genotype of the SNP-S0051 in the soybean genome to be tested is CC (i.e., the 4505th nucleotide of SEQ ID No.3 in the soybean genome is a homozygous type of CC); if the fluorescence signal data of the amplified product of the soybean to be tested is close to the Y-axis (FAM signal) after analysis by the genotyping software, the genotype of the SNP-S0051 in the soybean genome to be tested is AA (i.e., the 4505th nucleotide of SEQ ID No.3 in the soybean genome is a homozygous type of AA); if the fluorescence signal data of the amplified product of the soybean to be tested is located between the X-axis and the Y-axis (HEX and FAM signals) after analysis by the genotyping software, the genotype of the SNP-S0051 in the soybean genome to be tested is AC, i.e., the 4505th nucleotide of SEQ ID No.3 in the soybean genome is a homozygous type of AA. The 4505th nucleotide of No.3 is a heterozygous type of A and C. The sample shown in black in the lower left corner is a blank control.

[0141] 5) Marker typing data analysis

[0142] According to the method of step 4), the genotypes of soybean varieties Suiwuxingdou No. 2 (WX1), Suiwuxingdou No. 3 (WX2), Dongfudou No. 1 (WX3), Beidou 40 and Hefeng 50 were determined. The lipoxygenase deficiency of the soybean varieties to be tested was identified based on the genotype results.

[0143] If the genotype of SNP-S0003 of the soybean variety to be tested is AA, the soybean to be tested is a lipoxygenase LOX1 deficiency, i.e., a mutant type; if the genotype of SNP-S0003 of the soybean variety to be tested is TT or AT, the soybean variety to be tested contains a lipoxygenase LOX1 soybean variety, i.e., a wild type;

[0144] If the genotype of SNP-S0037 of the soybean variety to be tested is TT, the soybean to be tested is a soybean variety lacking lipoxygenase LOX2, i.e., a mutant type; if the genotype of SNP-S0037 of the soybean variety to be tested is CC or TC, the soybean to be tested is a soybean variety containing lipoxygenase LOX2, i.e., a wild type;

[0145] If the genotype of SNP-S0051 of the soybean variety to be tested is AA, the soybean to be tested is a soybean variety lacking lipoxygenase LOX3, that is, a mutant type. If the genotype of SNP-S0051 of the soybean variety to be tested is CC or AC, the soybean to be tested is a soybean variety containing lipoxygenase LOX3, that is, a wild type.

[0146] 9 Phenotypic Identification and Validation of Soybean Lipoxygenase

[0147] In order to verify whether the detection result of the molecular marker in step 3 is consistent with the soybean lipoxygenase deficiency phenotype, the traditional method for identifying the soybean lipoxygenase phenotype is used for verification. The specific method is as follows:

[0148] (1) Sample pretreatment: Take one seed for each sample, grind it into powder, take 8 mg and put it into a 2 mL centrifuge tube, add 1 mL of extraction buffer, stir evenly, extract by ultrasonic wave for 5-6 min, leave at room temperature for 20 min, stir again, centrifuge at 5000 r / min for 5 min, and take 10 ml of the supernatant for SDS-PAGE electrophoresis analysis.

[0149] (2) Prepare gel: Assemble glass plates and prepare separation gel and stacking gel. Add separation gel first, then add pure water until full, make the separation gel level, let it stand until solidified, pour out the pure water, absorb excess water, and then add stacking gel. Pour an appropriate amount of prepared electrophoresis buffer (SDS, Tris, glycine) into the vertical electrophoresis tank. Separation gel: 30% acrylamide, 1.0mol / L Tris-HCl (pH6.8), 10% SDS, distilled water, 10% ammonium persulfate (AP), TEMED. Stacking gel: 30% acrylamide, 1.5mol / LTris-HCl (pH8.8), 10% SDS, distilled water, 10% AP, TEMED.

[0150] (3) Electrophoresis conditions: voltage 100 V, electrophoresis time about 1 h. After the bromophenol blue indicator passes through the concentrated gel, increase the voltage to 150 V and electrophoresis time about 2 h. When the bromophenol blue indicator is about to pass through the bottom of the colloid, stop the electrophoresis and remove the colloid.

[0151] (4) Staining and destaining: Place the gel in staining solution and shake gently to stain for 1 hour. Take it out and place it in destaining solution and shake gently to destain for more than 4 hours. Under an ordinary fluorescent glass light box, identify whether lipoxygenase is missing.

[0152] The results are as follows Figure 2 As shown in the SDS-PAGE spectrum of soybean seed protein extract, line 1 is the lipoxygenase triple mutant Dongfudou No. 1, and there is no band in the lipoxygenase protein molecular weight region in the obtained protein electrophoresis spectrum. Line 2 is the wild-type Hefeng 50, with 3 bands corresponding to LOX3, LOX2 and LOX1 isozymes, and lines 3-9 are 7 F6 lines selected by sexual hybridization of Heinong 91×Dongfudou No. 1 by pedigree method, among which lines 3, 6, and 7 are wild-type banding types, which are normal materials, and lines 4, 5, 8, and 9 are triple mutant banding types, which are lipoxygenase-deficient materials. This result is consistent with the detection results of the self-developed SNP-S0003, SNP-S0037, and SNP-S0051 molecular markers.

[0153] Example 2 Application of three molecular markers SNP-S0003, SNP-S0037, SNP-S0051 in breeding for soybean lipoxygenase deficiency trait

[0154] The three SNP molecular markers SNP-S0003, SNP-S0037, and SNP-S0051 developed in Example 1 were used to perform molecular marker-assisted selection on soybean breeding intermediate materials.

[0155] The experimental materials were the following two groups: 2 lipoxygenase-deficient materials (mutants): Suiwuxindou No. 2 and Dongfudou No. 1; 2 normal materials (wild type): Beidou 40 and Hefeng 50, as control samples; the materials to be tested were 2541 F4 plants.

[0156] The materials to be tested are 2541 F4 individual plants, and the method of obtaining them is as follows:

[0157] The 2541 individual plants came from two combinations, namely Suiwuxindou 2×Hefeng 50, Beidou 40×Dongfudou 1, (Hefeng 50 was introduced from Jiamusi Branch of Heilongjiang Academy of Agricultural Sciences, Suiwuxindou 2 was introduced from Suihua Branch of Heilongjiang Academy of Agricultural Sciences, Beidou 40 was introduced from Hongxinglong Scientific Research Institute of Heilongjiang State Farm Bureau, and Dongfudou 1 was introduced from National Soybean Engineering Technology Research Center of Northeast Agricultural University). They were hybridized to obtain F1, and the pods were picked together in the F2 generation. One pod from each plant was taken for southern propagation. When the F3 was harvested from southern propagation, the one-seed transmission method was adopted. The F4 population was planted at Binxi Breeding Station of Beidahuang Kenfeng Seed Co., Ltd., with a total of 2541 individual plants in the two combinations.

[0158] 1 The total DNA of the control sample and the material to be tested was extracted using the SDS method in Example 1 above.

[0159] According to the method of step 8 of Example 1, PCR amplification was performed on the DNA of the sample to be tested using the genome-specific primer sets (SNP-S0003 primer set, SNP-S0037 primer set, SNP-S0051 primer set) of the three molecular markers SNP-S0003, SNP-S0037, and SNP-S0051 to obtain the polymorphism information of SNP-S0003, SNP-S0037, and SNP-S0051, thereby determining the genotype of the soybean sample to be tested, and the detection was performed using the QuatStudio 12k Flex 384-well module.

[0160] PCR amplification products were analyzed by genotyping.

[0161] The results showed that the PCR amplification product of soybean with SNP-S0003 genotype AA showed a blue fluorescent signal ( Figure 3 The blue fluorescence signal is consistent with the genotypes of Suiwuxindou 2 and Dongfudou 1, which are lipoxygenase LOX1 deficiency genotypes. In this experiment, there are 1015 soybeans with blue fluorescence signals; the PCR amplification product of soybeans with SNP-S0003 genotype TT has red fluorescence signals ( Figure 3 The red fluorescence signal is consistent with the genotypes of Beidou 40 and Hefeng 50, which is a homozygous wild genotype. There are 1029 soybeans with red fluorescence signals ( Figure 3 The PCR amplification product of soybean with SNP-S0003 genotype AT showed green fluorescence signal ( Figure 3 Circular data points are shown), green fluorescence signals are heterozygous genotypes, and there are 312 soybeans with green fluorescence signals in this experiment, and the next generation has separation of wild type and lipoxygenase deficiency mutant; the others are of uncertain genotype or missing samples.

[0162] The amplification product of soybean with SNP-S0037 genotype TT showed a blue fluorescent signal, which was consistent with the genotypes of Suiwushengdou No. 2 and Dongfudou No. 1, and was a lipoxygenase LOX2 deficiency genotype. In this experiment, there were 835 soybeans with blue fluorescent signals; the amplification product of soybean with SNP-S0037 genotype CC showed a red fluorescent signal, which was consistent with the genotypes of Beidou 40 and Hefeng 50, and was a wild genotype. There were 1120 soybeans with red fluorescent signals; the amplification product of soybean with SNP-S0037 genotype CT showed a green fluorescent signal, and the green fluorescent signal was a heterozygous genotype. In this experiment, there were 316 soybeans with green fluorescent signals, and the next generation had separation of wild type and lipoxygenase deficiency mutant; the others were of uncertain genotype or missing samples.

[0163] The amplification product of soybean with SNP-S0051 genotype AA showed a blue fluorescent signal, which was consistent with the genotype of Dongfu Bean No. 1 and was a lipoxygenase LOX3 deficiency genotype. A total of 1030 soybeans had blue fluorescent signals in this experiment; the amplification product of soybean with SNP-S0051 genotype CC showed a red fluorescent signal, which was consistent with the genotypes of Beidou 40 and Hefeng 50, and was a wild genotype. A total of 995 soybeans had red fluorescent signals; the amplification product of soybean with SNP-S0051 genotype CA showed a green fluorescent signal, which was a heterozygous genotype. A total of 386 soybeans had green fluorescent signals in this experiment, and the next generation had separation of wild type and lipoxygenase deficiency mutant; the others had uncertain genotypes or missing samples.

[0164] The phenotype of soybean lipoxygenase deficiency was detected using the traditional identification method in step 9 of Example 1. Some results are shown in Table 2.

[0165] Table 2. Phenotypic and genotypic information of 320 soybean lipoxygenase deficiency traits

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181] Note: The blank in genotype indicates an uncertain genotype, and the blank in lipoxygenase trait indicates a wild phenotype.

[0182] In breeding soybeans without fishy smell, it is best to select soybeans with SNP-S0003 genotype AA, SNP-S0037 genotype TT, SNP-S0051 genotype AA or soybeans containing two or three mutant genotypes as parents for breeding. The order of breeding material selection is: first select three genes mutated at the same time, followed by two genes mutated at the same time, and finally select materials with one gene mutation.

[0183] A total of 2541 samples were tested, with 3 markers and 7980 data points. A total of 310 strains with triple lipoxygenase deficiency were selected, 315 data points could not be determined, 220 abnormal points, and 357 data points were missing due to missing samples. This experiment successfully screened out lipoxygenase-deficient strains from a large number of intermediate breeding materials.

[0184] Example 3: Using three SNP molecular markers SNP-S0003, SNP-S0037, and SNP-S0051 to assist in selecting lipoxygenase-deficient materials and thereby improve the breeding of soybeans with no fishy smell traits

[0185] 1 Backcross Improved Prospect Detection

[0186] The combination was Heinong 91×Dongfu Bean No. 3, Kenbao No. 1×Dongfu Bean No. 3, Heihe 43×Dongfu Bean No. 5, and Hefeng 50×Dongfu Bean No. 5. DNA was extracted from the materials and parents for prospect selection using the SDS method, and the PCR amplification products were detected using the QuatStudio 12kFlex 384-well module genotyping analysis. Genotyping was determined based on the fluorescence signal (the specific steps were the same as in Example 2). The blue fluorescence signal was consistent with the genotypes of Dongfu Bean No. 3 and Dongfu Bean No. 5, which was a lipoxygenase-deficient genotype; the red fluorescence signal was consistent with the genotypes of Heinong 91, Kenbao No. 1, Heihe 43, and Hefeng 50, which was a wild type; the green fluorescence signal was a heterozygous genotype, and the next generation genotype was separated; the others were genotypes that were uncertain or samples were missing (see Figure 4 )

[0187] 2 Backcross Improved Background Detection

[0188] The technical route for backcrossing and background detection is shown in Figure 5 :

[0189] First, an initial hybridization is performed, that is, the soybean material lacking lipoxygenase is hybridized with the line to be improved to obtain the F1 generation.

[0190] The hybridization method is: female parent × male parent, specifically as follows: Heinong 91 × Dongfu Bean No. 3 (combination number 2019Z005), Kenbao No. 1 × Dongfu Bean No. 3 (combination number 2019Z006), Heihe 43 × Dongfu Bean No. 5 (combination number 2019Z008), Hefeng 50 × Dongfu Bean No. 5 (combination number 2019Z010).

[0191] The improved lines Heinong 91, Kenbao No. 1, Heinong 43 and Hefeng 50 were used as recurrent parents for backcrossing to obtain the BC1F1 generation. Three pairs of KASP markers were used for prospect selection, and the materials containing the target gene were subjected to 384 pairs of KASP background selection based on the whole genome (synthesized by LGC Company), as shown in Table 3. There were 4 combinations for background detection, and the combination numbers were 2019Z005, 2019Z006, 2019Z008 and 2019Z010, respectively. After the prospect detection, the number of single plants entering the background detection was 4, 5, 5 and 4, respectively. According to the number of polymorphic markers and the number of homozygous and heterozygous sites consistent with the recurrent parents, the highest background recovery rate was 2019Z006-2, reaching 79%, and the lowest background recovery rate was 2019Z008-4, which was 45.6%. Single plants with higher background recovery rates in the combination were preferentially selected for the next experiment.

[0192] Select the BC1F1 individual with the target locus, i.e., lipoxygenase-deficient genotype, and a high genetic background recovery rate, and backcross it with the recurrent parent to obtain BC2F1;

[0193] Plant BC2F1, select the foreground and background of BC2F1, select the BC2F1 plant with the target locus and a high genetic background recovery rate, and continue to backcross with the recurrent parent to obtain BC3F1;

[0194] Plant BC3F1, screen the foreground markers of the target site and select the background of BC3F1, obtain BC3F1 containing the site and with a high background recovery rate, and self-pollinate to obtain BC3F2;

[0195] Plant BC3F2, screen for promising markers of the lipoxygenase deficiency site, select BC3F2 homozygous for the site for continued planting, and then self-pollinate it to obtain a lipoxygenase-deficient strain, that is, an improved strain without fishy smell.

[0196] Table 3 Results of background recovery rate detection of lipoxygenase triple-deficient individual strains

[0197]

[0198] The soybean lipoxygenase deficiency phenotype was detected using the conventional identification method in step 9 of Example 1. Figure 6 As shown, line1-6 are the improved lipoxygenase LOX1, LOX2 and LOX3 triple-deficient mutant lines of BC1F1 individual plants 2019Z005-4, 2019Z006-2, 2019Z006-4, 2019Z008-3, 2019Z010-1 and 2019Z010-4, line7 is the triple-deficient mutant Dongfudou No. 1, and line8 is the wild-type Beidou 40. After identification, line1-6 are all lipoxygenase triple-deficient mutants, which is consistent with the results of molecular marker detection.

[0199] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of a substance for detecting the polymorphism or genotype of SNP-S0003 in identifying the soybean lipoxygenase deficiency trait or in preparing a product for identifying the soybean lipoxygenase deficiency trait, wherein SNP-S0003 is a SNP in the soybean genome, which is the 1112th nucleotide of SEQ ID No. 1 in the sequence list, and is A or T; The substance is a SNP-S0003 primer set, and the SNP-S0003 primer set consists of SNP-S0003-A, SNP-S0003-B, and SNP-S0003-C; The primer SNP-S0003-A is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No. 4 in the sequence list or a single-stranded DNA whose nucleotide sequence is the 22nd to 49th positions of SEQ ID No. 4 in the sequence list; The primer SNP-S0003-B is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No. 5 in the sequence list or a single-stranded DNA whose nucleotide sequence is the 22nd to 49th position of SEQ ID No. 5 in the sequence list; The primer SNP-S0003-C is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No. 6 in the sequence list.

2. Use of a substance for detecting the polymorphism or genotype of SNP-S0003 in soybean breeding or preparing soybean breeding products; the SNP-S0003 is a SNP in the soybean genome, which is the 1112th nucleotide of SEQ ID No.1 in the sequence list, which is A or T; The substance is a SNP-S0003 primer set, and the SNP-S0003 primer set consists of SNP-S0003-A, SNP-S0003-B, and SNP-S0003-C; The primer SNP-S0003-A is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No. 4 in the sequence list or a single-stranded DNA whose nucleotide sequence is the 22nd to 49th positions of SEQ ID No. 4 in the sequence list; The primer SNP-S0003-B is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No. 5 in the sequence list or a single-stranded DNA whose nucleotide sequence is the 22nd to 49th position of SEQ ID No. 5 in the sequence list; The primer SNP-S0003-C is a single-stranded DNA molecule whose nucleotide sequence is SEQ ID No.6 in the sequence list; The purpose of soybean breeding is to cultivate soybeans lacking lipoxygenase, that is, soybeans without fishy smell.

3. A method for identifying soybean lipoxygenase deficiency, the method comprising detecting the genotype of SNP-S0003 in the soybean to be tested, and identifying the soybean odor trait according to the genotype of the soybean to be tested: The soybean whose SNP-S0003 genotype is AA is or is a candidate for being soybean lacking lipoxygenase LOX1, and the soybean whose SNP-S0003 genotypes are TT and AT are both soybeans containing lipoxygenase LOX1; the SNP-S0003 genotype AA is a homozygous type in which the 1112th nucleotide of SEQ ID No.1 in the soybean genome is A, the SNP-S0003 genotype TT is a homozygous type in which the 1112th nucleotide of SEQ ID No.1 in the soybean genome is T, and the SNP-S0003 genotype AT is a heterozygous type in which the 1112th nucleotide of SEQ ID No.1 in the soybean genome is A and T respectively.

4. Use of the method according to claim 3 in soybean breeding, wherein the purpose of soybean breeding is to cultivate soybeans with lipoxygenase deficiency, i.e., soybeans without fishy smell.

5. A method for soybean breeding, comprising detecting the genotype of SNP-S0003 in the soybean genome, selecting soybeans with the genotype of SNP-S0003 being AA as parents for breeding, wherein the genotype of SNP-S0003 being AA is a homozygous type in which the nucleotide at position 1112 of SEQID No.1 in the soybean genome is A; the purpose of the soybean breeding is to cultivate soybeans with a lipoxygenase deficiency, i.e., soybeans without fishy smell.

6. The method according to claim 5, characterized in that: The method includes crossing the recipient parent as the recurrent parent.

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