A molecular marker closely linked to wheat powdery mildew resistance gene PmL424 and application thereof

Abstract

The application discloses a molecular marker closely linked to a wheat powdery mildew resistance gene PmL424, which is YTU024-L10; the nucleotide sequence of the upstream primer of the molecular marker is shown in SEQ ID NO:1; the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2; the marker primer of the molecular marker YTU024-L10 is used for carrying out PCR amplification on the wheat genomic DNA to be measured, and the corresponding amplification product with a molecular weight of 238 bp is obtained, that is, the molecular marker closely linked to the wheat powdery mildew resistance gene PmL424. The molecular marker provided by the application can more accurately and efficiently detect the genetic mapping population of PmL424, and is beneficial to the map-based cloning and fine mapping of PmL424; secondly, the molecular marker assisted selection of PmL424 using the marker can shorten the wheat breeding cycle and improve the 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 PmL424 and its application. Background Technology

[0002] Wheat powdery mildew is a major disease that seriously threatens wheat production. Its causative agent is *Pseudomonas aeruginosa*. Blumeria grassinis f. sp. wheat ( Bgt Powdery mildew in wheat can significantly reduce yields in affected fields, generally by 5-10%, and in severely affected areas, by as much as 30-50%. Studying the powdery mildew resistance genes carried by different wheat varieties and subsequently breeding superior resistant varieties is a crucial approach to controlling wheat powdery mildew. To date, more than 140 powdery mildew resistance genes have been reported for different wheat types. However, with the continuous evolution of pathogen races, many resistance genes have lost their efficacy. Therefore, to reduce the occurrence of powdery mildew and improve wheat yield and quality, it is necessary to continuously explore new sources of resistance. Rye ( Cereal rye L. , 2n = 2x = 14, RR), ice grass ( Leynus secalinus , 2n = 2x = 14, PP), tufted wheat ( Dasypyrum villous , 2n = 2x = 14, VV) and other wheat-related species, as well as Aegilops scabra ( Aegilops tauschii , 2n = 2x = 14, DD), Urartu wheat ( Wheat Urartu , 2n = 2x = 14, AA), a grain of wheat ( Wheat monococcous L, 2n = 2x = 14, AA), wild emmer wheat ( Wheat germ , 2n = 4x = 28, AABB), Timofewi wheat ( Timothy wheat Common wheat ancestral species such as 2n = 4x = 28 and AAGG are all valuable resources containing disease resistance characteristics. More than half of the recorded powdery mildew resistance genes originate from these species.

[0003] As a tetraploid species, cultivated emmer wheat ( Dicotyledonous wheat The emmer wheat (2n = 4x = 28, AABB) shares the A and B genomes with common wheat and is widely regarded as the ancestor of common wheat. This species possesses a wealth of desirable traits, especially in responding to biotic and abiotic stresses. For example, important genes such as Pm4a, Pm49, Pm50, and Pm71, which are resistant to powdery mildew, all originate from cultivated emmer wheat.

[0004] In recent years, wheat breeding technology has been continuously innovated, and molecular marker-assisted selection breeding has gradually become the core method for breeding wheat to resist powdery mildew. Having efficient, accurate, and easy-to-use molecular markers is fundamental to carrying out molecular marker-assisted selection of target genes. Tracking and detecting target genes through molecular markers can significantly shorten the breeding cycle and greatly improve breeding efficiency.

[0005] Cultivated emmer wheat L424 exhibits excellent agronomic traits and has demonstrated good resistance to powdery mildew in multi-year, multi-location field trials, making it an extremely superior wheat germplasm resource resistant to powdery mildew. Genetic analysis and molecular marker detection of powdery mildew resistance at the seedling stage revealed that, at this stage, L424's resistance to the prevalent powdery mildew strain E09 is controlled by a pair of dominant genes, PmL424, located on wheat chromosome 2BS. This gene is a newly discovered wheat powdery mildew resistance gene / allele. Developing molecular markers closely linked to gene PmL424 and applying them to marker-assisted selection breeding of the wheat powdery mildew resistance gene PmL424 is crucial for breeding powdery mildew-resistant wheat varieties and achieving effective control of powdery mildew. Summary of the Invention

[0006] The purpose of this invention is to provide a molecular marker closely linked to the wheat powdery mildew resistance gene PmL424 and its application, using this molecular marker for gene localization and detection of the wheat powdery mildew resistance gene PmL424. Using this marker for marker-assisted selection of PmL424 can shorten the breeding cycle and improve breeding efficiency.

[0007] This invention is achieved through the following method:

[0008] A molecular marker closely linked to the wheat powdery mildew resistance gene PmL424 is a codominant InDel marker YTU024-L10;

[0009] The upstream primer for the molecular marker YTU024-L10 is YTU024-L10-F, and its nucleotide sequence is as follows:

[0010] 5'-GCCTGGCTTCATATGCTGTG-3', as shown in SEQ ID NO: 1;

[0011] The downstream primer for the molecular marker YTU024-L10 is YTU024-L10-R, and its nucleotide sequence is as follows:

[0012] 5'-TTCTCGGATCAGTAGGCCCT-3', as shown in SEQ ID NO: 2;

[0013] The marker primer YTU024-L10 was used to amplify the wheat genomic DNA to be tested by PCR. The corresponding amplification product had a molecular weight of 238 bp, which is a molecular marker closely linked to the wheat powdery mildew resistance gene PmL424.

[0014] 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.

[0015] 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℃.

[0016] 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 220 V for 1.5-2 h. The gel is then stained with silver nitrate and photographed.

[0017] The present invention relates to the application of molecular markers closely linked to the wheat powdery mildew resistance gene PmL424 in gene localization, map-based cloning, and marker-assisted selection breeding of the wheat powdery mildew resistance gene PmL424.

[0018] The application described in this invention, which detects whether a wheat variety carries the powdery mildew resistance gene PmL424, mainly includes the following steps:

[0019] (1) Extract genomic DNA from fresh leaves of the wheat sample to be tested;

[0020] (2) The extracted wheat genomic DNA was amplified by PCR using primers with the molecular marker YTU024-L10 to obtain the amplification product;

[0021] (3) If a specific band of 238 bp can be amplified, it indicates that the powdery mildew resistance gene PmL424 exists in the wheat to be tested; otherwise, the powdery mildew resistance gene PmL424 does not exist in the wheat to be tested.

[0022] In the application described above, the primers for the molecular marker YTU024-L10 in step (2) include an upstream primer YTU024-L10-F and a downstream primer YTU024-L10-R. The nucleotide sequence of the upstream primer YTU024-L10-F is shown in SEQ ID NO:1, namely: YTU024-L10-F: 5'-GCCTGGCTTCATATGCTGTG-3'; the nucleotide sequence of the downstream primer YTU024-L10-R is shown in SEQ ID NO:2, namely: YTU024-L10-R: 5'-TTCTCGGATCAGTAGGCCCT-3'.

[0023] 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.

[0024] 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℃.

[0025] 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 220 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 the powdery mildew resistance gene PmL424 in the tested wheat germplasm. Otherwise, the powdery mildew resistance gene PmL424 was not present in the tested wheat germplasm.

[0026] This invention, through genetic analysis and molecular marker detection of seedling powdery mildew resistance, demonstrates that the resistance of cultivated emmer wheat L424 seedlings to the prevalent powdery mildew strain E09 is controlled by a single dominant gene, named PmL424. Polymorphism was detected using 155 pairs of molecular markers evenly distributed throughout the genome in resistant and susceptible populations comprised of 10 homozygous resistant and 10 homozygous susceptible families from cultivated emmer wheat L424, susceptible wheat Langdon (LDN), and the F2 population using L424×Langdon (LDN). 22 pairs of markers showed consistent polymorphism in both resistant and susceptible parents and in the resistant-susceptible populations. Subsequently, these markers were used to genotype 185 L424×Langdon (LDN) F2 populations, preliminarily locating PmL424 within the 21.62–21.81 Mb region of wheat 2BS chromosome. Based on the sequence of the Chinese spring wheat reference genome within this region, the insertion-deletion (INDEL) marker YTU024-L10, which is closely linked to the gene PmL424, was designed and screened using Primer 5.0 software. The molecular marker YTU024-L10 for the wheat powdery mildew resistance gene PmL424 provided by this invention, after genetic segregation population testing, showed a genetic distance of only 0.5 cM from the gene PmL424, indicating close linkage. This allows for more accurate and efficient detection of the genetic mapping population of PmL424, which is beneficial for map-based cloning and fine localization of PmL424.

[0027] This invention provides a molecular marker closely linked to the wheat powdery mildew resistance gene PmL424. When applied to powdery mildew resistant wheat breeding, it can not only greatly save costs and shorten the breeding cycle, but also be more accurate and efficient, and can be better applied to powdery mildew resistant wheat breeding work. Attached Figure Description

[0028] Figure 1 This is to label the partial amplification results of YTU024-L10 in an F2 population of 185 L424 × Langdon (LDN) derived from it.

[0029] In the figure, M: pUC19 Msp I; 1: L424 (resistant parent); 2: Langdon(LDN) (susceptible parent); 3-17: F2 population of L424×Langdon(LDN), where 3-7: homozygous resistant lineage, 8-12: resistant-susceptible segregating lineage, 13-17: homozygous susceptible lineage; white arrows are specific bands of pmL424. Detailed Implementation

[0030] 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.

[0031] Example 1: Development of the molecular marker YTU024-L10 for the wheat powdery mildew resistance gene PmL424.

[0032] 1. Materials

[0033] The resistant parent is cultivated emmer wheat L424, and the susceptible parent is durum wheat Langdon (LDN). L424 and Langdon (LDN) are crossed, and the resulting F1 generation is self-crossed to obtain the F2 population.

[0034] 2. Extraction of wheat genomic DNA

[0035] Wheat genomic DNA was extracted using the CTAB method, and the procedure is as follows:

[0036] 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;

[0037] 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;

[0038] 3) Add an equal volume of chloroform and mix on a shaker for 30 minutes;

[0039] 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.

[0040] 5) Centrifuge at 12000 rpm at room temperature for 10 min, discard the supernatant, and wash three times with 800 μL of 75% ethanol;

[0041] 6) Air dry the precipitate, then dissolve it in 50 μL of 1×TE or ddH2O;

[0042] 7) Dilute the DNA storage solution with sterile deionized water to 50 ng / μL as a working solution for later use.

[0043] 3. Identification of powdery mildew resistance in wheat seedlings and genetic analysis of resistance

[0044] Wheat seedling powdery mildew resistance identification was conducted in a greenhouse. The resistant parent L424, the susceptible parent Langdon (LDN), F1 hybrids, and F2 populations were planted in 128-cell trays (3.2 × 3.2 × 4.2 cm). At least 20 seeds from each parent and F1 were identified, and at least 25 seeds from each F2 population were identified. The susceptible control TN18 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 TN18 showed full disease development. 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.

[0045] The results showed that L424 exhibited high resistance to powdery mildew strain E09 (IT=0) and high susceptibility to Langdon (LDN) (IT=4). All F1 plants showed resistance (IT = 0-1), indicating that L424 carries a dominant resistance gene. Resistance identification of the F2 population of this combination showed a resistance-susceptibility segregation ratio of 127:58, which, according to the chi-square test, conformed to a segregation ratio of 3:1 for a single dominant gene (χ²). 2 =3.98, P =0.046). In summary, the resistance of L424 to the powdery mildew strain E09 is controlled by a single dominant gene, which is named PmL424.

[0046] 4. Fine localization of molecular markers for PmL424

[0047] Based on the 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 L424, durum wheat Langdon (LDN), and the resistant and susceptible pools using 155 pairs of molecular markers evenly distributed throughout the genome. 22 pairs of markers showed consistent polymorphism in both the resistant and susceptible parents and the resistant-susceptible pools. Subsequently, these markers were used to genotype 185 L424×Langdon (LDN) F2 populations, preliminarily locating PmL424 within the 21.62–21.81 Mb region of wheat 2BS chromosome.

[0048] 5. Development of molecular markers closely linked to PmL424

[0049] Based on the sequence information of the Chinese spring wheat reference genome within the candidate interval of 21.62-21.81 Mb, an Insertion-Deletion (INDEL) marker was designed using Primer 5.0 software. Genotyping was performed on the F2 population of L424×Langdon(LDN), and the INDEL marker YTU024-L10, which is closely linked to the gene PmL424, was obtained, with a genetic distance of only 0.5 cM.

[0050] The primers for the molecular marker YTU024-L10 include one upstream primer and one downstream primer:

[0051] The nucleotide sequence of the upstream primer YTU024-L10-F is: 5'-GCCTGGCTTCATATGCTGTG-3';

[0052] The nucleotide sequence of the downstream primer YTU024-L10-R is: 5'-TTCTCGGATCAGTAGGCCCT-3'.

[0053] 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.

[0054] 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℃.

[0055] The electrophoretic separation procedure for the amplified products was as follows: Electrophoresis was performed using an 8% non-denaturing polyacrylamide gel. The amplified products were 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 220 V for 1.5–2 h. After silver nitrate staining, photographs were taken. If a specific band of 238 bp was amplified, it indicates the presence of the powdery mildew resistance gene PmL424 in the tested wheat germplasm; otherwise, the powdery mildew resistance gene PmL424 is not present in the tested wheat germplasm.

[0056] Molecular marker detection results are shown below Figure 1 . Figure 1 This image shows partial amplification results of YTU024-L10 in 185 L424×Langdon(LDN) derived F2 populations. In the figure, M: pUC19. Msp1: L424 (resistant parent); 2: Langdon(LDN) (susceptible parent); 3-17: F2 populations of L424 × Langdon(LDN), where 3-7: homozygous resistant families, 8-12: segregating resistant and susceptible families, 13-17: homozygous susceptible families; white arrows indicate specific bands of PmL424. Amplification results showed that the marker YTU024-L10 amplified a specific band of 238 bp in the resistant parent L424 and resistant families, but not in the susceptible parent Langdon(LDN) and susceptible families.

[0057] The wheat powdery mildew resistance gene PmL424, derived from cultivated emmer wheat L424, is a novel gene exhibiting excellent resistance. Currently, there are no reports on its localization, map-based cloning, or application in molecular breeding. Using the molecular marker YTU024-L10 provided in this invention to detect large-scale genetically mapped populations facilitates the precise localization and map-based cloning of gene PmL424. Introducing PmL424 into major wheat varieties susceptible to powdery mildew, and utilizing the molecular marker YTU024-L10 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 PmL424. This is of great significance for the efficient transfer of gene PmL424 and for a deeper understanding of the disease resistance mechanism.

[0058] 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. The use of a molecular marker primer closely linked to the wheat powdery mildew resistance gene PmL424 in the detection of the wheat powdery mildew resistance gene PmL424, characterized in that, The molecular marker is YTU024-L10, primers of the molecular marker YTU024-L10 are composed of an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2, the primers of the molecular marker YTU024-L10 are used for PCR amplification on the wheat genomic DNA to be tested, and a corresponding amplification product with a molecular weight of 238 bp is obtained, that is, the molecular marker closely linked to the wheat powdery mildew resistance gene PmL424.

2. Use according to claim 1, characterized in that, The method comprises the following steps: (1) extracting genomic DNA of the wheat sample to be tested; (2) using primers of the molecular marker YTU024-L10 to perform PCR amplification on the extracted wheat genomic DNA to obtain an amplification product; (3) performing electrophoresis on the amplification product, and judging according to the electrophoresis result; if the wheat DNA can amplify a specific band of 238 bp, it is indicated that the wheat to be tested contains the powdery mildew resistance gene PmL424; otherwise, the wheat to be tested does not contain the wheat powdery mildew resistance gene PmL424.

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

4. Use according to claim 2, characterized in that, The PCR amplification program is: 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; 4℃ preservation.

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