Molecular marker fragment related to wheat phosphorus efficient utilization character and application of molecular marker fragment

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a molecular marker fragment related to wheat phosphorus efficient utilization characters and application of the molecular marker fragment. The invention provides a molecular marker fragment related to a wheat phosphorus efficient utilization character. The molecular marker fragment comprises a TaMYB4-7A gene promoter region; the nucleotide sequence of a promoter region of the TaMYB4-7A gene is as shown in SEQ ID NO. 1. The molecular marker fragment can be used for screening wheat varieties with efficient phosphorus utilization, the screening efficiency of wheat with efficient phosphorus utilization is improved, and wheat germplasm breeding is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a molecular marker fragment related to the trait of high phosphorus utilization in wheat and its application. Background Technology

[0002] Phosphorus is one of the three essential nutrients for crop growth and development, but the available phosphorus content in the soil often fails to meet the needs of normal crop growth and development. Phosphorus deficiency has become an important problem affecting crop yield and quality (Natr L. Mineral nutrients-Aubiquitous stress factor for photosynthesis[J]. Photosynthetica 1992,27:271–294.; Secco D, Jabnoune M, Walker H, et al. Spatio–temporal transcript profiling of rice roots and shoots in response to phosphate starvation and recovery[J]. Plant Cell 2013,25:4285–4304.; Lopez-Arredondo DL, Leyva-Gonzalez MA, Gonzalez-Morales SI, et al. Phosphate nutrition:improving low-phosphate tolerance in crops[J]. Annu Rev Plant Biol 2014,65:95–123.). In agricultural production, large amounts of phosphate fertilizer are often applied to increase the inorganic phosphorus content in the soil in order to ensure crop yield. However, due to the low absorption and utilization efficiency of phosphate fertilizer by crops, most of the phosphate fertilizer is wasted because it is not absorbed and utilized by crops (Li H, Huang G, Meng Q, et al. Integrated soil and plant phosphorus management for crop and environment in China. A review[J]. Plant Soil 2011,349:157–167.; Sattari SZ, Bouwman AF, Gillera KE, et al. Residual soil phosphorus as the missing piece in the global phosphorus crisis puzzle[J]. Proc Natl Acad Sci USA 2012,109:6348–6353.).

[0003] Wheat is the most widely distributed and cultivated grain crop in the world. However, wheat production also faces the problem of low phosphorus fertilizer utilization, with an average utilization efficiency of only 19% in the current season (Grierson CS, Barnes SR, Chase MW, et al. One hundred important questions facing plant science research[J]. New Phytologist, 2011, 192(1):6–12.). Currently, the molecular mechanisms of phosphorus absorption and utilization in wheat are unclear, and there is a lack of phosphorus-efficient candidate genes, which severely limits the breeding of new phosphorus-efficient wheat varieties. Therefore, identifying key genes involved in the efficient absorption and utilization of phosphorus in wheat, elucidating their molecular mechanisms, and breeding new phosphorus-efficient wheat varieties are crucial for improving phosphorus fertilizer utilization efficiency in wheat production. This is of great significance for achieving green and efficient wheat production, agricultural energy conservation and emission reduction, and the protection of phosphate resources and the environment.

[0004] Current research on transcriptional regulation of phosphorus uptake and utilization in plants mainly focuses on model plants such as Arabidopsis thaliana and rice. Due to the large size and complexity of the wheat genome, related research on wheat has lagged behind. Therefore, developing molecular markers associated with efficient phosphorus utilization traits in wheat holds promise for providing new theoretical basis and technical means for high-yield wheat breeding, and has significant scientific value and application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker fragment related to the phosphorus-efficient utilization trait in wheat and its application. The molecular marker fragment described in this invention can improve the screening efficiency of phosphorus-efficient wheat.

[0006] This invention provides a molecular marker fragment associated with the trait of high phosphorus utilization in wheat, the molecular marker fragment containing the promoter region of the TaMYB4-7A gene; the nucleotide sequence of the promoter region of the TaMYB4-7A gene is shown in SEQ ID NO.1.

[0007] The present invention also provides a primer pair for identifying the molecular marker fragment described in the above scheme, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.3.

[0008] The present invention also provides a kit for detecting the molecular marker fragments described in the above scheme, comprising the primer pairs and PCR amplification reagents described in the above scheme.

[0009] This invention also provides a method for screening phosphorus-efficient wheat varieties, including genomic selection.

[0010] Wheat samples containing the molecular marker fragments described in the above-mentioned scheme in their DNA.

[0011] As a preferred embodiment, the method includes using the primer pairs or kit described above to perform PCR amplification with the genomic DNA of a wheat sample as a template. When the length of the amplification product is 2113 bp, it indicates that the genomic DNA of the wheat sample contains the molecular marker fragment described above, which is a phosphorus-efficient wheat variety.

[0012] This invention also provides the application of the molecular marker fragment, primer pair, kit, or method described above in wheat breeding.

[0013] This invention also provides the application of the molecular marker fragment, primer pair, kit, or method described above in wheat-assisted breeding.

[0014] This invention also provides the application of the molecular marker fragment, primer pair, kit, or method described above in identifying traits of high phosphorus utilization efficiency in wheat.

[0015] This invention also provides the application of the molecular marker fragment, primer pair, kit, or method described above in screening phosphorus-efficient wheat.

[0016] This invention also provides a method for molecular marker-assisted breeding of phosphorus-efficient wheat varieties, which involves using the primer pairs or kits described above to perform molecular markers on target wheat, and then screening wheat germplasm containing the molecular marker fragments described above for subsequent screening.

[0017] This invention provides a molecular marker fragment associated with high phosphorus utilization efficiency in wheat. The molecular marker fragment contains the promoter region of the TaMYB4-7A gene; the nucleotide sequence of the TaMYB4-7A gene promoter region is shown in SEQ ID NO.1. This invention utilizes the molecular marker fragment to screen for high phosphorus utilization wheat varieties, improving the screening efficiency and benefiting wheat germplasm breeding. Using suitable primer pairs, this invention allows for sample identification through simple DNA extraction, PCR-specific amplification, and detection, facilitating the detection and screening of wheat varieties or lines with high phosphorus utilization efficiency, and significantly accelerating the breeding process of high-yielding wheat varieties. Attached Figure Description

[0018] 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.

[0019] Figure 1These are electrophoresis images of PCR amplification detection of the TaMYB4-7A coding region and promoter region; where A is the electrophoresis image of coding region amplification detection; and B is the electrophoresis image of promoter region amplification detection.

[0020] Figure 2 The allelic variation sites of the TaMYB4-7A promoter and coding region sequence in different wheat varieties and the two haplotypes (Hap1 and Hap2) formed therefrom;

[0021] Figure 3 This study analyzed the transcriptional initiation activities of different TamyB4-7A haplotypes (Hap1 and Hap2) promoters in tobacco leaves. A shows fluorescence images of *N. benthamiana* leaves 48 hours after infection with the recombinant vectors of the two promoters; red and blue represent strong and weak activities, respectively. B shows the ratio of LUC to REN activities 48 hours after infection with the recombinant vectors of the two promoters. "*" indicates p < 0.05.

[0022] Figure 4 The relative expression levels of TaMYB4-7A in two haplotype wheat varieties;

[0023] Figure 5 The low phosphorus tolerance coefficients for two haploid wheat plants;

[0024] Figure 6 Results of the development and testing of the TaMYB4-7A phosphorus-efficient molecular marker. Detailed Implementation

[0025]

[0026]

[0027] This invention also provides a primer pair for identifying the molecular marker fragment described in the above-described scheme, comprising an upstream primer (5′-TCTTGTCCGATGAGTACACGG A-3′) with the nucleotide sequence shown in SEQ ID NO.2 and a downstream primer (5′-CTGCTTGCTCTCTT GGGAAG-3′) with the nucleotide sequence shown in SEQ ID NO.3. The primer pair described in this invention can be used to amplify the TaMYB4-7A promoter region for screening phosphorus-efficient wheat.

[0028] This invention also provides a kit for detecting the molecular marker fragments described in the above-described scheme, comprising the primer pairs and PCR amplification reagents described in the above-described scheme. This invention does not specifically limit the PCR amplification reagents; conventional PCR amplification reagents in the art can be used.

[0029] This invention also provides a method for screening phosphorus-efficient wheat varieties, comprising selecting wheat samples whose genomic DNA contains the molecular marker fragments described in the above-described scheme. As one embodiment, this includes using the primer pairs or kit described in the above-described scheme to amplify the genomic DNA of the wheat sample as a template. When the length of the amplified product is 2113 bp, it indicates that the genomic DNA of the wheat sample contains the wheat sample, which is a phosphorus-efficient wheat variety. In a specific embodiment of this invention, the PCR amplification reaction system, in 50 μL increments, contains 25 μL of 2×ApexHF CLBuffer (Mg... 2+ The reagents included 1 μL of dNTP plus, 1 μL of LApexHF HSDNA Polymerase CL (1 U / μL), 1 μL each of forward and reverse primers (10 μM), 200 ng of DNA template, and ddH2O to make up the volume. The PCR amplification reaction program was as follows: Step 1, 94℃ for 1 min; Step 2, 98℃ for 10 s, 60℃ for 15 s, 68℃ for 90 s, for 35 cycles. This invention uses the primers to amplify the genomic DNA of wheat samples as a template. When the amplification product length is 2113 bp, it indicates that the genomic DNA of the wheat sample contains the molecular marker fragment described in the above scheme, indicating a phosphorus-efficient wheat variety; when the amplification product length is 1430 bp, it indicates that the genomic DNA of the wheat sample does not contain the molecular marker fragment described in the above scheme, indicating a non-phosphorus-efficient wheat variety.

[0030] This invention also provides the application of the molecular marker fragment, primer pair, kit, or method described above in wheat breeding.

[0031] This invention also provides the application of the molecular marker fragment, primer pair, kit, or method described above in wheat-assisted breeding.

[0032] This invention also provides the application of the molecular marker fragment, primer pair, kit, or method described above in identifying traits of high phosphorus utilization efficiency in wheat.

[0033] This invention also provides the application of the molecular marker fragment, primer pair, kit, or method described above in screening phosphorus-efficient wheat.

[0034] This invention also provides a method for molecular marker-assisted breeding of phosphorus-efficient wheat varieties, which involves using the primer pairs or kits described above to perform molecular markers on target wheat, and then screening wheat germplasm containing the molecular marker fragments described above for subsequent screening.

[0035] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0036] The high-fidelity PCR enzyme ApexHF HSDNA polymerase CL used in this invention for PCR amplification was purchased from Aikerui Biotechnology Co., Ltd. qRT-PCR experiments were performed using ChamQ SYBR qPCRMasterMix (HighROX Premixed). The zero-background ZT4 Simple-Blunt rapid cloning kit (without MCS) was purchased from Beijing Zhuangmeng Biotechnology Co., Ltd. Other reagents were domestically produced analytical grade. The pGreenII0800-LUC vector was used for promoter activity analysis.

[0037] Example 1

[0038] (1) Primer design: Based on the sequence differences of the three copies of the TaMYB4 gene, 7A (TraesCS7A02G272100), 7B (TraesCS7B02G169900), and 7D (TraesCS7D02G272400), in the wheat genome database (http: / / plants.ensembl.org / Triticum_aestivum / Info / Index), 7A-specific promoter region amplification primers TaMYB4-7A-Promoter-SF and TaMYB4-7A-Promoter-SR, and coding region amplification primers TaMYB4-7A-CDS-SF and TaMYB4-7A-CDS-SR, were designed. Primer synthesis and sequencing were performed by Henan Shangya Biotechnology Co., Ltd. The primer sequence information is shown below:

[0039] TaMYB4-7A-Promoter-SF (SEQ ID NO.2): 5′-TCTTGTCCGATGAGTACA CGGA-3′;

[0040] TaMYB4-7A-Promoter-SR (SEQ ID NO.3): 5′-CTGCTTGCTCTCTTGGGA AG-3′;

[0041] TaMYB4-7A-CDS-SF (SEQ ID NO.4): 5′-AGAGAGCAAGCAGATGAGA GG-3′;

[0042] TaMYB4-7A-CDS-SR (SEQ ID NO. 5): 5′-TACTAGTCTCCTCCTCACCA-3′.

[0043] (2) Extraction of total wheat DNA

[0044] This invention selected 255 common wheat varieties (see Table 1) and extracted total DNA from wheat using the CTAB method. First, wheat leaves or other tissues were rapidly frozen and ground in liquid nitrogen, and 1 mL of preheated CTAB extraction buffer was added. The mixture was then incubated at 65°C for 1 hour. After centrifugation at 12000g, the supernatant was collected and an equal volume of chloroform:isoamyl alcohol (24:1, v:v) was added and vortexed. The mixture was centrifuged at 12000g for 10 minutes, and the supernatant was transferred to a new tube. Two volumes of anhydrous ethanol were added and mixed, and the tube was incubated at -20°C for 30 minutes. The supernatant was then discarded by centrifugation, and the tube was washed twice with 75% ethanol. After drying, 100 μL of sterile water was added to dissolve the DNA, yielding wheat DNA.

[0045] (3) Amplification and sequencing of the promoter and coding region of the TaMYB4-7A gene

[0046] The target fragment was amplified using the high-fidelity PCR enzyme ApexHF HSDNA polymerase CL; the promoter and coding domain sequences of TaMYB4-7A were amplified using the wheat DNA in step (2) as a template with the two primer pairs TaMYB4-7A-Promoter-SF / R and TaMYB4-7A-CDS-SF / R.

[0047] The PCR system contains: 25 μL 2×ApexHFCLBuffer (Mg 2+The reagents included 1 μL of dNTP plus, 1 μL of LApex HFHS DNA Polymerase CL (1 U / μL), 1 μL each of forward and reverse primers (10 μM), 200 ng of DNA template, and ddH2O to a total volume of 50 μL. The PCR program for the TaMYB4-7A promoter region was as follows: Step 1, 94℃ for 1 min; Step 2, 98℃ for 10 s, 60℃ for 15 s, 68℃ for 90 s, for 35 cycles. The PCR program for the TaMYB4-7A coding region was as follows: Step 1, 94℃ for 1 min; Step 2, 98℃ for 10 s, 60℃ for 15 s, 68℃ for 1 min, for 35 cycles.

[0048] Amplification results as follows Figure 1 As shown, according to Figure 1 It can be seen that the promoter amplification fragment of TaMYB4-7A is 2113bp, and the coding region amplification fragment is 998bp.

[0049] The target fragments for PCR amplification of the above 255 wheat varieties were recovered and ligated into the ZT4 Simple-Blunt cloning vector, transformed into E. coli, and at least three positive clones were selected for sequencing using universal primers ZT4 Simple Forward Primer-23-mer and ZT4 Simple Reverse Primer-24-mer.

[0050] ZT4 Simple Forward Primer-23-mer (SEQ ID NO.6): 5′-CGACTCACTATAGGGAGAGCGTC-3′;

[0051] ZT4 Simple Reverse Primer-24-mer (SEQ ID NO. 7): 5′-AAGAACATCGCTTTTCGATGGCAG-3′.

[0052] (5) Sequence alignment and haplotype analysis of the promoter and coding region of the TaMYB4-7A gene

[0053] The sequencing results from step (4) were assembled and sequence aligned using Snapgene software. The sequence alignment revealed no sequence differences in the coding region of TaMYB4-7A across 255 wheat varieties. Five SNPs and four Indel polymorphic variants were found in the promoter region, tightly linked to form two haplotypes, named TaMYB4-7A-Hap1 and TaMYB4-7A-Hap2, respectively. (See [link to relevant documentation]). Figure 2The nucleotide sequence of TaMYB4-7A-Hap1 is shown in SEQ ID NO.1, and the nucleotide sequence of TaMYB4-7A-Hap2 is shown in SEQ ID NO.8. The haplotype results for wheat varieties are shown in Table 1.

[0054] Table 1. Haplotypes of different wheat varieties

[0055]

[0056]

[0057]

[0058]

[0059] (6) Activity determination of two TaMYB4-7A haplotype promoters

[0060] To detect differences in promoter activity among different TamYB4-7A haplotypes, hexaploid wheat DNA from both haplotypes was used as templates. The promoter fragments of haplotypes Hap1 and Hap2 were amplified using primers TamYB4-7A-promoter-LUC-F and TamYB4-7A-promoter-LUC-R. The promoter fragment of haplotype Hap1 was 2113 bp, and the promoter fragment of haplotype Hap2 was 1430 bp.

[0061] TaMYB4-7A-promoter-LUC-F (SEQ ID NO.9): 5′-TCACTATAGGGCGAATTGGGTACCCGAAGATGATCAGTGTAACG-3′;

[0062] TaMYB4-7A-promoter-LUC-R (SEQ ID NO. 10): 5′-GCTGCAGGAATTCGATATCAAGCTGGCCGATCTTTGATGGTGGT-3′.

[0063] The amplified Hap1 and Hap2 haplotype promoter fragments were inserted into the pGreenII 0800-LUC reporter plasmid, respectively, to obtain recombinant plasmids, which were then transformed into Agrobacterium GV3101 (pSoup-p19) strain for further infection of Tobacco Bengal leaves. Images were captured using an automated chemiluminescence imaging system (Tamon 5200, Shanghai, China). Finally, the relative LUC activity (LUC / REN) was measured using a dual-luciferase reporter gene kit. The method is described in [Shi HT; Lou, C.; Wang JF.; Dong D Q.; Yang LF.; Li GZ.; Tian Z Q.; Han Q X.; Wang P F.; Kang G Z. Identification of P-efficient elite allele of the TaPHT1;6 gene and development of its functional marker in common wheat (Triticum aestivum). L.).2024,24(5),1646-1655,DOI:10.1016 / j.jia.2024.09.009.】, the results are as follows Figure 3 As shown in Table 2.

[0064] Table 2. Relative LUC activity of different haplotypes of wheat

[0065] haplotype Hap1 Hap2 LUC / REN 1.00±0.05 0.47±0.05

[0066] according to Figure 3 As shown in Table 2, the fluorescence signal in the region infected by the Hap1 recombinant vector was significantly higher than that in the region infected by the Hap2 recombinant vector. After detecting their relative activities, it was found that the gene driving activity of the Hap1 promoter was also significantly higher than that of Hap2, which preliminarily proved that Hap1 of TaMYB4-7A is a phosphorus-efficient and excellent haplotype.

[0067] Example 2: Transcriptional levels of TaMYB4-7A in different haplotype wheat varieties under low phosphorus conditions

[0068] (1) Ten wheat varieties with different TaMYB4-7A haplotypes were selected, and wheat seedlings at the two-leaf-one-heart stage were cultured in Hogland nutrient solution under low phosphorus (10 μM Pi) and normal phosphorus (200 μM Pi) treatments, respectively. After 10 days of culture, RNA was extracted from leaves and roots and reverse transcribed to synthesize cDNA, using the same method as in Example 1. Among them, the wheat varieties with Hap1 haplotype were Zhongmai 998, Lunxuan 66, Shimai 15, Jinan 13, Baiquan 3039, Yumai 49, Yujiao 5, 92R137, Zhongmai 175, and Shimai 22; the wheat varieties with Hap2 haplotype were Jinfeng 3, Luohan 6, Luomai 4, Yannong 19, Shimai 14, Chuanmai 46, Lunxuan 154, Yumai 70, Taixue 7, and Shan 229.

[0069] (2) Based on the genome sequence of three copies of TaMYB4-7A, B and D, design 7A-specific quantitative primers TaMYB4-7A-qPCR-F and TaMYB4-7A-qPCR-R.

[0070] (3) Using the cDNA from step (2) as a template, a qPCR reaction was performed in a QuanStudio5 Real-time PCR System (Applied Biosystems, USA). The qPCR reaction system contained 10 μL of 2×ChamQ SYBR qPCR Master Mix (High ROX Premixed), 0.5 μL each of forward and reverse primers (10 μM), 200 ng of template, and ddH2O added to a final volume of 20 μL. The qPCR reaction program was as follows: 95℃ for 30 s, one cycle; 5℃ for 10 s, 60℃ for 30 s, 40 cycles; melting curve analysis program: 95℃ for 15 s, 55℃ for 15 s, 94℃ for 1 min. The TaActin gene was used as an internal reference gene, and 2 -ΔΔCT The method calculates the relative gene expression levels among different samples, and the results are as follows: Figure 4 As shown in Table 3.

[0071] The primer sequence information is shown below:

[0072] TaMYB4-7A-qPCR-F (SEQ ID NO.11): 5′-AGTTGAGTAAGCCGCCCAG GT-3′;

[0073] TaMYB4-7A-qPCR-R (SEQ ID NO. 12): 5′-CTCTCACTCTCAGGATGCTG CA-3′.

[0074] TaActin-F (SEQ ID NO. 13): 5′-CTATGTTCCCGGGTATTGCT-3′;

[0075] TaActin-R (SEQ ID NO. 14): 5'-AAGGGAGGCAAGAATCGAC-3'.

[0076] Table 3. Transcriptional levels of different haplotypes TaMYB4-7A

[0077] TaMYB4-7A transcriptional level Hap1 Hap2 blade 4.33±0.14 3.45±0.14 Root system 7.41±0.20 6.22±0.20

[0078] according to Figure 4 As shown in Table 3, under low phosphorus conditions, the TamYB4-7A transcription level in the Hap1 haplotype wheat variety was significantly higher than that in the Hap2 haplotype wheat variety.

[0079] Example 3: Determination of low phosphorus tolerance coefficients of different haplotype wheat varieties at the seedling and maturity stages.

[0080] This embodiment uses 255 wheat populations for the experiment, and the groups are as follows:

[0081] Hydroponic experimental group (HC): ① Wheat was placed in Hogland nutrient solution with normal phosphorus (200 μM Pi) for hydroponics; ② Wheat was placed in Hogland nutrient solution with low phosphorus (10 μM Pi) for hydroponics; other management measures were the same as for ordinary wheat.

[0082] Soil-based experimental group (SC): ① Wheat was subjected to field soil-based experiments under normal fertilization (+120 kg / ha P2O5); ② Wheat was subjected to field soil-based experiments under no phosphate fertilizer (+0 kg / ha P2O5); other management measures were the same as for ordinary wheat.

[0083] Three replicates were set up for each group. The total biomass (roots + leaves) of hydroponically grown wheat seedlings and the total aboveground biomass (stems + grains) of soil-grown wheat at maturity were measured. The low phosphorus tolerance coefficient (total biomass under low phosphorus conditions / total biomass under normal phosphorus conditions) was calculated. The results are as follows: Figure 5 As shown in Table 4, HC-R1 to HC-RC3 are three groups of hydroponic repeated experiments, and SC is a soil-cultured experiment.

[0084] Table 4 Low phosphorus tolerance coefficients of different haplotype wheat

[0085] Low phosphorus resistance coefficient HC-R1 HC-R2 HC-R3 SC Hap1 0.400±0.021 0.186±0.012 0.142±0.007 0.909±0.012 Hap2 0.187±0.032 0.062±0.012 0.079±0.015 0.761±0.095

[0086] according to Figure 5As shown in Table 4, in multiple experimental replicates, the low phosphorus tolerance coefficient of haplotype Hap1 wheat plants was significantly higher than that of haplotype Hap2 wheat plants, proving that Hap1 is a haplotype with excellent phosphorus absorption, which is consistent with the results of promoter activity analysis.

[0087] Example 4: Development of Highly Efficient Phosphorus Molecular Markers

[0088] Because of the large indels between the promoters of the two haplotypes TaMYB4-7A, specific amplification primers TaMYB4-7A-Promoter-SF (SEQ ID NO.2) and TaMYB4-7A-Promoter-SR (SEQ ID NO.3) can be designed based on these differences. The intensity of the PCR amplified bands can be used to distinguish between the two haplotypes. Using DNA from two wheat varieties (five varieties selected for each haplotype) as templates, the TaMYB4-7A promoter fragments in both haplotypes of wheat were amplified, and single amplified bands were obtained in both cases. The PCR reaction system and conditions were the same as in Example 1. The wheat varieties for haplotype Hap1 were Zhongmai 998, Lunxuan 66, Shimai 15, Jinan 13, and Jimai 30; the wheat varieties for haplotype Hap2 were Jinfeng 3, Luohan 6, Luomai 4, Yannong 19, and Shimai 14.

[0089] Test results as follows Figure 6 As shown, electrophoresis results revealed that the amplification product of the Hap1 haplotype wheat variety had a band of 2113 bp, while the amplification product of the Hap2 haplotype wheat variety had a band of 1430 bp. Therefore, this specific primer can distinguish between Hap1 and Hap2 and can be applied to the molecular design breeding of new phosphorus-efficient wheat varieties.

[0090] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A molecular marker fragment associated with the trait of high phosphorus utilization efficiency in wheat, characterized in that, The molecular marker fragment contains the promoter region of the TaMYB4-7A gene; the nucleotide sequence of the promoter region of the TaMYB4-7A gene is shown in SEQ ID NO.

1.

2. A primer pair for identifying the molecular marker fragment of claim 1, characterized in that, This includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

3.

3. A kit for detecting the molecular marker fragment of claim 1, characterized in that, Includes the primer pair and PCR amplification reagents as described in claim 2.

4. A method for screening wheat varieties with high phosphorus utilization efficiency, characterized in that, This includes selecting wheat samples whose genomic DNA contains the molecular marker fragment described in claim 1.

5. The method according to claim 4, characterized in that, The method includes using the primer pair described in claim 2 or the kit described in claim 3 to perform PCR amplification with the genomic DNA of a wheat sample as a template. When the length of the amplification product is 2113 bp, it indicates that the genomic DNA of the wheat sample contains the molecular marker fragment described in claim 1, which is a phosphorus-efficient wheat variety.

6. The application of the molecular marker fragment of claim 1, the primer pair of claim 2, the kit of claim 3, or the method of claim 4 or 5 in wheat breeding.

7. The application of the molecular marker fragment of claim 1, the primer pair of claim 2, the kit of claim 3, or the method of claim 4 or 5 in wheat-assisted breeding.

8. The application of the molecular marker fragment of claim 1, the primer pair of claim 2, the kit of claim 3, or the method of claim 4 or 5 in identifying the trait of high phosphorus utilization efficiency in wheat.

9. The application of the molecular marker fragment of claim 1, the primer pair of claim 2, the kit of claim 3, or the method of claim 4 or 5 in screening phosphorus-efficient wheat.

10. A method for molecular marker-assisted breeding of wheat varieties with high phosphorus utilization efficiency, characterized in that, The target wheat was molecularly labeled using the primer pair described in claim 2 or the kit described in claim 3, and wheat germplasm containing the molecular marker fragment described in claim 1 was screened for subsequent screening.

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