Soybean stalk GmST80-Indel molecular marker and application thereof

Abstract

The invention belongs to the technical field of molecular marker assisted breeding, and particularly relates to a soybean stem GmST80-Indel molecular marker and application thereof. The invention provides a GmST80-Indel molecular marker related to morphological characters of soybean stalks, the GmST80-Indel molecular marker is located in a promoter region of a GmST80 gene, and the nucleotide sequence of the GmST80-Indel molecular marker is as shown in SEQ ID NO: 1. Effectiveness of the molecular marker is verified in different genetic backgrounds, it is found that the molecular marker can accurately distinguish flat-stem soybeans from round-stem soybeans, then the molecular marker is used for screening soybean stem morphology and assisting soybean breeding, soybeans with the stem morphology character being flat stems can be rapidly bred, and then screening of varieties with the high single soybean plant yield is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker-assisted breeding, and particularly relates to a soybean stem GmST80-Indel molecular marker and application thereof. Background Art

[0002] Soybean (Glycine max (L.) Merr) originated in China and is an important oilseed and cash crop. Soybean seeds are high in protein and fat, making them a major source of human food, edible oil, and animal feed. Currently, soybean yields per unit area are relatively low, leaving ample room for improvement. Increasing soybean yields per unit area is a key focus for boosting overall soybean production.

[0003] Currently, soybean breeding is mostly carried out through hybridization. However, hybridization is time-consuming and labor-intensive, requiring the timely identification of desirable traits. Compared to traditional breeding, molecular marker-assisted breeding is more efficient, rapid, and precise. With the advancement of soybean genomics, proteomics, metabolomics, and phenotyping, a large number of genes associated with important agronomic traits have been identified. By developing these genetic loci into molecular markers and combining them with molecular marker-assisted breeding, the desired soybean varieties can be rapidly obtained.

[0004] Flat stem is a soybean stalk morphology mutant characterized by numerous leaves arranged in whorls on the main stem, clustered flower pods at the top, and a significant increase in pod number. Therefore, the flat stem trait is highly valuable for increasing soybean yields and improving plant architecture. Numerous researchers have conducted in-depth research on the biological characteristics, genetics, and physiological properties of flat stem soybeans. However, because the flat stem trait is controlled by a recessive gene and linked to many desirable agronomic traits, it is difficult to stabilize after multiple generations of continuous selection. Flat stem soybeans also suffer from weaknesses such as soft stems. The ability to stably inherit and detect the flat stem trait and improve its utilization directly impacts the scope and value of flat stem soybeans. Therefore, accurately, quickly, and simply using the soybean flat stem gene for molecular marker breeding is a pressing challenge in soybean breeding. Summary of the Invention

[0005] In order to solve the problem that the existing technology lacks the use of soybean flat stem gene for molecular marker breeding, the present invention provides soybean stem molecular marker GmST80 and its application, which specifically includes the following technical solutions:

[0006] The present invention provides a GmST80-Indel molecular marker related to soybean stalk morphological traits. The GmST80-Indel molecular marker is located in the promoter region of the GmST80 gene. The nucleotide sequence of the GmST80-Indel molecular marker is shown in SEQ ID NO: 1.

[0007] Preferably, the GmST80-Indel molecular marker is located at -2487 to -2541 bp in the promoter region of the soybean GmST80 gene.

[0008] Preferably, the GmST80 gene promoter region is shown in SEQ ID NO: 2

[0009] The present invention also provides a primer set for amplifying the GmST80-Indel molecular marker, comprising a forward primer having a nucleotide sequence as shown in SEQ ID NO: 4 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 5.

[0010] The present invention also provides a kit for detecting flat-stem soybean, comprising the primer set described above.

[0011] Preferably, the kit further comprises a Glycine max amplified fragment standard; the Glycine max amplified fragment standard is shown in SEQ ID NO: 1.

[0012] The present invention also provides the use of a substance or primer set or kit for detecting the GmST80-Indel molecular marker in detecting soybean stalk phenotypes.

[0013] Preferably, the soybean stem phenotype includes soybean flat stem and soybean round stem.

[0014] The present invention also provides a method for detecting soybean stalk morphological traits, comprising the following steps:

[0015] Extracting genomic DNA from the sample to be tested;

[0016] Using the genomic DNA as a template, performing PCR amplification on the genomic DNA using the primer set according to claim 3 to obtain a PCR amplification product; judging the soybean stalk morphology based on the size of the PCR amplification product;

[0017] When the size of the PCR amplification product is only 408 bp, the sample to be tested is round-stem soybean;

[0018] When the sizes of the PCR amplification products are 408 bp and 353 bp, the sample to be tested is round-stem soybean;

[0019] When the size of the PCR amplification product is only 353 bp, the sample to be tested is flat-stem soybean.

[0020] The present invention also provides application of a substance, primer set, kit or method for detecting the GmST80-Indel molecular marker in soybean breeding.

[0021] The beneficial effects of the present invention are:

[0022] The present invention provides a GmST80-Indel molecular marker associated with soybean stalk morphology, wherein the GmST80-Indel molecular marker is located in the promoter region of the GmST80 gene, and the nucleotide sequence of the GmST80-Indel molecular marker is shown in SEQ ID NO: 1. The GmST80-Indel molecular marker of the present invention can be used to quickly and accurately distinguish round-stem soybeans from flat-stem soybeans. The present invention verifies the effectiveness of the GmST80-Indel molecular marker in different genetic backgrounds. The GmST80-Indel molecular marker can accurately distinguish flat-stem soybeans from round-stem soybeans, and is then used for screening soybean stalk morphology, assisting soybean breeding, and can quickly select soybeans with flat-stem morphology, thereby facilitating the screening of soybean varieties with high single-plant yield.

[0023] The present invention designs PCR primers and kits based on the GmST80-Indel molecular marker. Through simple PCR amplification, soybean stalk morphological traits can be predicted and identified quickly, accurately, efficiently, and at low cost. The identification efficiency of soybean flat stems can reach up to 96.78%. Detection of substances detected by the GmST80-Indel molecular marker of the present invention can be used to predict soybean stalk morphology and distinguish between flat and round soybean stems. Furthermore, screening for the flat soybean stem trait can further select soybean varieties with high per-plant yield. The GmST80-Indel molecular marker of the present invention can assist in functional molecular breeding of soybeans, further shortening the breeding process and offering the advantages of high efficiency and convenience. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0025] Figure 1 is the mutation site of the GmST80 gene of the flat-stem soybean and the round-stem soybean in Example 1 of the present invention;

[0026] Figure 2 The difference sequence of the GmST80 gene between the flat-stem soybean and the round-stem soybean in Example 1 of the present invention;

[0027] Figure 3 This is the agarose gel electrophoresis diagram of primer In800F / R in Example 2 of the present invention. DETAILED DESCRIPTION

[0028] The present invention provides a GmST80-Indel molecular marker related to soybean stem morphology, wherein the GmST80-Indel molecular marker is located in the promoter region of the GmST80 gene, and the nucleotide sequence is shown in SEQ ID NO: 1. SEQ ID NO: 1: 5'-AAGTCCTAGTATAAGAGACTCCGGCTGCATAAATTATTTGAACTTGTCTTTAACT-3'.

[0029] In one embodiment, the GmST80-Indel molecular marker is located at -2487 to -2541 bp of the soybean GmST80 gene promoter region. In one embodiment, the GmST80-Indel molecular marker is located at -2487 to -2541 bp of SEQ ID NO: 2.

[0030] The reference genome for the GmST80-Indel molecular marker of the present invention is the GmST80 sequence of Glycine max Wm82.a4.v1. The sequence of the GmST80 gene is shown in SEQ ID NO:2.

[0031]

[0032]

[0033]

[0034] Among them, the bold part is the GmST80-Indel molecular marker.

[0035] The GmST80-Indel molecular marker of the present invention has a deletion polymorphism of the sequence shown in SEQ ID NO: 1 at 2541 bp upstream of the GmST80 gene promoter.

[0036] As an embodiment, the nucleotide sequence of the deletion polymorphism shown in SEQ ID NO: 1, which is present 2541 bp upstream of the promoter of the GmST80 gene, is shown in SEQ ID NO: 3.

[0037]

[0038]

[0039]

[0040] As an embodiment, the GmST80-Indel molecular marker of the present invention is a double-stranded DNA molecule in Glycine max, the nucleotide sequences of both chains being as shown in SEQ ID NO: 14;

[0041] The GmST80-Indel molecular marker in Glycine max is a double-stranded DNA molecule with a nucleotide sequence of both chains as shown in SEQ ID NO: 15, or a double-stranded DNA molecule with a nucleotide sequence of one chain as shown in SEQ ID NO: 14 and a nucleotide sequence of the other chain as shown in SEQ ID NO: 15.

[0042] The GmST80-Indel molecular marker disclosed in this invention is closely linked to the soybean flat stem trait and can be used to identify flat stem material and assist in soybean breeding. Molecular marker-assisted breeding is widely used in crop breeding, shortening the breeding process. This invention develops molecular markers for soybean stem morphology and performs genotyping. The sample genotyping results are significant, allowing accurate, efficient, and low-cost prediction of soybean stem morphology, thus advancing soybean breeding.

[0043] The present invention also provides primers for amplifying the GmST80-Indel molecular marker described above, comprising a forward primer as shown in SEQ ID NO: 4 and a reverse primer as shown in SEQ ID NO: 5.

[0044] SEQ ID NO:4(In800-F): 5'-GAACTTGTCTTTTTTTTATGTGGGA-3';

[0045] SEQ ID NO: 5 (In800-R): 5'-CGTGCTTAGTGAGGGTTTCTTTTTT-3'.

[0046] The present invention also provides a kit for detecting flat-stem soybean, comprising the forward primer and reverse primer as described above.

[0047] As an embodiment, the kit of the present invention includes a PCR kit. As an embodiment, the kit also includes a PCR amplification reagent. As an embodiment, the PCR amplification reagent includes: 2 μL of 50 ng / μL DNA, 10 μL of 2×Easytaq PCR SuperMix, 0.5 μL of primers In800-F (10 μM), 0.5 μL of In800-R (10 μM), and 7 μL of ddH2O, for a total of 20 μL. As an embodiment, the kit also includes a flat-stemmed soybean amplified fragment standard; the flat-stemmed soybean amplified fragment standard is shown in SEQ ID NO: 1.

[0048] The present invention also provides the use of the GmST80-Indel molecular marker or a substance, primer or kit for detecting the GmST80-Indel molecular marker in detecting soybean stalk phenotypes.

[0049] As an embodiment, the soybean stem phenotype includes soybean flat stem and soybean round stem.

[0050] The present invention also provides a method for detecting flat-stem soybean based on the GmST80-Indel molecular marker, comprising the following steps:

[0051] Extracting genomic DNA from the sample to be tested;

[0052] Using the genomic DNA as a template, performing PCR amplification on the genomic DNA using the forward primer and reverse primer set described above to obtain a PCR amplification product; judging the soybean stalk morphology based on the size of the PCR amplification product;

[0053] When the size of the PCR amplification product is only 408 bp, the sample to be tested is round-stem soybean;

[0054] When the sizes of the PCR amplification products are 408 bp and 353 bp, the sample to be tested is round-stem soybean;

[0055] When the size of the PCR amplification product is only 353 bp, the sample to be tested is flat-stem soybean.

[0056] In one embodiment, the PCR amplification system (20 μL) comprises 2 μL of 50 ng / μL DNA, 10 μL of 2×Easytaq PCR SuperMix, 0.5 μL of primers In800-F (10 μM), 0.5 μL of In800-R (10 μM), and 7 μL of ddH2O. In one embodiment, the amplification reaction procedure is as follows: pre-denaturation at 95°C for 3 minutes; 34 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds; extension at 72°C for 5 minutes; and 12°C for ∞. In one embodiment, the present invention can determine the size of the PCR amplification product by electrophoresis detection technology, gene sequencing technology, or real-time fluorescence quantitative PCR technology.

[0057] The present invention also provides the use of the above-mentioned molecular markers, primers, kits or methods in soybean breeding.

[0058] As an embodiment, the breeding includes soybean high-yield breeding. As an embodiment, the soybean high-yield breeding includes breeding to increase soybean yield per plant.

[0059] To further illustrate the present invention, the soybean stem GmST80-Indel molecular marker and its application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1 Screening of Soybean Stalk-Related Molecular Markers

[0061] (1) Mutation sites of the GmST80 gene between flat-stem soybean and round-stem soybean

[0062] In this example, flat-stem soybeans and round-stem soybeans produced by phenotypic separation during the breeding process of the flat-stem soybean variety with a soybean resource uniform number of "ZDD23811" were used as test samples, and four pairs of primers were designed based on the GmST80 sequence of the reference genome Glycine maxWm82.a4.v1. The primers are shown in Table 1.

[0063] Table 1 Four pairs of primer sequences designed based on the GmST80 sequence

[0064]

[0065]

[0066] PCR amplification was performed on the flat-stem soybean and round-stem soybean using the four primer pairs described in Table 1. The PCR amplification products were sequenced by Sanger sequencing. The gene structure of the GmST80 gene in the reference genome was mapped as follows: Figure 1 and Figure 2 As shown, it was found that the gene had no variation in the coding region, and there was an Indel variation in the promoter region of the GmST80 gene, 2541 bp upstream of ATG. The variation at -2541 bp was: compared with the round-stem soybean, the flat-stem soybean had a 55 bp deletion. Therefore, the Indel variation site was developed as a molecular marker.

[0067] (2) Development of primers for amplifying the GmST80 gene Indel molecular marker

[0068] Based on the sequence specificity of the Indel mutation site found in (1), a primer set was designed and used as the detection primer. The primer names and sequences are shown in Table 2.

[0069] Table 2 Molecular marker primer set information

[0070]

[0071] PCR amplification of soybean samples using the primer set in Table 2 can obtain products with sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15. The product shown in SEQ ID NO: 14 is 353 bp long, and the soybean stalk appears flat at this time. The product shown in SEQ ID NO: 15 is 408 bp long, and the soybean stalk appears round at this time. When the products shown in SEQ ID NO: 14 and SEQ ID NO: 15 are amplified simultaneously in soybeans, the soybean stalk appears round.

[0072]

[0073]

[0074] The sequence in bold is the sequence shown in SEQ ID NO.1.

[0075] Example 2 Molecular marker-assisted identification of the genotype of the flat stem segregating population

[0076] (1) Extraction of genomic DNA from tested soybeans

[0077] The flat stem segregant population produced by natural variation during the seed propagation process was used as the soybean to be tested. The leaves of the tested soybeans were placed in a 2 mL centrifuge tube, and the genomic DNA was extracted using the classic CTAB method. The specific operation method is as follows:

[0078] The preserved soybean leaves to be tested were taken out and placed in numbered 2 mL centrifuge tubes. Then, a steel ball was added to each centrifuge tube. The centrifuge tubes were placed in liquid nitrogen and sampled using a sampler for 30 seconds.

[0079] Add 800 μL of CTAB extraction solution (preheated at 65°C, 2% CTAB, add 2% β-mercaptoethanol before use) to the centrifuge tube, mix thoroughly, and place in a 65°C water bath for 45 minutes, turning it upside down every 10 minutes. After 45 minutes, add 5 μL of RNase and incubate in a 37°C water bath for 1 hour.

[0080] After the water bath, the centrifuge tube was taken out and 800 μL of a mixture of phenol and chloroform was added to the centrifuge tube (the volume ratio of phenol to chloroform in the mixture was 25:24), mixed thoroughly, and centrifuged at 4°C and 12,000 rpm for 10 min.

[0081] After centrifugation, the supernatant was transferred to a new set of 2 mL centrifuge tubes, 600 μL of chloroform was added to the centrifuge tubes, the tubes were mixed by inversion, and centrifuged again at 4°C, 12,000 rpm for 10 min.

[0082] After centrifugation, the supernatant was transferred to another set of new 2 mL centrifuge tubes. 500 μL of pre-cooled isopropanol was added to the centrifuge tubes, mixed well, and placed in a -20 °C refrigerator for 1 h to precipitate the DNA.

[0083] Centrifuge the tube at 4°C, 12,000 rpm for 10 minutes, and discard the supernatant. Rinse the pellet three times with 75% ethanol. Air-dry the DNA pellet at room temperature and dissolve it in an appropriate amount of ddH2O. Finally, test the DNA quality using 1% agarose gel and determine the extracted DNA concentration using NanodropOne. Dilute the extracted DNA to a concentration of 50 ng / μL to obtain the soybean genomic DNA to be tested. Store the DNA at -20°C until ready for use.

[0084] (2) Marker identification

[0085] The genomic DNA of the soybean to be tested obtained in step (1) is used as a template, and PCR amplification is performed on the genomic DNA using the In800-F / R primer set described in the invention.

[0086] The amplification system (20 μL) is:

[0087] 50ng / μL DNA 2μL, 2×Easytaq PCR SuperMix 10μL, primer In800-F (10μM) 0.5μL, In800-R (10μM) 0.5μL, ddH2O 7μL.

[0088] The amplification reaction procedure is:

[0089] Pre-denaturation at 95°C for 3 min; 34 cycles: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s; extension at 72°C for 5 min; 12°C, ∞.

[0090] The amplified products obtained by PCR amplification using the In800-F / R primer set were separated by 2% agarose electrophoresis and then detected and recorded using Bio-Rad gel imaging.

[0091] The marker band patterns corresponding to the PCR products amplified by the In800-F / R primer set are summarized as A, B, and H. Marker band pattern A corresponds to a product length of 408 bp, indicating a round stem phenotype; H corresponds to product lengths of 353 bp and 408 bp, indicating a round stem phenotype; and B corresponds to a product length of 353 bp, indicating a flat stem phenotype. The detection efficiency for flat stems was as high as 96.78%.

[0092] The flat stem segregation population consists of 311 individual plants. Using their genomic DNA as a template, the amplified product lengths are 353 bp for the In800-F / R flat stem soybean and 408 bp for the round stem soybean, or both. Based on the different band types, the stem morphology of the isolated individual plants was determined. The agarose test results for some soybeans are randomly displayed, as shown in the figure below. Figure 3 shown.

[0093] The band types of 311 samples of the flat stem segregation population were counted, and it was found that there were 61 samples of band type A, 28 samples of band type H, and 222 samples of band type B in the flat stem segregation population.

[0094] Example 3 Accuracy detection of molecular markers of flat stem segregating population

[0095] To verify the accuracy of molecular markers, the plant stem morphology of 311 soybeans was investigated in the field, of which 212 had flat stems and 99 had round stems. The marker band type matched the phenotype of 301 individual plants, and the accuracy of molecular marker detection was as high as 96.78%, indicating that the molecular markers in the present invention can accurately predict the stem morphology of soybeans, thereby facilitating the screening of soybean varieties with high single-plant yield.

[0096] Table 3 Statistics of marker banding patterns of isolated populations

[0097] Marking tape type flat stem round stem total A 0 61 61 H 0 28 28 B 212 10 222 total 212 99 311

[0098] Example 4 Identification of genotypes in variety resources using the GmST80-Indel marker

[0099] In order to verify the accuracy of molecular markers and identify the genotypes of soybean resource varieties, this example uses 24 variety resources. The unified resource numbers or variety names of the 24 soybeans are: ZDD23893, ZDD23811, ZDD23806, ZDD23808, ZDD22761, ZDD24636, ZDD23809, ZDD22975, 24HDS242, 24HDS107, ZDD30818, ZDD30812, ZDD23829, ZDD22761, ZDD30816, ZDD31328, ZDD24329, ZDD03257, ZDD00326, ZDD24685, ZDD23876, Zhongyin 1106, Jack and Williams82. Using the genomic DNA of the above 24 soybean varieties as templates, the amplification system and procedure were the same as in Example 2. The final amplified products were genotyped into A and B, with 14 genotypes A and 10 genotypes B. The stem morphology of different varieties was determined based on different genotypes, as shown in Table 4.

[0100] Table 4 Genotype statistics of different varieties

[0101] Marking tape type flat stem round stem total A 0 14 14 B 10 0 10 total 10 14 24

[0102] In summary, the GmST80-Indel molecular marker provided by the present invention can quickly and accurately distinguish round-stem soybeans from flat-stem soybeans, and is used for screening soybean stem morphology. It can quickly select soybeans with flat stem morphology, which is conducive to screening soybean varieties with high single-plant yield.

[0103] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments like this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A GmST80-Indel molecular marker related to soybean stem morphology, characterized in that: The GmST80-Indel molecular marker is located in the promoter region of the GmST80 gene, and the nucleotide sequence of the GmST80-Indel molecular marker is shown in SEQ ID NO:

1.

2. The GmST80-Indel molecular marker according to claim 1, characterized in that The GmST80-Indel molecular marker is located at -2487 to -2541 bp in the soybean GmST80 gene promoter region.

3. The GmST80-Indel molecular marker according to claim 1 or 2, characterized in that The GmST80 gene promoter region is shown in SEQ ID NO:

2.

4. A primer set for amplifying the GmST80-Indel molecular marker according to any one of claims 1 to 3, characterized in that: It includes a forward primer with a nucleotide sequence as shown in SEQ ID NO: 4 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

5.

5. A kit for detecting flat-stem soybeans, characterized in that: The method comprises the primer set according to claim 4.

6. The kit according to claim 5, wherein The kit further comprises a standard product of amplified fragments of Glycine max; the standard product of amplified fragments of Glycine max is shown in SEQ ID NO:

1.

7. Use of the substance for detecting the GmST80-Indel molecular marker according to any one of claims 1 to 3, the primer set according to claim 4, or the kit according to claim 5 or 6 in detecting soybean stalk phenotypes.

8. The use according to claim 7, characterized in that The soybean stem phenotype includes soybean flat stem and soybean round stem.

9. A method for detecting soybean stalk morphological traits, characterized in that: The steps include: Extracting genomic DNA from the sample to be tested; Using the genomic DNA as a template, performing PCR amplification on the genomic DNA using the primer set of claim 3 to obtain a PCR amplification product; determining the soybean stalk morphology based on the size of the PCR amplification product; When the size of the PCR amplification product is only 408 bp, the sample to be tested is round-stem soybean; When the sizes of the PCR amplification products are 408 bp and 353 bp, the sample to be tested is round-stem soybean; When the size of the PCR amplification product is only 353 bp, the sample to be tested is flat-stem soybean.

10. Use of a substance for detecting the GmST80-Indel molecular marker according to any one of claims 1 to 3, or the primer set according to claim 4, or the kit according to claim 5 or 6, or the method according to claim 9 in soybean breeding.

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