Star p molecular marker of wheat drought-resistance key gene tabhlh324 and application thereof

Abstract

The application discloses a STARP molecular marker of a wheat drought-resistant key gene TabHLH324 and application thereof, and belongs to the technical field of crop selection and cultivation. The drought-resistant key gene TabHLH324 comprises two haplotypes, the STARP molecular marker can be amplified by primers shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, when the amplification product only has a DNA fragment with a size of 193bp, the wheat haplotype is TabHLH324-I, and when the amplification product has two DNA fragments with sizes of 193bp and 201bp respectively, the wheat haplotype is TabHLH324-II; the TabHLH324-II haplotype wheat has a higher survival rate under drought stress. The application has the advantages of improved detection compatibility, greatly reduced detection cost and stronger typing specificity.

Description

Technical Field

[0001] This invention relates to a molecular marker and its application, specifically to the STARP molecular marker of the wheat drought-resistant key gene TabHLH324 and its application in wheat-assisted breeding, belonging to the field of crop selection and breeding technology. Background Technology

[0002] Wheat survival rate under drought conditions is a key indicator for evaluating the drought resistance of varieties, field cultivation management levels, and regional ecological adaptability of varieties, directly reflecting their ability to adapt to drought environments. Understanding the dynamic changes in wheat survival rate under drought stress is crucial for screening superior drought-resistant germplasm, optimizing planting density and water and fertilizer management techniques, and providing data for regional variety introduction, early warning of agricultural drought disasters, and ensuring stable grain yields. This is of great significance for strengthening food security and improving the economic benefits of crop farming. Therefore, identifying key drought-resistant genes that regulate wheat survival and developing related molecular markers can enable precise and rapid screening of superior drought-resistant germplasm, targeted aggregation of multiple desirable traits, significantly shortening the breeding process, and efficiently cultivating new drought-resistant and stable-yielding wheat varieties.

[0003] Currently, large-scale commercial wheat drought-resistant breeding generally uses KASP markers to complete high-throughput rapid genotyping of large numbers of progeny. However, the genotyping system relies on commercially available specific fluorescent premixes, has a single primer modification mode, and only supports fluorescence microplate detection, resulting in high detection costs and high equipment barriers. In contrast, STARP markers do not rely on dedicated commercial fluorescent kits. Their double mismatch primer design can effectively reduce non-specific amplification caused by homologous sequences in hexaploid wheat, and they combine both high-throughput fluorescence detection and gel electrophoresis genotyping. This makes them suitable for large-scale germplasm screening in breeding laboratories at different levels, and they are more suitable for large-scale genotyping identification and molecular marker-assisted breeding of superior drought-resistant wheat germplasm. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a STARP molecular marker for the key wheat drought resistance gene TabHLH324 and its application, thereby offering a low-cost and low-impact detection method for the genetic improvement of wheat yield traits.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: Application of the STARP molecular marker of the drought-resistant key gene TabHLH324 in the breeding of TabHLH324-II haplotype wheat. The drought-resistant key gene TabHLH324 includes two haplotypes, TabHLH324-I and TabHLH324-II. The STARP molecular marker can be amplified by specific primers shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. The amplification products are separated by electrophoresis using a gel. When the amplification product contains only a 193 bp DNA fragment, the wheat haplotype is TabHLH324-I. When the amplification product contains two DNA fragments of 193 bp and 201 bp, respectively, the wheat haplotype is TabHLH324-II. The survival rate of TabHLH324-II haplotype wheat under drought stress is higher than that of TabHLH324-I haplotype wheat.

[0006] The advantages of this invention are: (1) Improved detection compatibility: KASP can only be detected by fluorescence instrument, while STARP can be directly detected by gel electrophoresis for small-scale field screening, which lowers the equipment threshold and makes the detection mode more flexible; (2) Detection costs are significantly reduced: The high-priced KASP-specific fluorescent Master Mix (containing fluorescence quenching probes) is eliminated, reducing the cost of large-scale breeding population screening by more than 40%; (3) Stronger typing specificity: STARP double-site mismatch primers significantly reduce non-specific amplification of homologous sequences in hexaploid wheat, and the typing accuracy of heterozygous materials is higher than that of KASP markers at the same site. Attached Figure Description

[0007] Figure 1 This is a graph showing the results of detecting the expression level of the TabHLH324 gene under drought stress. Figure 2 This is a 10% non-denaturing polyacrylamide gel electrophoresis image of some wheat varieties; Figure 3 This is a graph showing the association between haplotypes of the TabHLH324 gene and wheat survival rate under drought stress. Detailed Implementation

[0008] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0009] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0010] I. Cloning the TabHLH324 gene Download the DNA sequence (SEQ ID NO: 1) of the TabHLH324 gene (gene number: TraesZ8425B5B01G261700) from the WheatOmics website.

[0011] Specific primers for amplifying the TabHLH324 gene were designed using DNAMAN software. The design results are as follows: TabHLH324-F: 5'-GTAAACATCCGCCATTATAAATACC-3' (SEQ ID NO: 3); TabHLH324-R: 5'-GATTTTATTCCGGGGCAC-3' (SEQ ID NO: 4).

[0012] DNA was extracted from the common wheat variety Fielder using the CTAB method. Using this DNA as a template, the DNA sequence of the TabHLH324 gene was amplified using specific primers TabHLH324-F / R.

[0013] The PCR amplification system is as follows: 0.2 μL each of 2 μmol / L primers, 1 μL of 100 ng / μL DNA template, 5 μL of 2×Taq MasterMix (without fluorescence quenching probe), and 3.6 μL of ddH2O.

[0014] The PCR amplification program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 15 s, 58℃ annealing for 20 s, 72℃ extension for 1 min, 35 cycles; store at 4℃ protected from light.

[0015] The amplified PCR products were sampled and detected using a 1% agarose gel. The electrophoresis apparatus was set to 150V and the electrophoresis time was 15min. After electrophoresis, the products were observed under a gel imaging system. The clear, bright bands of the correct length were cut off and purified using the FastPure Gel DNA Extraction Mini Kit from Nanjing Vazyme Biotechnology Co., Ltd.

[0016] After purification, the PCR products were ligated into the pCE2 TA / Blunt-Zero vector using the 5min TA / Blunt-ZeroCloning Kit from Nanjing Vazyme Biotechnology Co., Ltd.

[0017] Subsequent transformation experiments were performed using Fast-T1 competent E. coli cells, followed by single-clone sequencing. The single-clone sequencing results are shown in SEQ ID NO: 2.

[0018] II. Detection of TabHLH324 gene expression level under drought stress Seedlings of Qingmai 11 with uniform growth and free from pests and diseases were selected and transferred to uniformly sized hydroponic boxes for cultivation in 1 / 5 Hoagland nutrient solution. After the seedlings reached the two-leaf-one-heart stage, 250 mmol / L PEG 6000 was added to the 1 / 5 Hoagland nutrient solution for drought stress treatment. At 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h after drought stress treatment, using the wheat TaActin gene as an internal control and leaf cDNA of Qingmai 11 as a template, quantitative real-time PCR was performed using the ChamQ SYBR Color qPCR Master Mix kit produced by Nanjing Vazyme Biotechnology Co., Ltd., employing the following primer pairs. Two... −ΔΔCt The expression level of the TabHLH324 gene was analyzed by relative quantification.

[0019] The primer pairs used for real-time PCR are: qRT-TabHLH324-F: 5'-CGAAGCACCAGTGACCGT-3' (SEQ ID NO: 5); qRT-TabHLH324-R: 5'-GTACATCCCCCACCCAACA-3' (SEQ ID NO: 6); TaActin-F: 5'-TATGCCAGCGGTCGAACAAC-3' (SEQ ID NO: 7); TaActin-R: 5'-GGAACAGCACCTCAGGGCAC-3' (SEQ ID NO: 8).

[0020] The expression level of the TabHLH324 gene under drought stress is shown in the following results. Figure 1 .Depend on Figure 1 It can be seen that in the early stage of drought stress (3 to 12 hours of drought stress treatment), the expression of the TabHLH324 gene was significantly upregulated. As the duration of drought stress increased (24 to 48 hours of drought stress treatment), the expression of the TabHLH324 gene decreased due to the activation of negative feedback regulatory pathways.

[0021] Therefore, it is determined that the wheat TabHLH324 gene is an important gene in response to drought stress.

[0022] III. Identifying SNP variation sites in the key wheat drought-resistance gene TabHLH324 By performing PCR amplification and resequencing on the TabHLH324 gene of wheat germplasm with different genetic backgrounds, a substitution mutation was detected at 2210 bp downstream of the start codon ATG in different wheat varieties, changing the base G to the base C. When the base at 2210 bp downstream of the start codon ATG is G, the haplotype corresponding to the TabHLH324 gene is denoted as TabHLH324-I (SEQ ID NO: 9), and when the base at 2210 bp is C, the haplotype corresponding to the TabHLH324 gene is denoted as TabHLH324-II (SEQ ID NO: 10).

[0023] IV. Development of STARP Molecular Markers To target the G / C variation at 2210 bp downstream of the start codon ATG in the TabHLH324 gene, a STARP molecular marker was developed, and STARP-specific amplification primers (STARP-TabHLH324-F1 / F2 / R) were designed for the detection and identification of the wheat TabHLH324 gene.

[0024] STARP-TabHLH324-F1: 5'-AATCAAATCAACCATCCCCTG-3' (SEQ ID NO: 11); STARP-TabHLH324-F2: 5'-ACGACTGCAATCAAATCAACCATCCTC-3' (SEQ ID NO: 12); STARP-TabHLH324-R: 5'-GAGATTTTATTCCGGGGCAC-3' (SEQ ID NO: 13).

[0025] V. Group Validation DNA from different wheat varieties was extracted using the CTAB method. Using wheat DNA as a template, PCR amplification was performed using STARP-specific amplification primers (STARP-TabHLH324-F1 / F2 / R).

[0026] The PCR amplification system is as follows: 2 μL of 2 μmol / L mixed primers, 1.5 μL of 100 ng / μL DNA template, 5 μL of 2×TaqMaster Mix (without fluorescence quenching probe), and 1.5 μL of ddH2O.

[0027] Preparation method of 2μmol / L mixed primers: Mix STARP-TabHLH324-F1, STARP-TabHLH324-F2 and STARP-TabHLH324-R, each with a stock solution concentration of 100μmol / L, at a volume ratio of 1:1:2, and then dilute with enzyme-free water 50 times.

[0028] The target fragment was amplified by falling PCR: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing at 65℃ down to 56℃ for 30 s in each cycle, decreasing by 1℃ per cycle, extension at 72℃ for 15 s, for 10 cycles; 95℃ denaturation for 30 s, annealing at 59℃ for 30 s, extension at 72℃ for 15 s, for 25 cycles; extension at 72℃ for 10 min; storage at 4℃.

[0029] The amplification products were separated by electrophoresis in a 10% non-denaturing polyacrylamide gel. The electrophoresis buffer was 1×TBE, and the gel was kept at a constant voltage of 150V for 5 hours. Silver staining was then performed. The developed gel was placed on a viewing lamp, and gel images were taken and the band patterns recorded. Figure 2 As shown.

[0030] Depend on Figure 2 It can be seen that the developed STARP molecular marker (STARP-TabHLH324) exhibits significant polymorphism among different wheat varieties, with differences in the number and size of the amplified specific bands. Specifically, the amplification product of haplotype TabHLH324-Ⅰ contains only a 193bp (SEQ ID NO: 14) DNA fragment, while the amplification product of haplotype TabHLH324-Ⅱ contains two DNA fragments with sizes of 201bp (SEQ ID NO: 15) and 193bp (SEQ ID NO: 14), respectively.

[0031] VI. Analysis of the significant differences in wheat survival rates under drought stress Genotyping of 104 wheat varieties was performed using the STARP molecular marker (STARP-TabHLH324) of the TabHLH324 gene.

[0032] The genotyping results of 104 wheat samples are shown in Tables 1-1 and 1-2.

[0033] Table 1-1 Genotyping results of 104 wheat samples (Part 1)

[0034] Table 1-2 Genotyping results of 104 wheat samples (Part II)

[0035] Sixty plump, uniformly sized seeds of each variety were selected and cultured under suitable conditions until they began to germinate. Nutrient soil and vermiculite were thoroughly mixed at a volume ratio of 3:1 and then divided into uniform black culture pots (length × width × height: 6.5cm × 6.5cm × 7.5cm). The culture pots were neatly arranged in a tray, and tap water was slowly added using a bottom-drip irrigation method. After the substrate was fully saturated, excess water was poured out to prevent excessive humidity and seed rot. Four seeds with consistent germination were transplanted into the culture pots, sown at a depth of 2-3cm below the substrate surface. After sowing, the substrate was gently pressed to ensure close contact between the seeds and soil. All culture pots were placed in an artificial climate chamber and cultured under uniformly suitable conditions. The control group received conventional water management; the treatment group underwent natural drought stress treatment after watering was stopped when the plants reached the two-leaf-one-heart stage. Rehydration was initiated after the leaves had completely turned yellow and was continued for 10 days. The survival rate of each plant strain was then recorded. Independent samples t-tests were used, and statistical analysis was performed using SPSS software. The significance level was set at P<0.001.

[0036] The association analysis results between haplotypes of the key wheat drought resistance gene TabHLH324 and wheat survival rate under drought stress are shown in Table 2 and 3. Figure 3 .

[0037] Table 2. Association analysis results of haplotypes of the key drought resistance gene TabHLH324 in wheat and wheat survival rate under drought stress.

[0038] Note: *** represents P<0.001 (significant difference).

[0039] From Table 2 and Figure 3 It can be seen that under drought stress, the average survival rate of TabHLH324-II wheat was significantly higher than that of TabHLH324-I wheat (P<0.001).

[0040] The above research results indicate that the STARP marker (STARP-TabHLH324) developed in this invention is significantly associated with the survival rate of wheat under drought stress.

[0041] In summary, haplotype TabHLH324-II wheat has a higher survival rate under drought stress than haplotype TabHLH324-I wheat. Haplotype TabHLH324-II is an excellent haplotype for wheat and has potential value in the breeding of high-yielding varieties.

[0042] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. The application of primers for detecting the STARP molecular marker of the key drought-resistance gene TabHLH324 in the breeding of TabHLH324-II haplotype wheat, characterized by: The sequence of the key drought-resistant gene TabHLH324 is shown in SEQ ID NO: 1; The primers are: STARP-TabHLH324-F1:5'-AATCAAATCAACCATCCCCTG-3'; STARP-TabHLH324-F2: 5'-ACGACTGCAATCAAATCAACCATCCTC-3'; STARP-TabHLH324-R: 5'-GAGATTTTATTCCGGGGCAC-3'; The drought-resistant key gene TabHLH324 includes two haplotypes, TabHLH324-I and TabHLH324-II. Wheat with haplotype TabHLH324-II has a higher survival rate under drought stress than wheat with haplotype TabHLH324-I. The amplification products of the primers were separated by electrophoresis using a gel. When the amplification product contained only a DNA fragment of 193 bp, the haplotype of wheat was TabHLH324-I. When the amplification product contained two DNA fragments of 193 bp and 201 bp, respectively, the haplotype of wheat was TabHLH324-II.

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