A molecular marker tightly linked to the wheat powdery mildew resistance gene PmJ684 and its application

Abstract

This invention discloses a gene for wheat powdery mildew resistance. PmJ684 This invention relates to the field of bioengineering technology and its application of a tightly linked molecular marker. The molecular marker is YTULL-40; the upstream primer nucleotide sequence is shown in SEQ ID NO:1; the downstream primer nucleotide sequence is shown in SEQ ID NO:2; PCR amplification of wheat genomic DNA using this molecular marker yielded an amplification product with a molecular weight of 172 bp. This invention provides a gene associated with wheat powdery mildew resistance. PmJ684 The tightly linked molecular marker YTULL-40 can accurately detect... PmJ684 Genetic mapping populations are beneficial PmJ684 Precise localization and cloning of this marker. Furthermore, using this marker for assisted breeding allows for the precise selection of individuals carrying the powdery mildew resistance gene. PmJ684 This provides germplasm materials, shortens the breeding cycle, and facilitates wheat breeding.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a gene for wheat resistance to powdery mildew. PmJ684 Tightly linked molecular markers and their applications. Background Technology

[0002] Wheat powdery mildew is caused by the grass family fungus *Brucea fulvidraco*. Blumeria graminis f. sp. tritici ( Bgt Powdery mildew is a major disease that causes wheat to produce wheat rapidly and severely impacts wheat yield. Wheat infected with powdery mildew can typically experience a yield reduction of 5-20%, and in severe cases, up to 30-50%. There are many measures to control wheat powdery mildew, such as agricultural control, chemical control, biological control, and planting resistant varieties. Among these, planting resistant varieties is one of the most effective and convenient methods. By selecting and breeding highly resistant varieties, the impact of powdery mildew on wheat yield can be effectively mitigated. To date, more than 100 powdery mildew (Pm) resistance genes or alleles have been discovered in wheat and its closely related genera. However, with the passage of time and the continuous evolution of powdery mildew, many powdery mildew resistance genes have become ineffective. Therefore, finding new resistance genes has become a top priority in powdery mildew control.

[0003] Cultivated emmer wheat ( Triticum dicoccum Emelia (2n = 4x = 28, AABB) is an ancient tetraploid wheat, a key intermediate in the evolution of the genus *Wheat*, and plays an important role in the formation of modern hexaploid common wheat (such as bread wheat). Cultivated emeliads carry multiple disease resistance genes (such as powdery mildew resistance genes). Pm3h , Pm4a , Pm49 and Pm68 , Pm71 Erin wheat has been widely used in modern wheat disease resistance breeding. Cultivating erin wheat is not only a "living fossil" of the origin of agriculture, but also an irreplaceable genetic resource for modern wheat breeding.

[0004] Traditional breeding relies primarily on phenotypic observation and selection. This method is not only time-consuming and labor-intensive but also susceptible to environmental factors, leading to low selection accuracy. In contrast, marker-assisted selection (MAS) enables direct detection and targeted selection of genotypes by identifying molecular markers closely linked to or co-segregating with target genes. The advantages of this technology are twofold: firstly, it can rapidly and accurately identify resistance genes carried in different germplasm materials, significantly improving screening efficiency; secondly, it helps breeders precisely identify germplasm resources with desirable traits such as powdery mildew resistance, thereby accelerating the breeding process and promoting the dissemination of superior varieties. Therefore, MAS technology provides a highly efficient and precise molecular breeding method for modern crop breeding.

[0005] Cultivated emmer wheat AABB684 exhibits outstanding overall performance in the field, with continuous multi-location trials demonstrating excellent agronomic traits and strong tolerance to powdery mildew, making it a highly promising disease-resistant donor material. We crossed cultivated emmer wheat AABB684 with AABB620 to construct a genetically segregating population. After infection with the powdery mildew-endemic strain E09, we found that the resistance ratio in the secondary segregating population conformed to 3:1, consistent with Mendelian inheritance laws. Therefore, we believe that the resistance of AABB684 is due to a dominant gene. PmJ684 This gene, belonging to a newly discovered wheat powdery mildew resistance gene / allele, controls [the disease]. We have developed [a method / technology] that [controls / controls] [the disease]. PmJ684 The tightly linked molecular marker YTULL-40, which is not only for PmJ684 Map-based cloning laid the foundation and can also be used for screening high-quality disease-resistant wheat resources, accelerating the breeding cycle and improving breeding efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a gene for resistance to powdery mildew in wheat. PmJ684 Tightly linked molecular markers and their applications to utilize these markers for targeting wheat powdery mildew resistance genes. PmJ684 Gene localization and detection are performed. This marker is used for... PmJ684 Marker-assisted selection can shorten the breeding cycle, improve breeding efficiency, and facilitate wheat disease resistance breeding.

[0007] This invention is achieved through the following method: a gene associated with wheat powdery mildew resistance. PmJ684 The molecular marker is tightly linked and is a codominant InDel marker, YTULL-40.

[0008] The upstream primer for the molecular marker YTULL-40 is YTULL-40-F, and its nucleotide sequence is as follows:

[0009] 5'-ACACCAGGGGATGAAATGAAAC-3', as shown in SEQ ID NO: 1;

[0010] The downstream primer for the molecular marker YTULL-40 is YTULL-40-R, and its nucleotide sequence is as follows:

[0011] 5'-CGGGTTAGATGCGAAAACTGC-3', as shown in SEQ ID NO: 2;

[0012] The wheat genomic DNA to be tested was amplified by PCR using primers with the molecular marker YTULL-40. The corresponding amplification product band was 172 bp, which corresponds to the wheat powdery mildew resistance gene. PmJ684 Tightly linked molecular markers.

[0013] The appropriate PCR amplification system for this molecular marker is 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.

[0014] The applicable PCR amplification program for this molecular marker is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; store at 4℃.

[0015] The electrophoretic separation procedure for the amplification products applicable to this molecular marker is as follows: electrophoresis is performed using an 8% non-denaturing polyacrylamide gel. The amplification product is mixed with 2.5 μL of 10× Loading Buffer, and 1.2 μL of the mixture is loaded onto the gel. Electrophoresis is performed at a constant voltage of 140 V for 1.5-2 h. The gel is then stained with silver nitrate and photographed.

[0016] The wheat powdery mildew resistance gene provided by this invention PmJ684 Tightly linked molecular markers in wheat powdery mildew resistance genes PmJ684 The application of gene localization and molecular marker-assisted selection breeding.

[0017] The application described in this invention is to detect whether a test variety carries a wheat powdery mildew resistance gene. PmJ684 The main steps include:

[0018] (1) Extract genomic DNA from fresh leaves of the wheat sample to be tested;

[0019] (2) The extracted wheat genomic DNA was amplified by PCR using primers with the molecular marker YTULL-40 to obtain the amplification product;

[0020] (3) If a specific band of 172 bp can be amplified, it indicates that the wheat being tested contains a gene for resistance to powdery mildew. PmJ684 Otherwise, the wheat sample tested does not contain the wheat powdery mildew resistance gene. PmJ684 .

[0021] In the application described above, the primers for the molecular marker YTULL-40 in step (2) include an upstream primer YTULL-40-F and a downstream primer YTULL-40-R. The nucleotide sequence of the upstream primer YTULL-40-F is shown in SEQ ID NO:1, namely: YTULL-40-F: 5'-ACACCAGGGGATGAAATGAAAC-3'; the nucleotide sequence of the downstream primer YTULL-40-R is shown in SEQ ID NO:2, namely: YTULL-40-R: 5'-CGGGTTAGATGCGAAAACTGC-3'.

[0022] The application of this molecular marker is based on a 10 μL PCR amplification system, which includes: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.

[0023] The applicable PCR amplification program for this marker is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; store at 4℃.

[0024] Detection of PCR amplification products: Electrophoresis was performed using an 8% non-denaturing polyacrylamide gel. The amplification product was mixed with 2.5 μL of 10× Loading Buffer, and 1.2 μL of this mixture was loaded onto the gel. Electrophoresis was carried out at a constant voltage of 140 V for 1.5–2 h. The gel was then stained with silver nitrate and photographed. The electrophoresis results were used for interpretation. If a specific band of 172 bp was amplified, it indicated the presence of a powdery mildew resistance gene in the tested wheat germplasm. PmJ684 Otherwise, the wheat germplasm tested does not contain the wheat powdery mildew resistance gene. PmJ684 .

[0025] This invention, through genetic analysis and molecular marker detection of powdery mildew resistance in seedlings, demonstrates that the resistance of cultivated emmer wheat AABB684 seedlings to the prevalent powdery mildew strain E09 is controlled by a single dominant gene, which is named... PmJ684300 pairs of molecular markers evenly distributed throughout the genome were used to target cultivated emmer wheat AABB684, susceptible wheat AABB620, and the F1 generation of AABB684×AABB620. 2:3 Polymorphism was detected in disease-resistant and disease-susceptible pools composed of 10 homozygous resistant and 10 homozygous susceptible families. Fourteen pairs of markers showed consistent polymorphism in both the resistant and susceptible parents and the resistant-susceptible pools. These markers were then used to analyze the F1 chromosomes of 600 AABB684×AABB620 lines. 2:3 Genotyping of family pedigrees PmJ684 Preliminary localization was within the region of 16.28-17.01 Mb on wheat chromosome 2BS. Further, based on the sequence within this region of the durum wheat reference genome, Primer 5.0 software was used to design and screen for sequences related to the gene. PmJ684 The tightly linked insertion-deletion (InDel) marker YTULL-40. This invention provides a wheat powdery mildew resistance gene. PmJ684 The molecular marker YTULL-40 was analyzed in a genetic segregating population and found to be compatible with the gene. PmJ684 The genetic distance was only 0.4 cM, compared with PmJ684 Closely linked systems allow for more accurate and efficient detection. PmJ684 Genetic mapping populations are beneficial PmJ684 Map-based cloning and fine-tuning.

[0026] This invention provides a gene for resistance to powdery mildew in wheat. PmJ684 Tightly linked molecular markers, when applied to wheat breeding for resistance to powdery mildew, can not only greatly save costs and shorten the breeding cycle, but are also more precise and efficient, and can be better applied to wheat breeding for resistance to powdery mildew. Attached Figure Description

[0027] Figure 1 This study identifies partial amplification results of YTULL-40 in 600 AABB684 × AABB620 derived F2:3 families.

[0028] M in the image: pUC19 Msp I; 1: AABB684 (disease-resistant parent); 2: AABB620 (disease-susceptible parent); 3-17: F of AABB684 × AABB620 2:3 Family pedigrees, where 3-7: homozygous resistant pedigrees, 8-12: segregating resistant pedigrees, and 13-17: homozygous susceptible pedigrees; white arrows indicate... PmJ684 Specific bands. Detailed Implementation

[0029] The following examples are provided to better understand and use the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials and reagents used in the examples are commercially available.

[0030] Example 1: Wheat powdery mildew resistance gene PmJ684 Development of the molecular marker YTULL-40

[0031] Material

[0032] The resistant parent is cultivated emmer wheat AABB684, and the susceptible parent is cultivated emmer wheat AABB620. AABB684 and AABB620 are crossed, and the resulting F1 generation is self-crossed to obtain the F2 generation. 2:3 Family lineage.

[0033] 2. Extraction of wheat genomic DNA

[0034] Wheat genomic DNA was extracted using the CTAB method, and the procedure is as follows:

[0035] 1) Take tender fresh leaves of the wheat to be tested, freeze them quickly with liquid nitrogen, grind them into powder, and put them into 2 mL EP tubes;

[0036] 2) Add 600-800 μL of CTAB extraction solution and incubate in a 65°C water bath for 1 h, inverting the container every ten minutes during the incubation period;

[0037] 3) Add an equal volume of chloroform and mix on a shaker for 30 minutes;

[0038] 4) Centrifuge at 8000 rpm at room temperature for 10 min, aspirate 400 μL of supernatant into a 1.5 mL EP tube, add 3 times the volume of pre-cooled 95% ethanol, mix well, and allow to settle at -20°C for 0.5 h.

[0039] 5) Centrifuge at 12000 rpm at room temperature for 10 min, discard the supernatant, and wash three times with 800 μL of 75% ethanol;

[0040] 6) Air dry the precipitate and dissolve it in 50 μL of 1×TE or ddH2O.

[0041] 7) Dilute the DNA storage solution with sterile deionized water to 50 ng / μL as a working solution for later use.

[0042] 3. Identification of powdery mildew resistance in wheat seedlings and genetic analysis of resistance

[0043] Wheat seedling powdery mildew resistance identification was completed in a greenhouse. The resistant parent AABB684, the susceptible parent AABB620, F1 hybrids, and F2... 2:3The families were planted in 128-cell seed trays (3.2×3.2×4.2 cm). Both parents and F1 strains were identified as having at least 20 seeds per cell. 2:3 Each family was identified with at least 25 seeds. The susceptible control, Tai Nong 18, was randomly sown and tagged for identification. Greenhouse conditions were controlled at 18-20°C, 80% relative humidity, and a photoperiod of 14 h light / 10 h dark. Powdery mildew strain E09 was inoculated using the sweeping method at the one-leaf stage. Phenotypic assessment was conducted 10-14 days later, when the susceptible control, Tai Nong 18, was fully infected. Infection type (IT) was recorded according to a 0-4 grade standard. Disease resistance grades were divided as follows: 0-2 for resistant types, and 3-4 for susceptible types.

[0044] The results showed that AABB684 exhibited high resistance to powdery mildew strain E09 (IT=0), while AABB620 showed high susceptibility (IT=4). All F1 plants showed resistance (IT=0-1), indicating that AABB684 carries a dominant resistance gene. Resistance identification of F2 plants from this combination showed a resistance-susceptibility segregation ratio of 188:60, which, according to the chi-square test, conformed to a segregation ratio of 3:1 for a single dominant gene (χ²=0). 2 =0.086, P =0.769). Further analysis of its derived F 2:3 Family pedigree analysis showed a segregation ratio of 68:120:60 (χ²) between homozygous resistant families, segregating families, and homozygous susceptible families. 2 1:2:1 =0.77, P =0.68). In summary, the resistance of AABB684 to powdery mildew strain E09 is controlled by a single dominant gene, and this powdery mildew resistance gene is named... PmJ684 .

[0045] 4. PmJ684 fine localization of molecular markers

[0046] Based on phenotypic identification results, 10 homozygous resistant families and 10 homozygous susceptible families were selected to construct resistant and susceptible pools, respectively. Polymorphism detection was performed on cultivated emmer wheat AABB684, cultivated emmer wheat AABB620, and the resistant and susceptible pools using 300 pairs of molecular markers evenly distributed throughout the genome. Fourteen pairs of markers showed consistent polymorphism in the resistant and susceptible parents and the resistant-susceptible pools. Subsequently, these markers were used to analyze the F1 generation of 600 AABB684×AABB620 lines. 2:3 Genotyping of family pedigrees PmJ684 It was initially located within the 16.28-17.01 Mb region of wheat chromosome 2BS.

[0047] 5. with PmJ684 Development of tightly linked molecular markers

[0048] Based on the sequence information of the durum wheat reference genome within the candidate region of 16.28-17.01 Mb, insertion-deletion (InDel) markers were designed using Primer 5.0 software, and F-type markers for AABB684×AABB620 were selected. 2:3 Genotyping of the family pedigree yielded results related to the genes. PmJ684 The closely linked InDel marker YTULL-40 has a genetic distance of only 0.4 cM.

[0049] The primers for the molecular marker YTULL-40 consist of one upstream primer and one downstream primer:

[0050] The nucleotide sequence of the upstream primer YTULL-40-F is: 5'-ACACCAGGGGATGAAATGAAAC-3';

[0051] The nucleotide sequence of the downstream primer YTULL-40-R is: 5'-CGGGTTAGATGCGAAAACTGC-3'.

[0052] The applicable PCR amplification system for this marker is 10 μL, including: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.

[0053] The applicable PCR amplification program for this marker is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃.

[0054] The electrophoretic separation procedure for the amplified products is as follows: Electrophoresis is performed using an 8% non-denaturing polyacrylamide gel. The amplified product is mixed with 2.5 μL of 10× Loading Buffer, and 1.2 μL of this mixture is loaded onto the gel. Electrophoresis is carried out at a constant voltage of 140 V for 1.5–2 h. The sample is then stained with silver nitrate and photographed. If a specific band of 172 bp is amplified, it indicates the presence of a powdery mildew resistance gene in the tested wheat germplasm. PmJ684 Otherwise, the wheat germplasm tested does not contain the wheat powdery mildew resistance gene. PmJ684 .

[0055] Molecular marker detection results are shown below Figure 1 .in Figure 1 To label YTULL-40 in 600 AABB684×AABB620 derived F 2:3Partial amplification results from the family. M in the figure: pUC19 Msp I; 1: AABB684 (disease-resistant parent); 2: AABB620 (disease-susceptible parent); 3-17: F of AABB684 × AABB620 2:3 Family pedigrees, where 3-7: homozygous resistant pedigrees, 8-12: segregating resistant pedigrees, and 13-17: homozygous susceptible pedigrees; white arrows indicate... PmJ684 The specific band was observed. Amplification results showed that the marker YTULL-40 amplified a specific band of 172 bp in the resistant parent AABB684 and the resistant family, but did not amplify the target band in the susceptible parent AABB620 and the susceptible family.

[0056] The wheat material AABB684 is a cultivated emmer wheat that carries a novel gene for resistance to powdery mildew. PmJ684 It exhibits outstanding resistance, but no publicly available research has been conducted on its fine mapping, cloning, and breeding applications. The specific marker YTULL-40 designed in this invention can be accurately screened in genetically segregating populations, providing... PmJ684 This lays the foundation for precise positioning and map-based cloning. PmJ684 Introducing resistance genes into wheat resources with superior traits can better promote powdery mildew resistance breeding. Using molecular markers can significantly improve the efficiency and accuracy of transferring resistance fragments, thereby accelerating the process. PmJ684 Its application in breeding.

[0057] The above embodiments are optimized implementations of the present invention and are used only to illustrate the present invention, not to limit it. Modifications or equivalent substitutions made by those skilled in the art without departing from the spirit and principles of the embodiments of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. An amplified gene associated with wheat powdery mildew resistance PmJ684 Primer pair of tightly linked molecular marker YTULL-40 in wheat powdery mildew resistance gene PmJ684 Gene mapping and selection of powdery mildew resistance genes PmJ684 In its application in wheat, the upstream primer nucleotide sequence is shown in SEQ ID NO:1, and the downstream primer nucleotide sequence is shown in SEQ ID NO:

2. Using primers with the molecular marker YTULL-40, PCR amplification of the wheat genomic DNA to be tested was performed, yielding a 172 bp amplification product band, which corresponds to the wheat powdery mildew resistance gene. PmJ684 Tightly linked molecular markers.

2. The application according to claim 1, wherein the PCR amplification system is 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.

3. According to claim 1, the PCR amplification program is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃.

4. The application according to claim 1, detecting whether the test variety carries the wheat powdery mildew resistance gene. PmJ684 The main steps include: (1) Extract genomic DNA from the wheat sample to be tested; (2) The wheat genomic DNA to be tested was amplified by PCR using primers with the molecular marker YTULL-40 to obtain the amplification product; (3) Perform electrophoresis on the amplified products and judge based on the electrophoresis results. If the wheat DNA can amplify a specific band of 172 bp, it indicates that the wheat being tested contains a powdery mildew resistance gene. PmJ684 Otherwise, the wheat sample tested does not contain the wheat powdery mildew resistance gene. PmJ684 .

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