A wheat ear sprouting resistance related CAPs molecular marker BM_phs2_CAPs and application thereof

Abstract

The application belongs to the field of molecular breeding, and particularly relates to a wheat ear sprouting resistance related CAPs molecular marker BM_phs2_CAPs and application thereof. A wheat ear sprouting resistance related core candidate gene is obtained from a wheat ear sprouting resistant variety Bimei No. 6, and is named as Taphs2-6A-BM. The gene sequence is significantly different among different haplotypes. Based on the variation, specific amplification primers are designed for a CDS1 region, and an efficient CAPs molecular marker for distinguishing ear sprouting resistance is developed by using ApaI enzyme cutting technology, and is named as BM_phs2_CAPs. The CAPs marker is applied to 296 white grain wheat varieties, and the result shows that the detection result of the BM_phs2_CAPs marker is highly consistent with the multi-year and multi-point field ear sprouting resistance phenotype data.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding, specifically relating to a molecular marker BM_phs2_CAPs related to wheat ear sprouting resistance and its application. Background Technology

[0002] Wheat is one of the world's most important food crops. Pre-harvest sprouting (PHS) often leads to decreased seed yield, starch degradation, and deterioration of processing quality, causing serious economic losses in major white wheat producing areas.

[0003] Currently, although some genes regulating wheat spike germination (such as TaMKK3-A, TaSdr, and TaVp-1) have been cloned, most of these genes are located on chromosomes 3 and 4, and some resistance alleles are linked to the red grain trait, making them difficult to directly apply to resistance improvement in white-grained wheat varieties. Although studies have shown the existence of QTL signals controlling spike germination on chromosome 6A, the complex genome structure has long lacked functional molecular markers that can be precisely located and used for large-scale high-throughput breeding screening. Therefore, identifying resistance variation sites on chromosome 6A and developing efficient, low-cost molecular markers is a crucial problem urgently needing to be solved in white-grained wheat resistance breeding. Summary of the Invention

[0004] To address the shortcomings of existing methods for screening white-grained wheat for germination resistance, such as low accuracy and a lack of specific markers targeting major variant sites on chromosome 6A, this invention aims to provide a CAPs molecular marker developed based on key variants of the Taphs2-6A gene. This marker can rapidly and accurately identify wheat germination resistance haplotypes through a simple PCR and enzyme digestion process, providing a reliable molecular method for breeding resistant varieties.

[0005] The present invention specifically adopts the following technical solution:

[0006] 1. Cloning of resistance genes: This invention first obtained the core candidate gene related to wheat ear sprouting resistance from the excellent wheat variety Bima 6 through homologous cloning, and named it Taphs2-6A-BM.

[0007] 2. Bioinformatics alignment analysis: The cloned Taphs2-6A-BM sequence was aligned with the Chinese Spring 1.0 version reference genome sequence in the WheatOmics database (http: / / wheatomics.sdau.edu.cn / ). The results showed that the gene sequence exhibited significant differences among different haplotypes.

[0008] 3. Discovery of major variant sites: The comparison results revealed that, compared with the susceptible haplotypes (such as 'China Spring'), the disease-resistant haplotypes (such as 'Bima 6') have a key 12bp deletion and other base variations in the first coding region (CDS1) of the Taphs2-6A-BM gene.

[0009] 4. Development of CAPs Markers: Further sequence analysis revealed that this 12bp deletion variant resulted in the loss of the ApaI restriction enzyme recognition site (GGGCCC) originally present in the susceptible haplotype sequence. Based on this significant mutation, this invention designed specific amplification primers for the CDS1 region and developed highly efficient CAPs molecular markers for distinguishing budding resistance using ApaI enzyme digestion technology, named BM_phs2_CAPs.

[0010] 5. Application of CAPs markers: This invention applied CAPs markers to 296 white-grained wheat varieties (core germplasm resources). The results showed that the detection results of BM_phs2_CAPs markers had a very high agreement rate with the field spike germination phenotype data from multiple years and locations.

[0011] Based on the above, in a first aspect, this invention provides the application of the Taphs2-6A gene in regulating wheat ear germination resistance, wherein the nucleotide sequence of the Taphs2-6A gene is shown in SEQ ID NO:1, and the Taphs2-6A gene shown in SEQ ID NO:1 positively regulates wheat ear germination resistance. Compared with the susceptible haplotype, the resistant haplotype (Taphs2-6A-BM) has a critical 12bp deletion in the first coding region of the Taphs2-6A gene, as well as other base variations. The nucleotide sequence of the susceptible haplotype Taphs2-6A gene (Taphs2-6A-CS) is shown in SEQ ID NO:2.

[0012] Secondly, the present invention provides a product for detecting the molecular marker BM_phs2_CAPs, said product being a reagent or kit, said product comprising a primer pair and a restriction endonuclease ApaI, said primer pair comprising:

[0013] F: 5'-CGGAGTCGTACCCCGCT-3' (SEQ ID NO: 3);

[0014] R: 5'-TGGCCGGGAGGAATCACAAAT-3' (SEQ ID NO:4); This primer pair is used to amplify the first coding region of the Taphs2-6A gene.

[0015] Thirdly, the present invention provides the application of the molecular marker BM_phs2_CAPs or products that detect the molecular marker BM_phs2_CAPs in any of the following, wherein the molecular marker BM_phs2_CAPs is located in the first coding region of the Taphs2-6A gene, the disease-resistant haplotype Taphs2-6A-BM gene sequence is shown in SEQ ID NO:1, and the disease-susceptible haplotype Taphs2-6A-CS sequence is shown in SEQ ID NO:2, both of which are located on wheat chromosome 6A;

[0016] The applications include:

[0017] A: To identify or assist in the identification of wheat ear germination resistance;

[0018] B: Breeding for resistance to wheat ear sprouting.

[0019] Fourthly, the present invention provides a method for identifying or assisting in the identification of wheat spike germination resistance, comprising the following steps: detecting the Taphs2-6A gene haplotype in the genomic DNA of the wheat to be identified, wherein the average spike germination index of the wheat population to be identified with the Taphs2-6A gene haplotype shown in SEQ ID NO:1 is significantly lower than the average spike germination index of the wheat population to be identified with the Taphs2-6A gene haplotype shown in SEQ ID NO:2.

[0020] In a further embodiment, the present invention provides a method for identifying or assisting in the identification of wheat ear germination resistance, comprising the following steps:

[0021] (1) Using the wheat genomic DNA to be identified as a template, PCR amplification was performed using the primer pair to obtain the PCR product;

[0022] (2) The PCR product was digested with the restriction endonuclease ApaI;

[0023] (3) Identify wheat ear sprouting resistance based on the characteristic bands after enzyme digestion: those containing a 385 bp characteristic band after enzyme digestion are ear sprouting resistance haplotypes, and those containing 257 bp and 146 bp characteristic bands after enzyme digestion are ear sprouting disease susceptible haplotypes.

[0024] This invention provides a method for breeding wheat with resistance to germination in wheat ears, comprising:

[0025] (1) Use the method described above to detect wheat ear germination resistance;

[0026] (2) Select appropriate wheat parents for breeding according to the breeding objectives.

[0027] The wheat spike germination resistance-related CAPs molecular markers BM_phs2_CAPs developed in this invention have the following significant advantages compared with existing technologies:

[0028] 1. High detection sensitivity and extremely significant band differentiation.

[0029] The molecular marker BM_phs2_CAPs was designed based on a significant sequence variation (a 12 bp deletion) in the CDS1 region of the Taphs2-6A-BM gene. Because the resistant and susceptible haplotypes differ significantly in the number of ApaI restriction sites in the amplified fragments, the characteristic bands after restriction enzyme digestion are 385 bp (resistant) and 257 bp + 146 bp (susceptible), respectively. This multi-site, highly differentiated banding characteristic makes the electrophoretic detection results clear and easily distinguishable, greatly reducing the probability of human error and improving the reliability of the detection.

[0030] 2. It possesses extremely strong varietal universality and phenotypic correlation.

[0031] This invention utilizes this marker to conduct large-scale validation on 296 white-grained wheat varieties (core germplasm resources) in Henan Province. The experimental results show that the detection results of the BM_phs2_CAPs marker have an extremely high agreement rate with multi-year, multi-location field data on pre-harvest sprouting resistance phenotypes. This result proves that this marker can accurately reflect the contribution of the Taphs2-6A-BM gene to pre-harvest sprouting resistance and is one of the most effective molecular diagnostic tools currently available for targeting major resistance loci on wheat chromosome 6A.

[0032] 3. Highly specific amplification of complex hexaploid genomes was achieved.

[0033] The wheat genome is extremely large and highly homologous (subgenomes A, B, and D). This invention, through careful design and screening of primers F: 5'-CGGAGTCGTACCCCGCT-3' and R: 5'-TGGCCGGGAGGAATCACAAAT-3', successfully achieved specific amplification of the target gene CDS1 region on chromosome 6A, effectively avoiding interference from homologous sequences on chromosomes 6B and 6D. This ensures stable and unique amplification products can be obtained in wheat materials with different genetic backgrounds.

[0034] 4. Low testing cost, suitable for large-scale high-throughput screening in breeding.

[0035] This invention employs conventional PCR combined with restriction endonuclease treatment. Compared to expensive SNP chip detection or large-scale gene sequencing, BM_phs2_CAPs marker detection has low requirements for equipment, is easy to operate, and has extremely low cost per sample. This makes it possible for breeding bases to conduct large-scale, high-throughput screening of resistant materials in early generations (such as F2 and F3 generations), significantly shortening the breeding cycle of resistant varieties.

[0036] 5. It has significant value in breeding applications and contributes to the improvement of white-grained wheat.

[0037] For a long time, there has been a lack of efficient functional markers for improving the germination resistance of white-grained wheat. This invention directly targets the CDS region of functional genes and is closely linked to the resistance-superior haplotype ('Bima 6' type). In marker-assisted selection (MAS) or backcross breeding, this marker can be used to precisely introduce the resistance haplotype into high-yielding but disease-susceptible white-grained wheat varieties, which has significant economic and social value for improving the stress resistance and quality safety of white-grained wheat in my country. Attached Figure Description

[0038] Figure 1 The image shows the sequence alignment of PCR amplification using CAPs-marked primers for Bima 6 and Chinese Spring, and the full-length DNA sequence alignment of the Taphs2-6A gene.

[0039] Figure 2 Agarose gel electrophoresis image of the enzyme digestion products labeled with CAPs from a portion (96 samples) of wheat.

[0040] Figure 3 Association analysis of different haplotypes of the Taphs2-6A gene with wheat ear sprouting resistance. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0042] Example 1: Development of molecular markers BM_phs2_CAPs

[0043] 1. Candidate gene discovery and homologous cloning

[0044] This invention first observed in the model plant Arabidopsis thaliana that core components of the auxin signaling pathway (such as Aux / IAA repressor proteins and ARF transcription factors) play a crucial role in regulating ABI3 gene expression and thus influencing seed dormancy and germination. Based on the nucleotide sequence of this key gene in Arabidopsis thaliana (accession number: NC_003076), homologous sequence alignment was performed in the "Chinese Spring" reference genome in the WheatOmics database using the BLAST bioinformatics tool, successfully locating the homologous gene sequence Taphs2-6A-CS on wheat chromosome 6A. Subsequently, using the genomic DNA of the superior wheat variety 'Bima 6' with excellent resistance to pre-ear germination as a template, specific primers were designed according to the "Chinese Spring" reference sequence, and homologous cloning technology was used to successfully amplify and clone the resistance allelic variant of this gene, naming it Taphs2-6A-BM.

[0045] 2. Sequence alignment and discovery of core variant sites

[0046] The cloned 'Bima 6' Taphs2-6A-BM gene sequence was compared with the Taphs2-6A-CS sequence of the susceptible variety 'Zhongguo Chun' using multiple alignment (e.g., ...). Figure 1 (As shown). Sequence alignment results revealed a critical 12 bp deletion (and adjacent single nucleotide substitution) in the first coding region (CDS1) of the Taphs2-6A-BM gene. Further restriction site prediction analysis showed that this 12 bp deletion resulted in the loss of a specific recognition site (GGGCCC) for the restriction endonuclease ApaI that was originally present in the susceptible haplotype ('Chinese Spring' type) sequence.

[0047] 3. Design of CAPs molecular markers

[0048] Based on the significant mutations discovered in the CDS1 region, specific amplification primer pairs (F: 5'-CGGAGTCGTACCCCGCT-3'; R: 5'-TGGCCGGGAGGAATCACAAAT-3') were designed for this region and its flanking sequences. Combined with the ApaI restriction endonuclease, a CAPs molecular marker capable of efficiently distinguishing this resistant / susceptible haplotype was successfully developed and named BM_phs2_CAPs.

[0049] Example 2: Application of molecular markers BM_phs2_CAPs

[0050] 1. Experimental materials

[0051] Wheat samples to be analyzed: 296 white-grained wheat materials, including: wheat spike sprouting resistance control: the superior wheat variety Bima 6, carrying the resistance gene haplotype Taphs2-6A-BM; wheat spike sprouting susceptibility control: Chinese Spring.

[0052] Reagent: 2×Takara Ex Premier TM (Takara Bio, Dalian, China)、QuickCut TM ApaI endonuclease (Takara Bio, Dalian, China).

[0053] 2. Detection primers for molecular marker BM_phs2_CAPs

[0054] Based on the analysis of variant sites in the CDS1 region of the Taphs2-6A-BM gene, the following specific primers were designed:

[0055] Forward Primer (F): 5'-CGGAGTCGTACCCGCT-3';

[0056] Reverse Primer (R): 5' -TGGCCGGGAGGAATCACAAAT-3'.

[0057] 3. Testing Procedures

[0058] Genomic DNA extraction: Genomic DNA was extracted from wheat leaves using the conventional CTAB method.

[0059] PCR amplification:

[0060] Reaction system (20 μL): 1.0 μL DNA template (approximately 50 ng), 1.0 μL each of forward and reverse primers (10 μM), 2×Takara Ex Premier TM Mix 10.0 μL, then add ddH2O to bring the total volume to 20 μL.

[0061] Amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 67.5℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ final extension for 10 min.

[0062] ApaI enzyme digestion reaction:

[0063] PCR product 10 μL, 10× QuickCut Green Buffer 2 μL, QuickCut TMAdd 1 μL of Apa I endonuclease (Takara Bio, Dalian, China) to a final volume of 20 μL with ddH2O, and digest at 37°C for 2-4 hours.

[0064] Electrophoresis detection: The enzyme digestion products were detected by electrophoresis on a 1.5% agarose gel at 120V for 20-30 minutes.

[0065] 4. Result Determination

[0066] Based on the size of the amplified fragment and the distribution of ApaI restriction sites, the judgment criteria are as follows:

[0067] Resistance haplotype (R type): The PCR amplification product is 511 bp in length. Due to a base deletion in the CDS1 region, this fragment contains only one ApaI restriction site (located at 385 bp). After digestion, two bands are generated: 385 bp and 126 bp. Characteristic band: 385 bp.

[0068] The susceptible haplotype (S type) has a PCR amplification product length of 529 bp. This sequence contains two ApaI restriction sites (located at 146 bp and 403 bp, respectively). After restriction, three bands are generated: 146 bp, 257 bp, and 126 bp. Characteristic bands are observed at 257 bp and 146 bp.

[0069] Hybrid type (RS type): Characteristic bands of 385 bp, 257 bp and 146 bp appear simultaneously.

[0070] 5. Test Results

[0071] 296 white-grained wheat samples were tested, and the agarose gel electrophoresis images of some of the wheat CAPs-labeled enzyme digestion products are shown below. Figure 2 As shown in the figure. The results showed that materials with a 385 bp characteristic band after enzyme digestion of the amplified product had a low field germination index; while materials with 257 bp and 146 bp bands were susceptible to disease. These results indicate that the BM_phs2_CAPs marker can accurately and rapidly achieve molecular-assisted selection for wheat spike germination resistance.

[0072] 6. Association analysis between marker genotypes and multi-year field ear budding phenotypes

[0073] To further verify the reliability of the BM_phs2_CAPs marker in breeding practice, this invention collected field spike sprouting index (PHS index) phenotypic data of the aforementioned 296 white-grained wheat materials in 2023 and 2024, and performed a correlation analysis based on the CAPs marker genotyping results (resistant haplotype R, heterozygous RS, and susceptible haplotype S). The results showed that, under both 2023 and 2024 environmental conditions, the average spike sprouting index of wheat populations carrying the resistant haplotype (R type, characteristic band 385 bp) was significantly lower than that of wheat populations carrying the susceptible haplotype (S type, characteristic band 257 bp + 146 bp). The spike sprouting index of the heterozygous (RS type) population was between the two, significantly lower than that of the susceptible haplotype (R type, characteristic band 257 bp + 146 bp). Figure 3 This stable phenotypic difference across years fully demonstrates that the BM_phs2_CAPs marker can accurately capture the true resistance effect of the Taphs2-6A gene, and can serve as an efficient and reliable tool for marker-assisted selection (MAS) breeding of white-grained wheat to resist pre-sprouting.

[0074] Table 1. Detailed list of 296 white-grained wheat germplasm samples from Henan Province

[0075]

[0076]

[0077]

[0078]

[0079] Table 2. Genotyping of BM_phs2_CAPs markers in 296 natural wheat germplasm populations.

[0080]

[0081]

[0082]

[0083]

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the Taphs2-6A gene in regulating wheat ear germination resistance, characterized in that, The nucleotide sequence of the Taphs2-6A gene is shown in SEQ ID NO:

1. The Taphs2-6A gene shown in SEQ ID NO:1 positively regulates wheat ear germination resistance.

2. A product for detecting the molecular marker BM_phs2_CAPs, characterized in that, The product is a reagent or kit, comprising primer pairs and the restriction endonuclease ApaI, wherein the primer pairs include: F: 5'-CGGAGTCGTACCCCGCT-3'; R: 5'-TGGCCGGGAGGAATCACAAAT-3'.

3. The application of the molecular marker BM_phs2_CAPs or products that detect the molecular marker BM_phs2_CAPs in any of the following ways, characterized in that: The molecular marker BM_phs2_CAPs is located in the first coding region of the Taphs2-6A gene; the applications include: A: To identify or assist in the identification of wheat ear germination resistance; B: Breeding for resistance to wheat ear sprouting.

4. A method for identifying or assisting in the identification of wheat ear germination resistance, characterized in that, Includes the following steps: The Taphs2-6A gene haplotype in the genomic DNA of the wheat to be identified was detected. The average germination index of the wheat population with the Taphs2-6A gene haplotype shown in SEQ ID NO:1 was significantly lower than that of the wheat population with the Taphs2-6A gene haplotype shown in SEQ ID NO:

2.

5. A method for identifying or assisting in the identification of wheat ear germination resistance, characterized in that, Includes the following steps: (1) Using the wheat genomic DNA to be identified as a template, PCR amplification is performed using the primer pair described in claim 2 to obtain PCR products; (2) The PCR product was digested with the restriction endonuclease ApaI; (3) Identify wheat ear sprouting resistance based on the characteristic bands after enzyme digestion: those containing a 385 bp characteristic band after enzyme digestion are ear sprouting resistance haplotypes, and those containing 257 bp and 146 bp characteristic bands after enzyme digestion are ear sprouting disease susceptible haplotypes.

6. A method for breeding wheat with resistance to germination at the ear, characterized in that, include: (1) Detecting wheat ear germination resistance using the method described in claim 4 or 5; (2) Select appropriate wheat parents for breeding according to the breeding objectives.

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