Functional molecular markers, detection methods and applications for detecting wheat stem rust resistance gene Sr8155B1

Abstract

The present invention provides a functional molecular marker for detecting wheat stem rust resistance gene Sr8155B1, a detection method and an application thereof. The functional molecular marker comprises: an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2. The method can solve the problem in the prior art that it is difficult to accurately detect whether wheat stem rust resistance gene Sr8155B1 exists in plants, and is applicable to the field of molecular genetic breeding.

Description

Technical Field

[0001] The present invention relates to the field of molecular genetic breeding, and in particular to a functional molecular marker for detecting wheat stem rust resistance gene Sr8155B1, a detection method and an application thereof. Background Art

[0002] Wheat (Triticum aestivum) stem rust is an airborne fungal disease caused by wheat stem rust (Pucciniagraminis f. sp. tritici, Pgt), which seriously threatens the safe production of wheat worldwide. In 1998, the highly toxic physiological race Ug99 was first discovered, and then a large-scale outbreak caused a large area of ​​wheat to fail. It is reported that about 90% of wheat varieties in the world are highly susceptible to Ug99, so the emergence of this race poses a serious threat to global food security. In recent years, with global warming, the mutation and spread of this race have further accelerated. In 2023, the Ug99 mutant strain has spread to the border of Nepal (https: / / doi.org / 10.1094 / PDIS-03-24-0644-PDN), posing a great hidden danger to the safe production of wheat. Therefore, it is urgent to take effective measures to prevent and control wheat stem rust.

[0003] Breeding new wheat varieties using disease-resistant genes is the most economical, safe and effective way to prevent and control wheat stem rust. Nirmala et al. identified the wheat stem rust resistance gene Sr8155B1 in the tetraploid durum wheat line 8155-B1. This gene has full growth period resistance and resistance to multiple Ug99 variants, including TTKTT, TTTSK, TTKSF+, TTKST, etc. After the gene was introduced into hexaploid wheat, it can significantly improve the resistance of wheat to stem rust, which has important breeding value (https: / / doi:10.1534 / g3.117.300209). Wang et al. identified the resistance spectrum of the material and found that Sr8155B1 showed immunity or near immunity to the six Chinese Pgt subspecies tested (https: / / doi.org / 10.1016 / j.cj.2023.09.006). The current resistance spectrum identification results show that Sr8155B1 exhibits immunity or near-immunity to at least 12 domestic and foreign physiological races of stem rust. Therefore, this gene has important application prospects in breeding.

[0004] Since Sr8155B1 has not been isolated and cloned before, only the linked markers of this gene can be used for molecular marker-assisted selection in breeding. The lack of functional molecular markers within the gene makes it difficult to accurately and efficiently detect the wheat stem rust resistance gene Sr8155B1, which greatly limits the application of this gene in wheat breeding. Therefore, it is urgent to clone this gene and develop efficient diagnostic functional molecular markers within the gene. Summary of the invention

[0005] The main purpose of the present invention is to provide a functional molecular marker, a detection method and an application thereof for detecting the wheat stem rust resistance gene Sr8155B1, so as to solve the problem in the prior art that it is difficult to accurately detect whether the wheat stem rust resistance gene Sr8155B1 exists in plants.

[0006] To achieve the above object, according to the first aspect of the present invention, a functional molecular marker for detecting wheat stem rust resistance gene Sr8155B1 is provided, the functional molecular marker comprising: an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2.

[0007] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, a kit for detecting the wheat stem rust resistance gene Sr8155B1 is provided, the kit comprising the above-mentioned functional molecular marker, and the kit further comprising any one or more of the following: reagents for PCR amplification, reagents for DNA extraction, or reagents for PCR product analysis.

[0008] To achieve the above object, according to the third aspect of the present invention, a method for detecting the wheat stem rust resistance gene Sr8155B1 is provided, the detection method comprising: amplifying the wheat genome using the functional molecular marker or the kit, and detecting the amplified product.

[0009] Furthermore, the detection method includes: a) using the genomic DNA of the wheat to be tested as a template, performing PCR amplification using functional molecular markers, and obtaining a PCR product system; b) detecting the PCR product system to determine whether it contains a PCR amplification product; if there is a PCR amplification product, the wheat to be tested contains the wheat stem rust resistance gene Sr8155B1.

[0010] Further, b) includes: if the PCR product system contains a PCR amplification product with a length of 465 bp, the wheat to be tested contains the wheat stem rust resistance gene Sr8155B1.

[0011] Further, b) includes: testing the PCR product system, if there is no PCR amplification product with a length of 465 bp, the tested wheat does not contain the wheat stem rust resistance gene Sr8155B1.

[0012] Further, b) includes: testing the PCR product system, if there is no electrophoresis band, the tested wheat does not contain the wheat stem rust resistance gene Sr8155B1.

[0013] Furthermore, the detection method in b) includes detecting the PCR product by gel electrophoresis.

[0014] Further, the PCR amplification program in a) is: 94°C for 3 minutes; 94°C for 30 seconds, 65°C for 30 seconds, -0.5°C / cycle, 72°C for 20 seconds, for a total of 10 cycles; 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 20 seconds, for a total of 28 cycles; 72°C for 10 minutes.

[0015] In order to achieve the above-mentioned purpose, according to the fourth aspect of the present invention, there is provided a functional molecular marker for detecting the wheat stem rust resistance gene Sr8155B1, or a kit for detecting the wheat stem rust resistance gene Sr8155B1, or a method for detecting the wheat stem rust resistance gene Sr8155B1 in the breeding of wheat resistant to stem rust.

[0016] Beneficial technical effect: By applying the technical scheme of the present invention, a functional molecular marker for detecting the wheat stem rust resistance gene Sr8155B1 is provided. The above-mentioned functional molecular marker comprises: an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2. The above-mentioned functional molecular marker is used to perform PCR on the genomic DNA of the wheat to be tested, that is, whether the wheat to be tested contains the wheat stem rust resistance gene Sr8155B1 can be detected by whether the PCR amplification product fragment is obtained. The detection method is simple, fast and has low detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 The schematic diagram of Example 1 of the present invention shows the phenotypes of the durum wheat disease-resistant parent 8155-B1, the disease-susceptible parent Rusty and the F2 segregating population produced by hybridization after inoculation with stem rust race 34MKGQM and culturing for 14 days.

[0019] Figure 2 The schematic diagram of the structure of the stem rust resistance gene Sr8155B1 according to Example 2 of the present invention is shown.

[0020] Figure 3The sequence alignment diagram of different haplotypes (Hap1-Hap7) of the Sr8155B1 protein according to Example 2 of the present invention at the functional molecular marker position is shown, wherein only haplotype Hap 1 is a disease-resistant type, namely Sr8155B1.

[0021] Figure 4 The PCR electrophoresis gel image of the molecular markers amplified according to Example 3 of the present invention in the F2 segregating population generated by hybridization of 8155-B1 and Rusty is shown.

[0022] Figure 5 The PCR electrophoresis gel image of the molecular markers amplified in partial diploid, hexaploid and tetraploid wheat varieties according to Example 3 of the present invention is shown. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0024] As mentioned in the background technology, the wheat stem rust resistance gene Sr8155B1 has resistance throughout the growth period and is resistant to multiple Ug99 variants. It is an excellent rust resistance gene resource and has great application prospects in wheat rust resistance breeding. However, since it has not been successfully isolated and cloned, it is difficult to accurately and efficiently detect the Sr8155B1 gene in wheat. Therefore, in this application, the inventors attempt to develop a functional molecular marker that can detect the wheat stem rust resistance gene Sr8155B1, and on this basis, propose a series of protection schemes of this application.

[0025] In a first typical embodiment of the present application, a functional molecular marker for detecting wheat stem rust resistance gene Sr8155B1 is provided, and the functional molecular marker comprises: an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2.

[0026] SEQ ID NO: 1: TTCGGTGATATAGAGGAGTCAGA.

[0027] SEQ ID NO: 2: GCTTGCACTGTAGCTCAGACC.

[0028] The above functional molecular markers are used to perform PCR amplification on wheat genomic DNA. If a PCR product can be obtained, and the detection method of the PCR product includes but is not limited to agarose gel electrophoresis, it means that the wheat being tested contains the Sr8155B1 gene. On the contrary, if no PCR amplification product is detected, it means that the wheat being tested does not contain the Sr8155B1 gene. The above functional molecular markers can be used to simply and quickly detect whether wheat contains this stem rust resistance gene. This functional molecular marker can also be regarded as a primer set.

[0029] In a second typical embodiment of the present application, a kit for detecting the wheat stem rust resistance gene Sr8155B1 is provided, the kit comprising the above-mentioned functional molecular marker, and the kit further comprising any one or more of the following: reagents for PCR amplification, reagents for DNA extraction, or reagents for PCR product analysis.

[0030] In the above-mentioned kit, reagents used for PCR amplification include but are not limited to DNA polymerase, PCR amplification buffer and other reagents; reagents used for DNA extraction include but are not limited to reagents used in common DNA extraction methods in the prior art such as phenol-chloroform extraction method, silica gel membrane column method, magnetic bead method, etc.; reagents used for PCR product analysis include but are not limited to reagents used in methods such as gel electrophoresis, fluorescent probe capture or sequencing.

[0031] In a third typical embodiment of the present application, a method for detecting the wheat stem rust resistance gene Sr8155B1 is provided, characterized in that the detection method comprises: amplifying the wheat genome using the above-mentioned functional molecular marker or the above-mentioned kit, and detecting the amplified product.

[0032] In the above detection method, the above functional molecular marker or kit is used to perform PCR amplification on the wheat to be tested, and the PCR amplification product is detected, so as to detect whether the wheat to be tested contains the wheat stem rust resistance gene Sr8155B1.

[0033] In a preferred embodiment, the detection method includes: a) using the genomic DNA of the wheat to be tested as a template, using primers to perform PCR amplification to obtain a PCR product system; b) detecting the PCR product system to determine whether it contains a PCR amplification product; if there is a PCR amplification product, the wheat to be tested contains the wheat stem rust resistance gene Sr8155B1.

[0034] In a preferred embodiment, b) includes: if the PCR product system contains a PCR amplification product with a length of 465 bp, the wheat to be tested contains the wheat stem rust resistance gene Sr8155B1.

[0035] Preferably, the sequence of the PCR amplification product with a length of 465 bp is shown in SEQ ID NO:3.

[0036] SEQ ID NO: 3:

[0037] TTCGGTGATATAGAGGAGTCTGAATGCAAGGCCCTGATGGAATCTCTGTGCAACCTGCACAAGATCCATAGTCTGACAGTTGTGGATGATTGTTATGATCGTGAGTATTCGGTTGTCCCCAACCATAGCTTGGAAGACTTGGCACCCTGTACAAAGCTGTACGAGTTAGGTCTGTTTAGCATAGTTATACCGAGGGTGCCATCATGGATCAATCAGTTAAGTGTTCCACTCCT TTCTCATCTATGTCTGCATGTGGATGCGGTAGAAGTTCGGGATGTTCAAACCATCGGCAGGTTGCCGTCCCTCCTCGTCCTTTTTCTCTGGAGTAAGGATGAAAAGAACATATCATATACTTTTGGCAGCAATGAGTTTCACAAGCTGAGAGTCTTGTGGACGAAGAAGATAGAGATTGCTGTTGGAGAAGGAGCATTGCCGATGCTTGAAGGGCTGAGCTACAGTGCAAGC.

[0038] In a preferred embodiment, b) includes: testing the PCR product system, if there is no PCR amplification product with a length of 465 bp, the tested wheat does not contain the wheat stem rust resistance gene Sr8155B1.

[0039] In a preferred embodiment, b) comprises: testing the PCR product system, if there is no electrophoresis band, the tested wheat does not contain the wheat stem rust resistance gene Sr8155B1.

[0040] The above functional molecular markers of the present application are used to perform PCR amplification on the wheat genome to be tested. If the wheat genome to be tested does not contain the wheat stem rust resistance gene Sr8155B1, no new DNA fragments will be amplified in the PCR amplification system except for the primer dimer, so no electrophoretic bands will appear in the electrophoresis detection, especially no electrophoretic bands will appear in the range of 100bp-2000bp. In a preferred embodiment, the detection method in b) includes detecting the PCR product by gel electrophoresis.

[0041] It should be noted that the detection method in the present application uses the above-mentioned functional molecular markers and uses the genomic DNA of the wheat to be tested as a template for PCR amplification. If a 465 bp electrophoresis band that is clearly visible in gel electrophoresis can be amplified, it means that the wheat to be tested contains the wheat stem rust resistance gene Sr8155B1. Therefore, the above-mentioned detection method can clearly determine whether the wheat contains the Sr8155B1 gene, and the detection can be achieved without a more accurate analysis of the exact length of the electrophoresis band and the specific sequence of the product.

[0042] In a preferred embodiment, the PCR amplification program in a) is: 94°C for 3 minutes; 94°C for 30 seconds, 65°C for 30 seconds, -0.5°C / cycle, 72°C for 20 seconds, for a total of 10 cycles; 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 20 seconds, for a total of 28 cycles; 72°C for 10 minutes.

[0043] In a fourth typical embodiment of the present application, there is provided a functional molecular marker for detecting the wheat stem rust resistance gene Sr8155B1, or a kit for detecting the wheat stem rust resistance gene Sr8155B1, or a method for detecting the wheat stem rust resistance gene Sr8155B1 in the breeding of wheat resistant to stem rust.

[0044] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0045] The experimental methods in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available unless otherwise specified.

[0046] The wheat leaf genome extraction method used in this application is the CTAB method, and the steps are as follows:

[0047] (1) Place an appropriate amount of leaves into a 2 mL round-bottom centrifuge tube, add steel beads, freeze in liquid nitrogen, and grind the leaves into powder using a grinder;

[0048] (2) Add 600 μL of CTAB extract and heat in a 65°C water bath for 1 hour, inverting and mixing twice;

[0049] (3) Remove the centrifuge tube, cool it to room temperature, add 600 μL of chloroform:isoamyl alcohol (24:1) mixture, shake well, let it stand for 15 minutes, and centrifuge at 12,000 rpm for 15 minutes;

[0050] (4) Take 600 μL of supernatant, add 600 μL of pre-cooled isopropanol, mix gently, let stand at 4°C for 5 minutes, and centrifuge at 12,000 rpm for 15 minutes;

[0051] (5) Discard the supernatant, add 600 μL 70% ethanol for washing, and centrifuge at 12,000 rpm for 10 minutes;

[0052] (6) Discard the supernatant, air-dry, and add 200 μL ddH2O to dissolve to obtain wheat leaf genomic DNA.

[0053] Those skilled in the art can also flexibly select other methods in the prior art to extract the wheat leaf genome.

[0054] Example 1 Identification of wheat stem rust resistance at the seedling stage

[0055] The disease-resistant durum wheat parent 8155-B1 and the susceptible parent Rusty to be identified were planted in an artificial climate chamber, as well as the F2 segregating population produced by the hybridization of the two. The conditions of the artificial climate chamber were set as follows: a photoperiod of 16 hours, 22°C during the day, 20°C at night, and a humidity of 80-90%. When the seedlings grew to the two-leaf and one-heart stage, fresh stem rust spores were inoculated onto the seedlings using the manual sweeping method. After inoculation, the seedlings were kept in darkness and kept moist for 24 hours. 14 days after inoculation, the material was identified for stem rust resistance. The resistance identification results are as follows: Figure 1 As shown in the figure, R means resistance and S means susceptibility. 14 days after inoculation, the strains without and with Sr8155B1 showed different phenotypes to stem rust race 34MKGQM. The strains with Sr8155B1 showed immunity or near immunity (R), while the strains without Sr8155B1 showed moderate or high susceptibility (S).

[0056] Example 2 Development of functional molecular markers for stem rust resistance gene Sr8155B1

[0057] In order to fine-tune the stem rust resistance gene Sr8155B1, the applicant used the Svevo reference genome (http: / / 202.194.139.32 / blast / blast.html) to develop InDel, CAPS or KASP markers on chromosome 6AS. By using the F2 segregation population of 8155-B1×Rusty containing 4600 individuals, the stem rust resistance gene Sr8155B1 was located in the 0.03 cM region between the CAPS marker pku2772 and the dominant marker pku6A3 (SEQ ID NO: 4: TATTCTCCAGCACAATCCG and SEQ ID NO: 5: AACAGGCTTCAGGTACTCACA). (For primer sequences, see Wang J, Li H, Shen T, et al. High-resolution genetic mapping and identification of candidate genesfor the wheat stem rust resistance gene Sr8155B1[J]. Crop Journal (2095-5421), 2023, 11(6). DOI:10.1016 / j.cj.2023.09.006.) The physical interval of this region in the Svevo reference genome is 295 Kb and contains only 6 genes, including 1 NLR gene (TRITD6Av1G001430) and 1 receptor protein kinase gene (TRITD6Av1G001460). We performed PacBio HiFi long-read sequencing on the tetraploid hybrid F3 family PKU81 containing Sr8155B1, assembled the data, and obtained a contig: ptg000390l (https: / / ngdc.cncb.ac.cn / bioproject / , accession number: PRJCA032807) containing the Sr8155B1 candidate interval. Sequence annotation showed that the PKU81 physical interval was highly colinear with Svevo, and the interval also contained only 6 high-confidence genes. Through transcriptional expression analysis, sequence polymorphism, and gene function annotation, it was inferred that CNL1 (TRITD6Av1G001430.5) was a candidate gene for Sr8155B1. Subsequently, we carried out functional verification of the candidate gene: using susceptible EMS mutants, gene editing knockout, and transgenic complementation experiments to prove that CNL1 is the stem rust resistance gene Sr8155B1.

[0058] The Sr8155B1 gene is 4448 bp in length and consists of 4 exons. The encoded protein contains 1043 amino acids. The gene structure is as follows: Figure 2As shown in the figure (boxes represent exons and solid lines represent introns). Using the amino acid sequence of the gene translated from the genome of diploid, tetraploid and hexaploid wheat, it was found that most proteins had a similarity of less than 93% with the Sr8155B1 protein, with only 6 haplotypes ( Figure 3 Hap 2-7) and Sr8155B1 ( Figure 3 The similarity between the two strains is above 96.8%, and the polymorphism is concentrated in the LRR domain. Figure 3 The sequence alignment diagram of different haplotypes (Hap 1-Hap7) of Sr8155B1 protein at the functional molecular marker position is shown in FIG. Figure 3 The results of partial sequence alignment of Hap 1-Hap 7 are shown in FIG. Among the seven haplotypes, the 699th and 926th amino acids are Sr8155B1 protein-specific amino acids ( Figure 3 The red arrow indicates the position). When analyzed together with the resequencing or exon sequencing data of about 1,500 tetraploid / hexaploid wheat accessions (https: / / triticeaetoolbox.org / wheat / ; http: / / wheatgenomics.plantpath.ksu.edu / 1000EC / ; https: / / doi.org / 10.1038 / s41588-019-0393-z), it was found that only the 699th amino acid was specific to the Sr8155B1 protein.

[0059] Using Primer 5 software, diagnostic functional molecular markers of the Sr8155B1 gene were designed at the 699th and 840th amino acid positions ( Figure 3 The blue arrow indicates the position). The forward primer is shown in SEQ ID NO: 1, the reverse primer is shown in SEQ ID NO: 2, and the diagnostic molecular marker is named 8155B1F1R1. The sequence of the amplified product is shown in SEQ ID NO: 3.

[0060] SEQ ID NO: 1: TTCGGTGATATAGAGGAGTCAGA.

[0061] SEQ ID NO: 2: GCTTGCACTGTAGCTCAGACC.

[0062] SEQ ID NO: 3:

[0063] TTCGGTGATATAGAGGAGTCTGAATGCAAGGCCCTGATGGAATCTCTGTGCAACCTGCACAAGATCCATAGTCTGACAGTTGTGGATGATTGTTATGATCGTGAGTATTCGGTTGTCCCCAACCATAGCTTGGAAGACTTGGCACCCTGTACAAAGCTGTACGAGTTAGGTCTGTTTAGCATAGTTATACCGAGGGTGCCATCATGGATCAATCAGTTAAGTGTTCCACTCCT TTCTCATCTATGTCTGCATGTGGATGCGGTAGAAGTTCGGGATGTTCAAACCATCGGCAGGTTGCCGTCCCTCCTCGTCCTTTTTCTCTGGAGTAAGGATGAAAAGAACATATCATATACTTTTGGCAGCAATGAGTTTCACAAGCTGAGAGTCTTGTGGACGAAGAAGATAGAGATTGCTGTTGGAGAAGGAGCATTGCCGATGCTTGAAGGGCTGAGCTACAGTGCAAGC.

[0064] Figure 3 The partial sequence of Hap 1 shown in is shown in SEQ ID NO: 6, Figure 3 The partial sequence of Hap 2 shown in is shown in SEQ ID NO: 7, Figure 3 The partial sequence of Hap 3 shown in is shown in SEQ ID NO: 8, Figure 3 The partial sequence of Hap 4 shown in is shown in SEQ ID NO: 9, Figure 3 The partial sequence of Hap 5 shown in is shown in SEQ ID NO: 10, Figure 3 The partial sequence of Hap 6 shown in is shown in SEQ ID NO: 11, Figure 3 The partial sequence of Hap 7 shown in is shown as SEQ ID NO:12.

[0065] SEQ ID NO: 6:

[0066] RCLWADKGTVMKGKIGKLTCLEELQLYTVEKCPNFFTEVGKLTELRLLKIFFGDIEESECKALMESLCNLHKIHSLTVVDDCYDREYSVVPNHSLEDLAPCTKLYELGLFSIVIPRVPSWINQLSVPLLSHLCLHVDAVEVRDVQTIGRLPSLLVLFLWSKDEKNISYTFGSNEFHKLRVLWTKKIEIAVGEGALPMLEGLSYSASAERKDAASLVPWRRNSCPLLKYVTCVLDCTNSSYREVEEAEEALRQASGTRPNAVYLDLDIEKENYDEEAGKFIDNLEWILRGLDRPEDVGRPAAHQEERTRRMIRSLERRLRD。

[0067] SEQ ID NO:7:

[0068] RCLWADKGTVMKGKIGKLTCLEELQLYTVEKCPNFFTEVGKLTELRLLKIFFGDIEESACKALMESLCNLHKIHSLTVVDDCYDREYSVVPNHSLEDLAPCTKLYELGLFSIVIPRVPSWINQLSVPLLSHLCLHVDAVEVRDVQTIGRLPSLLVLFLWSKDEKNISYTFGSNEFHKLRVLFTKKIEIAVGEGALPMLEVLSYSASAERKDAASLVPWRRNSCPLLKDVTCVLDCTNSSYREVEEAEEALRQASGTRPNAVYLDLDIEYENYDEEAGKFIDNLEWTLRGLDRPEDVGRPAAHQEERTRRMIRSLERRLRD。

[0069] SEQ ID NO:8:

[0070] RCLWADKGTVMKGKIGKLTCLEELQLYTVEKCPNFFTEVGKLTELRLLKIFFGDIEESACKALMESLCNLHKIHSLTVVDDCYDREYSVVPNHSLEDLAPCTKLYELGLFSIVIPRVPSWINQLSVPLLSHLCLHVDAVEVRDVQTIGRLPSLLVLFLWSKDEKNISYTFGSNEFHKLRVLFTKKIEIAVGEGALPMLEVLSYSASAERKDAASLVPWRRNSCPLLKDVTCVLDCTNSSYREVEEAEEALRQASGTRPNAVYLDLDIEYENYDEEAGKFIDNLEWTLRGLDRPEDVGRPAAHQEERTRRMIRSLERRLRD。

[0071] SEQ ID NO:9:

[0072] RCLWADKGTVMKGKIGKLTCLEELQLYTVEKCPNFFTEVGKLTELRLLKIFFGDIEESACKALMESLCNLHKIHSLTVVDDCYDREYSVVPNHSLEDLAPCTKLYELGLFSIVIPRVPSWINQLSVPLLSHLCLHVDAVEVRDVQTIGRLPSLLVLFLWSKDEKNISYTFGSNEFHKLRVLFTKKIEIAVGEGALPMLEVLSYSASAERKDAASLVPWRRNSCPLLDYVRCYLDCTNSSYREVEEAEEALRQASGTRPNAVYLDLDIEYENYDEEAGKFIDNLEWTLRGLDRPEDVGRPAAHQEERTRRMIRSLERRLRD。

[0073] SEQ ID NO:10:

[0074] RCLWADKGTVMKGKIGKLTCLEELQLYTVEKCPNFFTEVGKLTELRLLKIFFGDIEESACKALMESLCNLHKIHSLTVVDDCYDREYSVVPNHSLEDLAPCTKLYELGLFSIVIPRVPSWINQLSVPLLSHLCLHVDAVEVRDVQTIGRLPSLLVLFLWSKDEKNISYTFGSNEFHKLRGLFTKKIEIAVGEGALPMLEGLRYSASAERKDAASLVPWRRNSCPLLKDVTCVLDCTNSSYREVEEAKEALRQASGTRPNAVYLDLDIEYENYDEEAGKFIDKLEEVLGGLDRPEDVGRPAAHQEERTRRMIRSLERRLRD。

[0075] SEQ ID NO:11:

[0076] RCLWADKGTVMKGKIGKLTCLEELQLYTVEKCPNFFTEVGKLTELRLLKIFFGDIEESACKALMESLCNLHKIHSLTVMDDCYDPEYSVVSNHSLEDLAPCTKLYELSLFSIVIPRVPSWINQLTVPLLSRLCLHVDAVEVRDVQTIGRLPSLLDLFLWSKDEKNISYTFGSNEFHKLRWLWTKKIEIAVGEGALPMLEDLGYSASAERKDAASLVPWRRNSCPLLKHVTCFLDCTNSSYREVEEARQALRQASGTRPNAVYPDLDIEYENYDEEAGKFIDNLEWTLRGLDRPEDVARPAAYQEERTRRMIRSLERRLRD。

[0077] SEQ ID NO:12:

[0078] RCLWADKGTVMKGEIGKLTCLEELQLYSVEKCPNFFTEVGKLTELRVLGICFGDIEESACKALMESLCNLNKIHSLTVVDDCHDPEYSVVLNHSLEDLAPCTKLYKLILPIIVIPRVPSWINQLTVPLLSRLCLHVDAVEVRDVQTIGRLPSLLVLLLLLS KDEKNISYTFGSNEFHKLRGLWTKKIEIAVGEGALPMLEVLRYSASAERKDAASLVPWRRNSCPLLKYVGCYLDCTNSSYREVEEAAEALRQASGTRPNAVYLELYIEKENYDEEAGKFIDNLEWTLRGLDRPEDVARPAAYQEERTRRMIRSLERRLRD.

[0079] Example 3 Application of Sr8155B1 functional molecule marker

[0080] The functional molecular marker was used to identify the genotypes of 94 individual plants from the disease-resistant parent 8155-B1, the disease-susceptible parent Rusty, and the F2 segregation population produced by their hybridization.

[0081] Specifically, according to the designed molecular marker 8155B1F1R1, the genomic DNA of the wheat sample was extracted by CTAB method and PCR amplification was performed. The specific reaction system is as follows:

[0082] DNA template (50 ng / μL) 2.0 μL,

[0083] 2 × Rapid Taq Master Mix (Novozyme, P222) 12.5 μL,

[0084] 10 μM primer 0.5 μL,

[0085] Add ddH2O to make up to 25 μL.

[0086] PCR amplification reaction program: 94°C for 3 minutes; 94°C for 30 seconds, 65°C for 30 seconds, -0.5°C / cycle, 72°C for 20 seconds, for a total of 10 cycles; 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 20 seconds, for a total of 28 cycles; 72°C for 10 minutes.

[0087] The amplified products were analyzed by 1.5% agarose gel electrophoresis, the electrophoresis buffer was 1×TAE, and the constant voltage of the electrophoresis instrument was 100 V. The gel was developed with EB and photographed using a BIO-RAD imager. Some of the results are shown in Figure 4 shown. Figure 4Lane 16 is the resistant parent 8155-B1, lane 17 is the susceptible parent Rusty, lanes 1-15 are the individual plants 1-15 of the F2 segregation population, and lane M represents the marker. Among them, individual plants 1, 2, 3, 5, 6, 7, 9, 10, 11, 12, 14, and 15 amplified the same bands as 8155-B1 (lane 16), indicating that they contain the gene Sr8155B1; while individual plants 4, 8, and 13 are the same as Rusty (lane 17), with no PCR amplification products, indicating that they do not contain the gene Sr8155B1.

[0088] The disease resistance of the resistant and susceptible parents and the above 94 F2 plants was identified using stem rust race 34MKGQM. The results showed that the obtained phenotype was completely consistent with the genotype.

[0089] Molecular marker 8155B1F1R1 was used to identify genotypes of 80 diploid wheat accessions (T. monococcum and T. urartu), 98 tetraploid wheat accessions (T. dicoccon, T. dicoccoides and T. durum) and 138 hexaploid wheat accessions (T. aestivum) from different countries.

[0090] Specifically, according to the designed molecular marker 8155B1F1R1, the genomic DNA of the above wheat materials was extracted by CTAB method for PCR amplification. The specific reaction system is as follows:

[0091] DNA template (50 ng / μL) 2.0 μL,

[0092] 2 × Rapid Taq Master Mix (Novozyme, P222) 12.5 μL,

[0093] 10 μM primer 0.5 μL,

[0094] Add ddH2O to make up to 25 μL.

[0095] PCR amplification reaction program: 94°C for 3 minutes; 94°C for 30 seconds, 65°C for 30 seconds, -0.5°C / cycle, 72°C for 20 seconds, for a total of 10 cycles; 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 20 seconds, for a total of 28 cycles; 72°C for 10 minutes.

[0096] The amplified products were analyzed by 1.5% agarose gel electrophoresis, the electrophoresis buffer was 1×TAE, and the constant voltage of the electrophoresis instrument was 100 V. The gel was developed with EB and photographed using a BIO-RAD imager.

[0097] The statistical results are shown in Tables 1 and 2. The results showed that except for 8155-B1, the other tested materials did not amplify the 465 bp target band, indicating that the tested materials did not contain the Sr8155B1 gene (as shown in Table 1). Figure 5 Shown is the electrophoresis gel image of the molecular marker 8155B1F1R1 amplified in some diploid, hexaploid and tetraploid wheat varieties. Figure 5 Lanes 1-13 are diploid wheat PI 272560 (lane 1), PI 277130 (lane 2), G3116 (lane 3), DV92 (lane 4), hexaploid wheat Chinese Spring (lane 5), Avocet-S (lane 6), Neepawa (lane 7), Fielder (lane 8) and tetraploid wheat Kronos (lane 9), Line E (lane 10), Svevo (lane 11), Rusty (lane 12) and 8155-B1 (lane 13), respectively. Lane M indicates marker.

[0098] Only 8155-B1 amplified a 465 bp target band, and no bands were amplified in other materials, indicating that the molecular marker 8155B1F1R1 provided by the present invention can efficiently and accurately identify whether the wheat material contains the stem rust resistance gene Sr8155B1.

[0099] Table 1

[0100]

[0101] Table 2

[0102]

[0103] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present invention provides a functional molecular marker for detecting wheat stem rust resistance gene Sr8155B1, and the functional molecular marker includes: an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2. By using the upstream primer and the downstream primer, the genomic DNA of the wheat sample to be detected can be used as a template for amplification, so as to detect whether the wheat sample contains the wheat stem rust resistance gene Sr8155B1, and the detection difficulty is low, which solves the problem of difficulty in accurately identifying and effectively utilizing the wheat stem rust resistance gene Sr8155B1 in wheat breeding, and has broad application prospects and significant economic value.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting wheat stem rust resistance genes Sr8155B1 A functional molecular marker, characterized in that The functional molecular marker comprises: an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO:

2.

2. A method for detecting wheat stem rust resistance genes Sr8155B1 The kit is characterized in that The kit comprises the functional molecular marker according to claim 1, and the kit further comprises any one or more of the following: Reagents for PCR amplification, reagents for DNA extraction, or reagents for PCR product analysis.

3. A wheat stem rust resistance gene Sr8155B1 The detection method is characterized in that The detection method comprises: amplifying the wheat genome using the functional molecular marker described in claim 1 or the kit described in claim 2, and detecting the amplified product.

4. The detection method according to claim 3, characterized in that: The detection method comprises: a) using the genomic DNA of the wheat to be tested as a template and performing PCR amplification using the functional molecular marker to obtain a PCR product system; b) detecting the PCR product system to determine whether it contains PCR amplification products; If the PCR amplification product is present, the wheat to be tested contains the wheat stem rust resistance gene Sr8155B1 .

5. The detection method according to claim 4, characterized in that: b) includes: if the PCR product system contains the PCR amplification product with a length of 465 bp, then the wheat to be tested contains the wheat stem rust resistance gene Sr8155B1 .

6. The detection method according to claim 4, characterized in that: b) includes: testing the PCR product system, if there is no PCR amplification product with a length of 465 bp, then the wheat to be tested does not contain the wheat stem rust resistance gene Sr8155B1 .

7. The detection method according to claim 6, characterized in that: b) includes: testing the PCR product system, if there is no electrophoresis band, the tested wheat does not contain the wheat stem rust resistance gene Sr8155B1 .

8. The detection method according to any one of claims 4 to 7, characterized in that: The detection method in b) comprises detecting the PCR product by gel electrophoresis.

9. The method for detecting wheat stem rust resistance gene according to claim 1 Sr8155B1 or the wheat stem rust resistance gene detection method according to claim 2 Sr8155B1 A kit, or the wheat stem rust resistance gene according to any one of claims 4 to 8 Sr8155B1 Application of detection method in wheat breeding for stem rust resistance.

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