Molecular marker for identifying wheat starch branching enzyme gene SBEs and application thereof

Abstract

The invention discloses a molecular marker for identifying wheat starch branching enzyme genes SBEs and application of the molecular marker, and belongs to the technical field of molecular markers. The invention provides a KASP molecular marker aiming at wheat starch branching enzyme genes SBEIIa and SBEIIb. Two upstream primers and a downstream primer are respectively obtained aiming at three subgenomes of wheat A, B and D of the SBEIIa gene; the method comprises the following steps: respectively obtaining two upstream primers and a downstream primer aiming at two subgenomes A and B of wheat with the SBEIIb gene; the provided KASP molecular marker aiming at the wheat starch branching enzyme genes SBEIIa and SBEIIb can be used for accurately performing typing identification on the SBEIIa-A / B / D gene and the SBEIIb-A / B gene, has the advantages of easiness in distinguishing and high typing accuracy, is low in application cost, and can be used for successfully creating sweet wheat only by three generations by adopting the developed KASP molecular marker.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, in particular to a molecular marker for identifying wheat starch branching enzyme genes SBEs and an application thereof. Background Art

[0002] Wheat is one of the world's three major food crops, accounting for one-fifth of the world's crop planting area and serving as the staple food for 40% of the world's population. It is crucial for ensuring national food security and meeting market demand. Starch accounts for approximately 70% of the dry weight of wheat grains and is the primary component of the wheat endosperm. Wheat is an allohexaploid with three subgenomes: A, B, and D. Currently, two genes, SBEⅡa and SBEⅡb, are known to encode key enzymes in starch synthesis (starch branching enzymes), which are associated with the resistant starch content in wheat grains. Studies have shown that polymorphisms in the SBEⅡb gene (e.g., candidate differential sites at the carboxyl terminus of the coding region) significantly influence the resistant starch content among wheat varieties.

[0003] KASP (Kompetitive Allele-Specific PCR), short for competitive allele-specific PCR, is a biallelic typing technology based on allele-specific PCR. Its core principle involves designing two 3′-base-specific upstream primers (matching the wild-type and mutant forms, respectively) and a universal downstream primer for each SNP locus. Alleles are distinguished by fluorescent labeling, and genotypes are determined by detecting the different fluorescent signals. This allows for highly accurate typing of samples without the need for probes. KASP offers the advantages of high throughput, low cost, and applicability to a wide range of sample types.

[0004] In genetic research, KASP technology offers significant advantages when precisely distinguishing allelic differences between individuals is crucial: a single experiment can analyze specific genetic variants in hundreds of DNA samples in parallel, significantly improving genotyping efficiency and scalability. This technology directly determines allele type by differences in fluorescence signal intensity, significantly simplifying the workflow and making it more suitable for large-scale research scenarios such as crop genetic mapping, association analysis, and marker-assisted breeding. Compared to existing amplification technologies, KASP not only offers high throughput, enabling large-scale testing, but also offers low cost (KASP is approximately 60% less expensive than TaqMan, which is more expensive) and high typing accuracy (over 90%). Therefore, the development of KASP molecular markers targeting the starch branching enzyme (SBE) genes in wheat, which have three subgenomes, is of great significance for studying the mechanisms of starch synthesis in wheat, reducing breeding workload, and accelerating the breeding process. Summary of the Invention

[0005] The purpose of the present invention is to provide a molecular marker for identifying wheat starch branching enzyme genes SBEs and its application, and to provide a convenient KASP marker for SBEⅡa and SBEⅡb genes for the breeding of wheat varieties with high resistant starch content, so as to reduce the breeding workload and accelerate the breeding process.

[0006] To achieve the above objectives, the present invention provides a molecular marker for identifying wheat starch branching enzyme genes SBEs, wherein the wheat starch branching enzyme genes are SBEⅡa and SBEⅡb; the ID number of the SBEⅡa gene is GenBank AF338431, and the ID number of the SBEⅡb gene is GenBank AY740398; the molecular marker is a KASP marker.

[0007] Preferably, the upstream typing primers for the wheat A subgenome in the SBEⅡa gene are Sbe2a-AF and Sbe2a-AH, and the downstream typing primer is Sbe2a-AG; the upstream typing primers for the B subgenome are Sbe2a-BF and Sbe2a-BH, and the downstream typing primer is Sbe2a-BG; two upstream typing primers for the D genome are Sbe2a-DF and Sbe2a-DH, and the downstream typing primer is Sbe2a-DG.

[0008] Preferably, the sequence of Sbe2a-AF is shown as SEQ ID NO.1, the sequence of Sbe2a-AH is shown as SEQ ID NO.2, the sequence of Sbe2a-AG is shown as SEQ ID NO.11, the sequence of Sbe2a-BF is shown as SEQ ID NO.3, the sequence of Sbe2a-BH is shown as SEQ ID NO.4, the sequence of Sbe2a-BG is shown as SEQ ID NO.12, the sequence of Sbe2a-DF is shown as SEQ ID NO.5, the sequence of Sbe2a-DH is shown as SEQ ID NO.6, and the sequence of Sbe2a-DG is shown as SEQ ID NO.13.

[0009] Preferably, the upstream typing primers for the A subgenome in the SBEⅡb gene are Sbe2b-AF and Sbe2b-AH, and the downstream typing primer is Sbe2b-AG; the upstream typing primers for the B subgenome are Sbe2b-BF and Sbe2b-BH, and the downstream typing primer is Sbe2b-BG.

[0010] Preferably, the sequence of Sbe2b-AF is shown as SEQ ID NO.7, the sequence of Sbe2b-AH is shown as SEQ ID NO.8, the sequence of Sbe2b-AG is shown as SEQ ID NO.14, the sequence of Sbe2b-BF is shown as SEQ ID NO.9, the sequence of Sbe2b-BH is shown as SEQ ID NO.10, and the sequence of Sbe2b-BG is shown as SEQ ID NO.15.

[0011] A method for identifying wheat starch branching enzyme genes uses the above-mentioned molecular marker for identifying wheat starch branching enzyme genes SBEs, configures a reaction system, and performs PCR amplification.

[0012] Preferably, the reaction system comprises a primer mixture solution, an enzyme probe mixture and DNA of the plant to be tested; the primer mixture solution is a mixed solution of KASP upstream typing primers and downstream typing primers of the three subgenomes A to D; the probe is a mixture of FAM, HEX and Rox, and the final concentration ratio of FAM, HEX and Rox is 1:1:2.5.

[0013] Preferably, the PCR amplification program is 95°C for 10 min; 95°C for 20 s, 61-55°C for 40 s, 10 cycles, decreasing 0.6°C per cycle; 95°C for 20 s, 55°C for 40 s, 35 cycles; 25°C∞, after the amplification is completed, the end point fluorescence signal is read at 25-40°C.

[0014] A KASP molecular marker for identifying wheat starch branching enzyme genes SBEs as described above.

[0015] Therefore, the present invention provides a molecular marker for identifying wheat starch branching enzyme gene SBEs and its application, and its specific technical effects are as follows:

[0016] (1) The present invention provides KASP molecular markers for wheat starch branching enzyme genes SBEⅡa and SBEⅡb, wherein two upstream primers and one downstream primer are obtained for each of the three wheat subgenomes A, B, and D for the SBEⅡa gene; two upstream primers and one downstream primer are obtained for each of the two wheat subgenomes A and B for the SBEⅡb gene;

[0017] (2) The KASP molecular markers for wheat starch branching enzyme genes SBEⅡa and SBEⅡb provided by the present invention can accurately type and identify the SBEIIa gene and SBEIIb gene, and have the advantages of easy differentiation and high typing accuracy, and low application cost. By using the developed KASP molecular markers, "sweet wheat" can be successfully created in only three generations.

[0018] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0020] Figure 1 is the sensitivity test result in Example 2 of the present invention; wherein a is the typing result of the SBEIIa gene A subgenome; b is the typing result of the SBEIIa gene B subgenome; c is the typing result of the SBEIIa gene D subgenome; d is the typing result of the SBEIIb gene A subgenome; e is the typing result of the SBEIIb gene B subgenome;

[0021] Figure 2 is the first genotyping result in the repeatability test of Example 2 of the present invention; wherein a is the typing result of the SBEIIa gene A subgenome; b is the typing result of the SBEIIa gene B subgenome; c is the typing result of the SBEIIa gene D subgenome; d is the typing result of the SBEIIb gene A subgenome; and e is the typing result of the SBEIIb gene B subgenome;

[0022] Figure 3 is the second genotyping result in the repeatability test of Example 2 of the present invention; wherein a is the typing result of the SBEIIa gene A subgenome; b is the typing result of the SBEIIa gene B subgenome; c is the typing result of the SBEIIa gene D subgenome; d is the typing result of the SBEIIb gene A subgenome; and e is the typing result of the SBEIIb gene B subgenome;

[0023] Figure 4 is the result of wheat genetic diversity analysis in Example 4 of the present invention; wherein a is the typing result of the SBEIIa gene A subgenome; b is the typing result of the SBEIIa gene B subgenome; c is the typing result of the SBEIIa gene D subgenome; d is the typing result of the SBEIIb gene A subgenome; e is the typing result of the SBEIIb gene B subgenome;

[0024] Figure 5These are the PCR results of the 15 Wx protein-deficient strains in Example 5 of the present invention; wherein a is the genotyping result of the SBEIIa gene A subgenome in the Wx-abd strain; b is the genotyping result of the SBEIIa gene B subgenome in the Wx-abd strain; c is the genotyping result of the SBEIIa gene D subgenome in the Wx-abd strain; d is the genotyping result of the SBEIIb gene A subgenome in the Wx-abd strain; e is the genotyping result of the SBEIIb gene B subgenome in the Wx-abd strain; and f is the genotyping result of the SBEIIa gene D subgenome in the self-pollinated progeny of the Wx-abd-6 strain. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0026] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0027] The instruments, equipment, reagents and materials used in the examples were all obtained from commercial sources; the experimental methods and steps not described in detail are all conventional techniques in the art.

[0028] Example 1

[0029] Design KASP marker primers as follows:

[0030] (1) Upstream primers, design requirements: length 21-28 bp, GC content 40-60%, discard the region with 4 or more consecutive G / C, remove the unknown terminal base, and make the 3′ terminal base match the two alleles respectively, such as the last base of primer F corresponds to A, and the last base of primer H corresponds to C. At the same time, add a universal fluorescent label (FAM / HEX) and amplification primer region at the 5′ end.

[0031] For the SBEⅡa gene (GenBank AF338431), two upstream typing primers for wheat A subgenome were obtained: Sbe2a-AF (sequence shown in SEQ ID NO.1) and Sbe2a-AH (sequence shown in SEQ ID NO.2); two upstream typing primers for B subgenome: Sbe2a-BF (sequence shown in SEQ ID NO.3) and Sbe2a-BH (sequence shown in SEQ ID NO.4); two upstream typing primers for D genome: Sbe2a-DF (sequence shown in SEQ ID NO.5) and Sbe2a-DH (sequence shown in SEQ ID NO.6).

[0032] For the SBE IIb gene (GenBank AY740398), two upstream typing primers for the A subgenome, Sbe2b-AF (sequence shown in SEQ ID NO. 7) and Sbe2b-AH (sequence shown in SEQ ID NO. 8), and two upstream typing primers for the B subgenome, Sbe2b-BF (sequence shown in SEQ ID NO. 9) and Sbe2b-BH (sequence shown in SEQ ID NO. 10), were obtained. The regions in bold indicate the universal fluorescent tag and amplification primers.

[0033] SEQ ID NO.1:

[0034]

[0035] SEQ ID NO.2:

[0036]

[0037] SEQ ID NO.3:

[0038]

[0039] SEQ ID NO.4:

[0040]

[0041] SEQ ID NO.5:

[0042]

[0043] SEQ ID NO.6:

[0044]

[0045] SEQ ID NO.7:

[0046]

[0047] SEQ ID NO.8:

[0048]

[0049] SEQ ID NO.9:

[0050]

[0051] SEQ ID NO.10:

[0052]

[0053] (2) Downstream primers: The downstream primers only need to bind to the template in reverse complementation. For the SBEⅡa gene, a downstream typing primer for wheat A subgenome: Sbe2a-AG (sequence shown in SEQ ID NO.11), a downstream typing primer for wheat B subgenome: Sbe2a-BG (sequence shown in SEQ ID NO.12), and a downstream typing primer for wheat D subgenome: Sbe2a-DG (sequence shown in SEQ ID NO.13) were obtained. For the SBEⅡb gene, two downstream typing primers for the A subgenome: Sbe2b-AG (sequence shown in SEQ ID NO.14) and one downstream typing primer for the B genome: Sbe2b-BG (sequence shown in SEQ ID NO.15) were obtained.

[0054] SEQ ID NO.11:

[0055]

[0056] SEQ ID NO.12:

[0057]

[0058] SEQ ID NO.13:

[0059]

[0060] SEQ ID NO.14:

[0061]

[0062] SEQ ID NO.15:

[0063]

[0064] (3) The fluorescent probe (FAM / HEX) is complementary to the upstream primer tag, and the quencher probe suppresses nonspecific signals.

[0065] Example 2

[0066] The KASP primer construction system designed in Example 1 was used for verification, as follows:

[0067] (1) Sensitivity test. Prepare a 10 μL reaction system: 4 μL of HiGeno 2×Probe Mix B, 2 μL of mixed primers (10 μM of TaSBEIIa-A-KASP-F1, TaSBEIIa-A-KASP-F2, and TaSBEIIa-A-KASP-R, with a concentration ratio of 1:1:2.5), DNA template, and ddH2O to make up to 10 μL. The DNA template is the F1 generation obtained by hybridizing Shumai 1963 and SBE-ABD (standard sample DNA with all known genotypes being heterozygous). Shumai 1963 is used as the positive parent labeled with FAM, and high-amylose wheat SBE-ABD is used as the negative parent labeled with HEX. 5 μL of each is aspirated and some ddH2O is added to make the final concentration of the template DNA 200 ng / μL. The system without template DNA is used as the control, which is recorded as the NTC control.

[0068] Mix all the reaction mixtures thoroughly and place them in a fluorescent quantitative PCR instrument (supporting FAM / HEX channels). Perform PCR using the following protocol: 95°C for 10 min; 10 cycles of 95°C for 20 s, 61-55°C for 40 s, decreasing 0.6°C each cycle; 35 cycles of 95°C for 20 s, 55°C for 40 s; and 25°C for ∞. After completion of amplification, read the endpoint fluorescence signal at 25-40°C.

[0069] The results are as follows Figure 1 As shown, the fluorescent dots are divided into three groups. The parent Shumai 1963 represents the FAM-marked alleles (SBEIIa / SBEIIb gene expression), and the high-amylose wheat SBE-ABD group represents the HEX-marked alleles (SBEIIa / SBEIIb gene silencing), and the remaining green dots represent F1 generation heterozygous individuals.

[0070] (2) Repeatability test. In the process of creating wheat starch-specific materials, the F2 generation obtained by hybridization of Shumai 1963 and SBE-ABD was used. 53 DNA samples of unknown genotype were randomly selected from one of the segregating populations numbered 5-24. A 10 μL reaction system was prepared according to (1). The DNA template concentration was diluted to 200 ng / μL. The same PCR amplification procedure as in (1) was used to conduct two repeatability experiments and compare the two results.

[0071] The first genotyping diagram is as follows Figure 2 As shown,

[0072] The second genotyping diagram is as follows Figure 3 shown.

[0073] have Figure 2 and Figure 3 It can be seen that after two amplifications, the results were consistent, excluding the PCR experimental operation error. The results show that KASP primers are accurate, stable, and usable.

[0074] Example 4

[0075] The genetic diversity of wheat was analyzed on one of the BC1F2 lines numbered 5-32, which was a hybrid of Shumai 1963 and SBE-ABD. The details are as follows:

[0076] The CTAB method was used to extract DNA from 60 individual strains in the population. The DNA quality was tested and the DNA solutions that met the quality requirements were diluted to a concentration of 200 ng / μL.

[0077] Prepare a 10 μL reaction system: 4 μL HiGeno 2× Probe Mix B, 2 μL primer mix (10 μM each of TaSBEIIa-A-KASP-F1, TaSBEIIa-A-KASP-F2, and TaSBEIIa-AK ASP-R, with a FAM, HEX, and Rox concentration ratio of 1:1:2.5), 2 μL DNA template, and fill to 10 μL with ddH2O. A control without template DNA was used, designated as the NTC control.

[0078] Mix all the reaction mixtures thoroughly and place them in a fluorescent quantitative PCR instrument (supporting FAM / HEX channels). Perform PCR using the following protocol: 95°C for 10 min; 10 cycles of 95°C for 20 s, 61-55°C for 40 s, decreasing 0.6°C each cycle; 35 cycles of 95°C for 20 s, 55°C for 40 s; and 25°C for ∞. After completion of amplification, read the endpoint fluorescence signal at 25-40°C.

[0079] The five genotypes of the strain BC1F1 are: aa, Bb, Dd, aa, Bb. Figure 4 As shown in the figure, the five genotyping patterns of the BC1F2 generation segregating population conform to Mendelian inheritance. The polymorphism ratio of KASP markers is over 90%.

[0080] Example 5

[0081] The SBEIIa and SBEIIb genes of 15 wheat Wx protein-deficient strains from the F2 generation obtained by hybridizing SM969 and SBE-ABD were typed and identified using KASP markers. The parents were used as controls, and the system without template DNA was used as a control, which was recorded as NTC. Figure 5 As shown: The fluorescent scatter points are divided into three groups, including one group representing FAM-marked alleles (SBEIIa / SBEIIb gene expression) with SM969 waxy wheat, one group representing HEX-marked alleles (SBEIIa / SBEIIb gene silencing) with high-amylose wheat, and the remaining group representing the heterozygous type.

[0082] The results demonstrated that this KASP marker can accurately genotype the SBEIIa and SBEIIb genes. Among the 15 Wx-abd lines, line Wx-abd-6 exhibited recessive homozygous mutations in both SBEIIa and SBEIIb at the A and B genomic loci, with only the D genomic locus in SBEIIa being heterozygous. Line Wx-abd-6 was cultivated in a greenhouse and further characterized using the KASP marker for SBEIIa-D. Plants with the genotype Wx-aabbdd / SBEIIa-aabbdd / SBEIIb-aabbDD were identified as the sweet wheat developed in this study.

[0083] Therefore, the present invention provides KASP molecular markers for wheat starch branching enzyme genes SBEⅡa and SBEⅡb, wherein two upstream primers and one downstream primer are obtained for each of the three wheat subgenomes A, B, and D of the SBEⅡa gene; two upstream primers and one downstream primer are obtained for each of the two wheat subgenomes A and B of the SBEⅡb gene; the provided KASP molecular markers for wheat starch branching enzyme genes SBEⅡa and SBEⅡb can accurately type and identify the SBEIIa gene and the SBEIIb gene, have the advantages of easy distinction and high typing accuracy, and have low application cost. By using the developed KASP molecular markers, "sweet wheat" can be successfully created in only three generations.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A molecular marker for identifying wheat starch branching enzyme gene SBEs, characterized by: The wheat starch branching enzyme genes are SBEⅡa and SBEⅡb; the ID number of the SBEⅡa gene is GenBank AF338431, and the ID number of the SBEⅡb gene is GenBank AY740398; and the molecular marker is a KASP marker.

2. A molecular marker for identifying wheat starch branching enzyme gene SBEs according to claim 1, characterized in that: The upstream typing primers for the wheat A subgenome in the SBEⅡa gene are Sbe2a-AF and Sbe2a-AH, and the downstream typing primer is Sbe2a-AG; the upstream typing primers for the B subgenome are Sbe2a-BF and Sbe2a-BH, and the downstream typing primer is Sbe2a-BG; two upstream typing primers for the D genome are Sbe2a-DF and Sbe2a-DH, and the downstream typing primer is Sbe2a-DG.

3. A molecular marker for identifying wheat starch branching enzyme gene SBEs according to claim 2, characterized in that: The sequence of Sbe2a-AF is shown as SEQ ID NO.1, the sequence of Sbe2a-AH is shown as SEQ ID NO.2, the sequence of Sbe2a-AG is shown as SEQ ID NO.11, the sequence of Sbe2a-BF is shown as SEQ ID NO.3, the sequence of Sbe2a-BH is shown as SEQ ID NO.4, the sequence of Sbe2a-BG is shown as SEQ ID NO.12, the sequence of Sbe2a-DF is shown as SEQ ID NO.5, the sequence of Sbe2a-DH is shown as SEQ ID NO.6, and the sequence of Sbe2a-DG is shown as SEQ ID NO.

13.

4. A molecular marker for identifying wheat starch branching enzyme gene SBEs according to claim 1, characterized in that: The upstream typing primers for the A subgenome in the SBEⅡb gene are Sbe2b-AF and Sbe2b-AH, and the downstream typing primer is Sbe2b-AG; the upstream typing primers for the B subgenome are Sbe2b-BF and Sbe2b-BH, and the downstream typing primer is Sbe2b-BG.

5. A molecular marker for identifying wheat starch branching enzyme gene SBEs according to claim 4, characterized in that: The sequence of Sbe2b-AF is shown in SEQ ID NO.7, the sequence of Sbe2b-AH is shown in SEQ ID NO.8, the sequence of Sbe2b-AG is shown in SEQ ID NO.14, the sequence of Sbe2b-BF is shown in SEQ ID NO.9, the sequence of Sbe2b-BH is shown in SEQ ID NO.10, and the sequence of Sbe2b-BG is shown in SEQ ID NO.

15.

6. A method for identifying a wheat starch branching enzyme gene, characterized in that: The method comprises using a molecular marker for identifying wheat starch branching enzyme gene SBEs according to any one of claims 1 to 5, configuring a reaction system, and performing PCR amplification.

7. The method for identifying a wheat starch branching enzyme gene according to claim 6, wherein: The reaction system includes a primer mixture solution, an enzyme probe mixture and plant DNA to be tested; the primer mixture solution is a mixed solution of KASP upstream typing primers and downstream typing primers of three subgenomes A to D; the probe is a mixture of FAM, HEX and Rox, and the final concentration ratio of FAM, HEX and Rox is 1:1:2.

5.

8. The method for identifying a wheat starch branching enzyme gene according to claim 6, wherein: The PCR amplification program is 95°C for 10 min; 94°C for 20 s, 61-55°C for 40 s, 10 cycles, decreasing 0.6°C per cycle; 95°C for 20 s, 55°C for 40 s, 35 cycles; 25°C∞, after the amplification is completed, the end point fluorescence signal is read at 25-40°C.

9. Use of the molecular marker for identifying wheat starch branching enzymes (SBEs) according to any one of claims 1 to 5 in wheat genetic diversity analysis, genotyping and seed purity identification.

10. Use of the method for identifying wheat starch branching enzyme genes according to any one of claims 6 to 8 in wheat genetic diversity analysis, genotyping and seed purity identification.

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