Soybean stem Gmst80-indel molecular marker and application thereof

Abstract

The application 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 application provides a GmST80-Indel molecular marker related to a soybean stem morphological trait, wherein the GmST80-Indel molecular marker is located in a GmST80 gene promoter region, and a nucleotide sequence of the GmST80-Indel molecular marker is shown as SEQ ID NO:1. The application verifies effectiveness of the molecular marker in different genetic backgrounds, finds that the molecular marker can accurately distinguish flat-stemmed soybeans from round-stemmed soybeans, and is used for screening of soybean stem morphologies, assisting soybean breeding, and quickly breeding soybeans with flat-stemmed stem morphological traits, thereby facilitating screening of soybean varieties with high single-plant yield.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted breeding technology, specifically relating to the GmST80-Indel molecular marker in soybean stems and its application. Background Technology

[0002] Soybean [Glycine max (L.) Merr] originated in China and is an important oilseed and economic crop. Soybean seeds contain high levels of protein and fat, making them a major source of human food, edible oil, and animal feed. Currently, soybean yields are relatively low, leaving significant room for improvement; therefore, increasing soybean yield is a crucial focus for boosting overall soybean production.

[0003] Currently, hybridization breeding is the most common method used in soybean breeding. However, hybridization breeding is time-consuming and requires the timely identification of desirable traits, making it both time-consuming and labor-intensive. Compared to traditional breeding, molecular marker-assisted breeding technology is highly efficient, rapid, and precise. With the development of soybean genomics, proteomics, metabolomics, and phenomics, a large number of genes related to important agronomic traits have been identified. These genetic loci have been developed into molecular markers, and combined with molecular marker-assisted breeding, the desired soybean varieties can be obtained rapidly.

[0004] Flat-stem soybeans are a mutant of soybean stem morphology, characterized by a large number of leaves on the main stem with whorled arrangement, clustered flowers and pods at the top, and a significantly increased number of pods. Therefore, the flat-stem trait is highly valuable for improving soybean yield and plant architecture. Many scholars have conducted in-depth research on the biological characteristics, genetic patterns, and physiological properties of flat-stem soybeans. However, because the flat-stem trait is controlled by a recessive gene and linked to many superior agronomic traits, it is difficult to stabilize after multiple generations of continuous selection. Flat-stem soybeans also have weaknesses such as soft stems. Whether the flat-stem trait can be stably inherited and detected for improvement directly affects the scope and value of flat-stem soybeans. Therefore, how to accurately, quickly, and simply utilize the soybean flat-stem gene for molecular marker breeding is a pressing problem to be solved in the field of soybean breeding. Summary of the Invention

[0005] To address the lack of molecular marker breeding using soybean flat stem genes in existing technologies, this invention provides the soybean stem molecular marker GmST80 and its applications, specifically including the following technical solutions:

[0006] This invention provides a GmST80-Indel molecular marker associated with soybean stem morphology. 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.

[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 as 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 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.

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

[0011] Preferably, the kit further includes a standard for amplified fragments of flat-stemmed soybean; the standard for amplified fragments of flat-stemmed soybean is shown in SEQ ID NO:1.

[0012] This invention also provides the application of substances, primer sets, or kits for detecting the GmST80-Indel molecular marker in the identification of soybean stem phenotypes.

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

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

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

[0016] Using the genomic DNA as a template, the genomic DNA is amplified by PCR using the primer set described in claim 3 to obtain PCR amplification products; the morphology of soybean stems is determined based on the size of the PCR amplification products.

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

[0018] When the size of the PCR amplification product is 408bp and 353bp, the sample to be tested is round-stemmed soybean;

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

[0020] This invention also provides the application of substances, primer sets, kits, or methods for detecting the GmST80-Indel molecular marker in soybean breeding.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention provides a GmST80-Indel molecular marker associated with soybean stem morphology. The GmST80-Indel molecular marker is located in the promoter region of the GmST80 gene, and its nucleotide sequence is shown in SEQ ID NO:1. Using the GmST80-Indel molecular marker described in this invention, round-stemmed and flat-stemmed soybeans can be rapidly and accurately distinguished. This invention has verified the effectiveness of the GmST80-Indel molecular marker in different genetic backgrounds. The GmST80-Indel molecular marker can accurately distinguish between flat-stemmed and round-stemmed soybeans, and can therefore be used for screening soybean stem morphology, assisting in soybean breeding. It can rapidly breed soybeans with flat stem morphology, thereby facilitating the selection of soybean varieties with high yield per plant.

[0023] This invention designs PCR primers and kits based on the GmST80-Indel molecular marker. Through simple PCR amplification, it enables rapid, accurate, efficient, and low-cost prediction and identification of soybean stem morphology traits, achieving an identification efficiency of up to 96.78% for soybean flat stems. Detection of substances bearing the GmST80-Indel molecular marker of this invention can be used to predict soybean stem morphology, distinguish between flat and round stems, and further, screening for flat stem traits can help select soybean varieties with high yield per plant. The GmST80-Indel molecular marker of this invention can assist in functional molecular breeding of soybeans, further shortening the breeding process and offering advantages of high efficiency and convenience. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0025] Figure 1 These are the mutation sites in the GmST80 gene of flat-stemmed soybean and round-stemmed soybean in Example 1 of this invention;

[0026] Figure 2 The differential sequence of the GmST80 gene between flat-stemmed soybean and round-stemmed soybean in Example 1 of this invention;

[0027] Figure 3 This is an agarose gel electrophoresis image of primer In800F / R in Example 2 of the present invention. Detailed Implementation

[0028] This invention provides a GmST80-Indel molecular marker associated with soybean stem morphology. The GmST80-Indel molecular marker is located in the promoter region of the GmST80 gene, and its 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 in the promoter region of the soybean GmST80 gene. In another embodiment, the GmST80-Indel molecular marker is located at -2487 to -2541 bp in SEQ ID NO:2.

[0030] The reference genome for the GmST80-Indel molecular marker described in this 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] The bolded part represents the GmST80-Indel molecular marker.

[0035] The GmST80-Indel molecular marker described in this invention exhibits a deletion polymorphism, as shown in SEQ ID NO:1, at a position 2541 bp upstream of the GmST80 gene promoter.

[0036] As one implementation, the nucleotide sequence with deletion polymorphism, as shown in SEQ ID NO:1, exists 2541 bp upstream of the promoter of the GmST80 gene, as shown in SEQ ID NO:3.

[0037]

[0038]

[0039]

[0040] In one embodiment, the GmST80-Indel molecular marker of the present invention, when used in flat-stemmed soybean, is a double-stranded DNA molecule with a two-stranded nucleotide sequence as shown in SEQ ID NO:14;

[0041] The GmST80-Indel molecular marker in round-stem soybean is a double-stranded DNA molecule with two strands of nucleotide sequence as shown in SEQ ID NO:15, or a double-stranded DNA molecule with one strand of nucleotide sequence as shown in SEQ ID NO:14 and the other strand of nucleotide sequence as shown in SEQ ID NO:15.

[0042] The GmST80-Indel molecular marker disclosed in this invention is closely linked to the flat stem trait in soybean and can be used for the identification of flat stem materials, thus assisting soybean breeding. Molecular marker-assisted breeding is widely used in crop breeding to shorten the breeding process. This invention develops molecular markers for soybean stem morphology and performs genotyping. The genotyping effect of the samples is significant, and it can accurately, efficiently, and cost-effectively predict soybean stem morphology, thereby promoting the soybean breeding process.

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

[0047] In one embodiment, the kit of the present invention includes a PCR kit. In another embodiment, the kit further includes PCR amplification reagents. In one embodiment, the PCR amplification reagents include: 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 primers In800-R (10 μM), and 7 μL of ddH2O, totaling 20 μL. In another embodiment, the kit further includes a standard for amplified fragments of flat-stemmed soybean; the standard for amplified fragments of flat-stemmed soybean is shown in SEQ ID NO:1.

[0048] This invention also provides the application of the GmST80-Indel molecular marker, or substances, primers, or kits for detecting the GmST80-Indel molecular marker as described above, in the identification of soybean stem phenotypes.

[0049] In one embodiment, the soybean stem phenotype includes flat soybean stems and round soybean stems.

[0050] This invention also provides a method for detecting flat-stemmed soybeans based on GmST80-Indel molecular markers, comprising the following steps:

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

[0052] Using the genomic DNA as a template, PCR amplification was performed on the genomic DNA using the forward and reverse primers as described above to obtain PCR amplification products; the morphology of soybean stems was determined based on the size of the PCR amplification products.

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

[0054] When the size of the PCR amplification product is 408bp and 353bp, the sample to be tested is round-stemmed soybean;

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

[0056] In one embodiment, the PCR amplification system (20 μL) consists of 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 primers In800-R (10 μM), and 7 μL of ddH2O. In another embodiment, the amplification reaction program is as follows: 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, infinity. In yet another embodiment, the size of the PCR amplification product can be determined using electrophoresis, gene sequencing, or real-time quantitative PCR.

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

[0058] In one implementation, the breeding includes high-yield soybean breeding. In another implementation, the high-yield soybean breeding includes breeding to increase the yield per soybean plant.

[0059] To further illustrate the present invention, the following detailed description of the soybean stem GmST80-Indel molecular marker and its application, in conjunction with the accompanying drawings and embodiments, is provided but should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1: Screening of molecular markers related to soybean stems

[0061] (1) Variation sites of the GmST80 gene between flat-stemmed soybean and round-stemmed soybean.

[0062] In this embodiment, flat-stemmed soybeans and round-stemmed soybeans produced during the propagation of the flat-stemmed soybean variety with the unified soybean resource number "ZDD23811" were used as test samples. Four pairs of primers were designed based on the GmST80 sequence of the reference genome Glycine maxWm82.a4.v1, as 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 flat-stemmed soybean and round-stemmed soybean using the four primer pairs described in Table 1. The PCR products were then subjected to Sanger sequencing, and the gene structure of the GmST80 gene in the reference genome was plotted as follows: Figure 1 and Figure 2 As shown, no variation was found in the coding region of the gene, but there was an Indel variation 2541 bp upstream of the ATG promoter region of the GmST80 gene. The variation at -2541 bp is: compared with round-stemmed soybean, flat-stemmed soybean has a 55 bp deletion. Therefore, this Indel variation site was developed as a molecular marker.

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

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

[0069] Table 2 Information on molecular marker primer sets

[0070]

[0071] Using the primer sets in Table 2, PCR amplification of soybean samples yielded products with sequences as 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 stems appear as flat stems. The product shown in SEQ ID NO:15 is 408 bp long, and the soybean stems appear as round stems. When products as shown in SEQ ID NO:14 and SEQ ID NO:15 are amplified simultaneously in soybeans, the soybean stems appear as round stems.

[0072]

[0073]

[0074] The bolded part of the sequence is the sequence shown in SEQ ID NO.1.

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

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

[0077] The flat-stemmed segregating population resulting from natural variation during propagation was used as the soybean sample for testing. Leaves of the soybean sample were placed in 2 mL centrifuge tubes, and genomic DNA was extracted using the classic CTAB method. The specific procedure is as follows:

[0078] Take out the preserved soybean leaves and place them in numbered 2mL centrifuge tubes. Then add one steel ball to each centrifuge tube, place the centrifuge tubes in liquid nitrogen, and use a sampling machine to sample for 30 seconds.

[0079] Add 800 μL of CTAB extraction solution (preheated at 65°C, 2% CTAB, with 2% β-mercaptoethanol added before use) to a centrifuge tube, mix thoroughly, and incubate at 65°C for 45 min. Invert the tube every 10 min. After 45 min, add 5 μL of RNase and incubate at 37°C for 1 h.

[0080] After the water bath, remove the centrifuge tube and add 800 μL of a mixture of phenol and chloroform (the volume ratio of phenol to chloroform in the mixture is 25:24). Mix thoroughly and centrifuge at 12,000 rpm for 10 min at 4 °C.

[0081] After centrifugation, transfer the supernatant to a new 2mL centrifuge tube, add 600μL of chloroform to the centrifuge tube, mix by inverting, and centrifuge again at 4℃ and 12000rpm for 10min.

[0082] After centrifugation, transfer the supernatant to another set of new 2mL centrifuge tubes. Add 500μL of pre-chilled isopropanol to the centrifuge tubes, mix well, and place in a -20℃ freezer for 1 hour to precipitate the DNA.

[0083] Centrifuge the centrifuge tubes at 4°C and 12,000 rpm for 10 min, discarding the supernatant. Rinse the precipitate three times with 75% ethanol solution. Air-dry the DNA precipitate at room temperature, then dissolve it in an appropriate amount of ddH2O. Finally, use 1% agarose to check the DNA quality and determine the concentration of the extracted DNA using NanodropOne. Dilute the DNA to a concentration of 50 ng / μL to obtain the genomic DNA of the soybean to be tested, and store it at -20°C for later use.

[0084] (2) Marker identification

[0085] Using the genomic DNA of the soybean obtained in step (1) as a template, PCR amplification was performed on the genomic DNA using the In800-F / R primer set described in the invention.

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

[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 as follows:

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

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

[0091] The PCR amplification products amplified by the In800-F / R primer set were categorized into three band patterns: A, B, and H. Band pattern A corresponds to a product length of 408 bp and a round stem phenotype; band pattern H corresponds to product lengths of 353 bp and 408 bp, also with a round stem phenotype; and band pattern B corresponds to a product length of 353 bp, with a flat stem phenotype. The detection efficiency for flat stems was as high as 96.78%.

[0092] A total of 311 individual plants were isolated from the flat-stemmed soybean population. Using their genomic DNA as templates, the amplification products were 353 bp in length (In800-F / R) for flat-stemmed soybeans and 408 bp for round-stemmed soybeans, or a combination of both. Based on the different band types, the stem morphology of the isolated plants was determined. Agarose gelatin analysis results of some soybeans are randomly displayed, such as... Figure 3 As shown.

[0093] The banding patterns of 311 samples from the flat-stem segregation population were analyzed, and it was found that there were 61 samples with band A, 28 samples with band H, and 222 samples with band B in the flat-stem segregation population.

[0094] Example 3: Accuracy Detection of Molecular Markers in Flat-stem Separation Populations

[0095] To verify the accuracy of the molecular markers, the stem morphology of 311 soybean accessions was investigated in the field. Among them, 212 had flat stems and 99 had round stems. The marker banding matched the phenotype of 301 individual plants, and the accuracy of the molecular marker detection was as high as 96.78%. This indicates that the molecular markers in this invention can accurately predict the stem morphology of soybeans, which is beneficial for screening soybean varieties with high yield per plant.

[0096] Table 3. Statistics on banding patterns of segregating population markers

[0097] A 0 61 61 H 0 28 28 B 212 10 222 total 212 99 311

[0098] Example 4: Genotyping of GmST80-Indel Markers in Variety Resources

[0099] To verify the accuracy of the molecular markers and identify the genotypes of soybean varieties, this embodiment utilizes 24 varieties. The unified resource numbers or variety names of these 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 genotypes of the final amplification products were divided into A and B. There were 14 genotypes A and 10 genotypes B. Based on the different genotypes, the stem morphology of different varieties was determined as shown in Table 4.

[0100] Table 4. Statistics on genotypes of different varieties

[0101] A 0 14 14 B 10 0 10 total 10 14 24

[0102] In summary, the GmST80-Indel molecular marker provided by this invention can quickly and accurately distinguish between round-stemmed and flat-stemmed soybeans, and can be used for screening soybean stem morphology. It can quickly breed soybeans with flat stem morphology, which is beneficial for screening soybean varieties with high yield per plant.

[0103] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of substances with GmST80-Indel molecular markers in soybean stem phenotypic identification; 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 is located at -2487~-2541bp in the promoter region of the soybean GmST80 gene; the promoter region of the GmST80 gene is shown in SEQ ID NO:2; The soybean stem phenotype is either flat soybean stem or round soybean stem.

2. A method for detecting the morphological characteristics of soybean stems, characterized in that, Includes the following steps: Extract genomic DNA from the sample to be tested; Using the genomic DNA as a template, the genomic DNA was amplified by PCR using the primer set shown in SEQ ID NO:4 and SEQ ID NO:5 to obtain PCR amplification products; the morphology of soybean stems was determined based on the size of the PCR amplification products. When the size of the PCR amplification product is only 408 bp, the sample to be tested is round-stemmed soybean; When the size of the PCR amplification product is 408bp and 353bp, the sample to be tested is round-stemmed soybean; When the PCR amplification product is only 353 bp in size, the sample to be tested is flat-stemmed soybean.

3. The application of substances that detect GmST80-Indel molecular markers or the method described in claim 2 in soybean breeding; 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 is located at -2487~-2541bp in the promoter region of the soybean GmST80 gene; the promoter region of the GmST80 gene is shown in SEQ ID NO:2; The soybean breeding refers to soybean flat-stem or soybean round-stem breeding.

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