A molecular marker closely linked to the wheat powdery mildew resistance gene PmL555 and its application

Abstract

This invention discloses a molecular marker, YTUL62, tightly linked to the wheat powdery mildew resistance gene PmL555. 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 the marker primers of YTUL62, PCR amplification of the wheat genomic DNA to be tested yielded amplification products with molecular weights of 226 bp and 260 bp, which are the molecular markers tightly linked to the wheat powdery mildew resistance gene PmL555. The molecular marker provided by this invention can more accurately and efficiently detect the genetic mapping population of PmL555, which is beneficial for map-based cloning and fine localization of PmL555. Furthermore, using this marker for marker-assisted selection of PmL555 can shorten the breeding cycle and improve breeding efficiency.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to a molecular marker closely linked to the wheat powdery mildew resistance gene PmL555 and its application. Background Technology

[0002] wheat powdery mildew obligate parasite ( Blumeria grassinis f. sp. wheat, Bgt *Powdery mildew* is an obligate, live parasitic fungus, with a host range limited to plants of the *Wheat* genus. This fungus can cause global diseases affecting wheat. In the early stages of infection, chlorotic necrotic spots form on the leaf surface. As the disease progresses, these spots expand into elliptical or nearly circular shapes, covered with a white mycelial layer composed of conidiophores and chains of conidia. In the middle and late stages, the mycelial layer gradually changes from pure white to grayish-white, and later forms numerous grayish-brown cleistothecia containing asci and ascospores. In mild infections, the mycelium is scattered; in severe infections, the mycelial layer merges into patches, completely obscuring the leaf surface. The leaf tissue below the infected area rapidly turns chlorotic and yellow, accompanied by programmed early wilting. When the pathogen spreads to the stem and leaf sheath, the mechanical strength of the stem base decreases significantly, easily inducing lodging. Ultimately, the plants exhibited weakened growth, reduced plant height, and fewer effective tillers; the wheat ears were shortened, the grain filling rate decreased, and the thousand-grain weight was significantly reduced, resulting in severe yield losses.

[0003] To effectively control wheat powdery mildew and reduce its damage, various measures have been taken, including developing disease-resistant wheat varieties, optimizing field management, and implementing chemical control measures. Compared to traditional control methods using chemical and biological agents, breeding disease-resistant varieties is the preferred measure to reduce losses from this disease.

[0004] Wheat and its wild relatives are important sources of wheat powdery mildew resistance genes. Wild emmer wheat ( Wheat turgid ssp. Dicoccoid The hexaploid wheat (2n = 4X = 28, AABB) is an ancestral species of hexaploid wheat and an important gene donor for improving wheat resistance to powdery mildew. It contains abundant resistance gene resources, and exploring and utilizing its superior genes is an important way to enhance wheat disease resistance. Currently, officially named powdery mildew resistance genes that have been discovered in this species and successfully transferred into hexaploid wheat include Pm16, Pm26, Pm30, Pm36, Pm41, Pm42, Pm64, and Pm69.

[0005] Traditional breeding relies primarily on phenotypic identification to screen for superior genotypes. However, because phenotypes are easily influenced by environmental factors, this often leads to decreased selection accuracy, prolonged breeding cycles, and reduced efficiency. In contrast, marker-assisted selection (MAS) utilizes molecular markers that are tightly linked to or co-segregate with target genes to directly identify genotypes and perform targeted selection, thereby significantly improving breeding precision and genetic gain. Currently, MAS technology has demonstrated significant application value in wheat disease resistance breeding.

[0006] Studies have shown that wild emmer wheat L555 not only possesses excellent agronomic traits but also exhibits stable and broad-spectrum resistance to powdery mildew. Through multi-year, multi-location natural induction and artificial inoculation identification, combined with seedling genetic analysis and molecular marker genotyping, it was discovered that the resistance of this material to the dominant physiological race E09 is controlled by a single dominant gene PmL555 on chromosome 2BS. Therefore, developing usable molecular markers for PmL555 and verifying their effectiveness will provide important technical support for the breeding of powdery mildew-resistant wheat varieties. Summary of the Invention

[0007] The purpose of this invention is to provide a molecular marker closely linked to the wheat powdery mildew resistance gene PmL555 and its application, so as to use the molecular marker to locate and detect the wheat powdery mildew resistance gene PmL555.

[0008] This invention is achieved through the following method:

[0009] A molecular marker closely linked to the wheat powdery mildew resistance gene PmL555 is a codominant SSR marker YTUL62.

[0010] The upstream primer for the molecular marker YTUL62 is YTUL62-F, and its nucleotide sequence is as follows:

[0011] 5'-AGAGATTTCAATGCAGACCA-3', as shown in SEQ ID NO:1;

[0012] The downstream primer for the molecular marker YTUL62 is YTUL62-R, and its nucleotide sequence is as follows:

[0013] 5'-AGTAACGCAGACCACAAAAT-3', as shown in SEQ ID NO:2;

[0014] Using marker primers for the molecular marker YTUL62, PCR amplification was performed on the wheat genomic DNA to be tested. The corresponding amplification products had molecular weights of 226 bp and 260 bp, which are molecular markers closely linked to the wheat powdery mildew resistance gene PmL555.

[0015] 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.

[0016] 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℃.

[0017] 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 170 V for 1.5-2 h. The gel is then stained with silver nitrate and photographed.

[0018] The present invention also provides the application of the molecular markers closely linked to the wheat powdery mildew resistance gene PmL555 in gene localization, map-based cloning and marker-assisted selection breeding of the wheat powdery mildew resistance gene PmL555.

[0019] The application described in this invention, which detects whether a wheat variety carries the powdery mildew resistance gene PmL555, mainly includes the following steps:

[0020] (1) Extract genomic DNA from fresh leaves of the wheat sample to be tested;

[0021] (2) The extracted wheat genomic DNA was amplified by PCR using primers with the molecular marker YTUL62 to obtain the amplification product;

[0022] (3) If linkage-specific bands of 226 bp and 260 bp can be amplified, it indicates that the powdery mildew resistance gene exists in the wheat being tested. PmL555 Otherwise, the wheat sample does not contain the wheat powdery mildew resistance gene PmL555.

[0023] In the application described above, the primers for the molecular marker YTUL62 in step (2) include an upstream primer YTUL62-F and a downstream primer YTUL62-R. The nucleotide sequence of the upstream primer YTUL62-F is shown in SEQ ID NO:1, namely: YTUL62-F: 5'-AGAGATTTCAATGCAGACCA-3'; the nucleotide sequence of the downstream primer YTUL62-R is shown in SEQ ID NO:2, namely: YTUL62-R: 5'-AGTAACGCAGACCACAAAAT-3'.

[0024] 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.

[0025] 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℃.

[0026] 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 170 V for 1.5–2 h. The gel was then stained with silver nitrate and photographed. The electrophoresis results were used to determine the presence of linkage-specific bands of 226 bp and 260 bp in the tested wheat germplasm. Otherwise, the wheat germplasm did not contain the powdery mildew resistance gene PmL555.

[0027] This invention, through genetic analysis and molecular marker detection of seedling powdery mildew resistance, demonstrates that the resistance of wild emmer wheat L555 seedlings to the prevalent powdery mildew strain E09 is controlled by a single dominant gene, named PmL555. Using 173 pairs of molecular markers evenly distributed throughout the genome, the resistance of wild emmer wheat L555, susceptible wheat Langdon (LDN), and F1 cells derived from L555×LDN were compared. 2:3 Polymorphism detection was performed on disease-resistant and disease-susceptible pools composed of 10 homozygous resistant families and 10 homozygous susceptible families. Seventeen 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 generation of 162 L555×LDNs. 2:3Genotyping of the family pedigree initially located PmL555 within the 10.61-13.08 Mb region of wheat chromosome 2BS. Further, based on the sequence of the Chinese spring wheat reference genome within this region, the SSR marker YTUL62, closely linked to the PmL555 gene, was designed and screened using Primer 5.0 software. The molecular marker YTUL62 for wheat powdery mildew resistance gene PmL555 provided by this invention, after genetic segregation population analysis, showed a genetic distance of only 0.5 cM from the PmL555 gene, indicating close linkage. This allows for more precise and efficient detection of the genetic mapping population of PmL555, facilitating fine mapping and map-based cloning of PmL555.

[0028] The molecular marker provided by this invention, which is closely linked to the wheat powdery mildew resistance gene PmL555, can be applied to powdery mildew resistant wheat breeding. This not only greatly saves costs and reduces the breeding cycle, but is also more accurate and efficient, and can be better applied to powdery mildew resistant wheat breeding work. Attached Figure Description

[0029] Figure 1 This is to label the partial amplification results of YTUL62 in 162 L555 × Langdon (LDN) derived F2:3 families.

[0030] M in the image: pUC19 Msp I; 1: L555 (disease-resistant parent); 2: Langdon(LDN) (susceptible parent); 3-17: F of L555 × Langdon(LDN) 2:3 Family pedigrees, of which 3-7: homozygous resistant pedigrees, 8-12: resistant segregating pedigrees, 13-17: homozygous susceptible pedigrees; white arrows indicate specific bands of PmL555. Detailed Implementation

[0031] 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.

[0032] Example 1: Development of the molecular marker YTUL62 for the wheat powdery mildew resistance gene PmL555

[0033] 1. Materials

[0034] The resistant parent was wild emmer wheat L555, and the susceptible parent was durum wheat Langdon (LDN). L555 and Langdon (LDN) were crossed, and the resulting F1 generation was self-crossed to obtain the F1 generation. 2:3 Family lineage.

[0035] 2. Extraction of wheat genomic DNA

[0036] Wheat genomic DNA was extracted using the CTAB method, and the procedure is as follows:

[0037] 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;

[0038] 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;

[0039] 3) Add an equal volume of chloroform and mix on a shaker for 30 minutes;

[0040] 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.

[0041] 5) Centrifuge at 12000 rpm at room temperature for 10 min, discard the supernatant, and wash three times with 800 μL of 75% ethanol;

[0042] 6) Air dry the precipitate and dissolve it in 50 μL of 1×TE or ddH2O.

[0043] 7) Dilute the DNA storage solution with sterile deionized water to 50 ng / μL as a working solution for later use.

[0044] 3. Identification of powdery mildew resistance in wheat seedlings and genetic analysis of resistance

[0045] Wheat seedling powdery mildew resistance identification was completed in a greenhouse. The resistant parent L555, the susceptible parent Langdon (LDN), F1 hybrids, and F2 hybrids were used. 2:3 The 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 17 h light / 10 h dark. Powdery mildew strain E09 was inoculated using the sweeping method at the one-leaf stage. Phenotypic assessment was conducted 10-17 days later, when the susceptible control, Tai Nong 18, was fully infected. Infection type (IT) was recorded according to a 0-4 grade standard. Resistance levels were classified as follows: 0-2 for resistant types, and 3-4 for susceptible types.

[0046] The survey results showed that L555 exhibited high resistance to powdery mildew strain E09 (IT=0), high susceptibility to Langdon (LDN) (IT=4), and all F1 plants showed resistance (IT=0-1), indicating that L555 carries a dominant resistance gene; the F1 generation of this combination... 2:3 Family pedigrees underwent resistance testing, and the results showed a homozygous resistance: resistance to susceptibility segregation: homozygous susceptibility ratio of 41:84:37. The chi-square test confirmed a segregation ratio of 1:2:1 for a single dominant gene. 2 =0.42, P =0.81). In summary, the resistance of L555 to the powdery mildew strain E09 is controlled by a single dominant gene, which is named PmL555.

[0047] 4. Fine localization of molecular markers for PmL555

[0048] 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 wild emmer wheat L555, durum wheat Langdon (LDN), and the resistant and susceptible pools using 173 pairs of molecular markers evenly distributed throughout the genome. The 17 pairs of markers showed consistent polymorphism in both the resistant and susceptible parents and the resistant-susceptible pools. Subsequently, these markers were used to analyze the F1 generation of 162 L555×Langdon (LDN) lines. 2:3 Genotyping of the family revealed that PmL555 was initially located in the range of 10.61-13.08 Mb on the wheat 2BS chromosome.

[0049] 5. Development of molecular markers closely linked to PmL555

[0050] Based on the sequence information of the Chinese spring wheat reference genome within the candidate region of 10.61-13.08 Mb, SSR markers were designed using Primer 5.0 software, and the F sequence of L555×Langdon(LDN) was analyzed. 2:3 Genotyping of the family revealed the INDEL marker YTUL62, which is closely linked to gene PmL555, with a genetic distance of only 0.5 cM.

[0051] The primers for the molecular marker YTUL62 consist of one upstream primer and one downstream primer:

[0052] The nucleotide sequence of the upstream primer YTUL62-F is: 5'-AGAGATTTCAATGCAGACCA-3';

[0053] The nucleotide sequence of the downstream primer YTUL62-R is: 5'-AGTAACGCAGACCACAAAAT-3'.

[0054] 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.

[0055] 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℃.

[0056] 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 170 V for 1.5–2 h. After silver nitrate staining, photographs are taken. If linkage-specific bands of 226 bp and 260 bp are amplified, it indicates the presence of the powdery mildew resistance gene PmL555 in the tested wheat germplasm; otherwise, the powdery mildew resistance gene PmL555 is not present in the tested wheat germplasm.

[0057] Molecular marker detection results are shown below Figure 1 .in Figure 1 To label YTUL62 in 162 L555×Langdon(LDN) derived F 2:3 Partial amplification results from the family. M in the figure: pUC19 Msp I; 1: L555 (disease-resistant parent); 2: Langdon(LDN) (susceptible parent); 3-17: F of L555 × Langdon(LDN) 2:3 The families are shown in the diagram: 3-7: homozygous resistant families; 8-12: resistant-susceptible segregating families; 13-17: homozygous susceptible families; the white arrows indicate specific bands for PmL555. Amplification results showed that the marker YTUL62 amplified linkage-specific bands of 226 bp and 260 bp in the resistant parent L555 and the resistant families, but not in the susceptible parent Langdon (LDN) and the susceptible families.

[0058] The wheat powdery mildew resistance gene PmL555 originates from the wild emmer wheat L555 in my country. It is a novel gene exhibiting excellent resistance, but currently, there are no reports on its localization, map-based cloning, or application in molecular breeding. Using the molecular marker YTUL62 provided in this invention to detect large-scale genetic mapping populations is beneficial for achieving precise localization and map-based cloning of the PmL555 gene. Introducing PmL555 into major wheat varieties susceptible to powdery mildew, and utilizing the molecular marker YTUL62 developed in this invention, allows for efficient and accurate detection of large breeding populations, significantly improving the efficiency and accuracy of transferring the disease-resistant gene PmL555. This is of paramount importance for the efficient transfer of the PmL555 gene and for in-depth analysis of the disease resistance mechanism.

[0059] 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 PmL555 Primer pair of tightly linked molecular marker YTUL62 in wheat powdery mildew resistance gene PmL555 Gene mapping and selection of powdery mildew resistance genes PmL555 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 YTUL62, PCR amplification was performed on the wheat genomic DNA to be tested, yielding amplification products with molecular weights of 226 bp and 260 bp, which correspond to the wheat powdery mildew resistance gene. PmL555 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. PmL555 The main steps include: (1) Extract genomic DNA from the wheat sample to be tested; (2) The extracted wheat genomic DNA was amplified by PCR using primers with the molecular marker YTUL62 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 linkage-specific bands of 226 bp and 260 bp, it indicates that the wheat being tested contains a powdery mildew resistance gene. PmL555 Otherwise, the wheat sample tested does not contain the wheat powdery mildew resistance gene. PmL555 .

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