Functional molecular marker of wheat leaf rust resistance gene Lr.ace-4a, detection method and application thereof

Abstract

The application provides a functional molecular marker of a wheat leaf rust resistance gene Lr.ace-4A, a detection method and 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 application can solve the problem that the Lr.ace-4A gene in the wheat is difficult to detect rapidly in the prior art, and is suitable for the field of molecular genetic breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular genetics breeding, and in particular, to a functional molecular marker of a wheat leaf rust resistance gene Lr.ace-4A, a detection method and application thereof. BACKGROUND

[0002] Wheat leaf rust is a kind of fungal airborne disease caused by Puccinia triticina (Pt) infection, which mainly damages the leaves of wheat, destroys photosynthesis, and then causes the yield reduction of wheat. Generally, it can cause 5% to 15% yield reduction, and when the disease is serious, it can cause more than 40% yield reduction. In recent years, due to the continuous evolution of physiological races of Puccinia triticina, the resistance of commonly used Lr1, Lr3, Lr10, Lr13 and Lr26 specific disease resistance genes has been lost. At the same time, global warming and changes in farming system exacerbate the prevalence of wheat leaf rust, which seriously threatens the safe production of wheat. Therefore, the prevention and control of wheat leaf rust has become an important task in wheat production.

[0003] The wheat leaf rust resistance gene Lr.ace-4A is from a local variety PI192051 of T. durum in Portugal. Using a genetic segregation population, the gene is located on the 4A chromosome near the centromere region. Recent studies have shown that Lr.ace-4A shows near-immune resistance to most of the prevalent strong virulence physiological races of Puccinia triticina, and has good breeding application potential. At present, only the linked markers of Lr.ace-4A can be used for molecular marker assisted selection in breeding, and there is a lack of functional molecular markers inside the gene, which cannot accurately and quickly detect the wheat leaf rust resistance gene Lr.ace-4A, limiting the utilization of the gene in wheat disease resistance breeding. Therefore, it is urgent to develop functional molecular markers according to the gene sequence to realize early molecular assisted selection of the leaf rust resistance gene Lr.ace-4A and improve the efficiency of wheat disease resistance breeding. SUMMARY

[0004] The main purpose of the present application is to provide a functional molecular marker of a wheat leaf rust resistance gene Lr.ace-4A, a detection method and application thereof, so as to solve the problem that the Lr.ace-4A gene in wheat is difficult to detect quickly in the prior art.

[0005] In order to achieve the above purpose, according to a first aspect of the present application, a functional molecular marker for detecting a wheat leaf rust resistance gene Lr.ace-4A is provided, which comprises: an upstream primer having SEQ ID NO: 1 and a downstream primer having SEQ ID NO: 2.

[0006] In order to achieve the above-mentioned purpose, according to a second aspect of the present application, there is provided a kit for detecting the wheat leaf rust resistance gene Lr.ace-4A, the kit comprising the 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.

[0007] In order to achieve the above-mentioned purpose, according to a third aspect of the present application, there is provided a method for detecting the wheat leaf rust resistance gene Lr.ace-4A, the method comprising: amplifying the genome of wheat using the functional molecular marker or the kit, and detecting the amplified product.

[0008] Further, the method comprises: a) using the genomic DNA of the wheat to be tested as a template, performing PCR amplification using the functional molecular marker to obtain a PCR product system; b) detecting the PCR product system to determine whether the PCR amplification product is present; if the PCR amplification product is present, the wheat to be tested contains the wheat leaf rust resistance gene Lr.ace-4A.

[0009] Further, b) comprises: if the PCR product system contains a PCR amplification product with a length of 916 bp, the wheat to be tested contains the wheat leaf rust resistance gene Lr.ace-4A.

[0010] Further, b) comprises: detecting the PCR product system, and if there is no PCR amplification product with a length of 916 bp, the wheat to be tested does not contain the wheat leaf rust resistance gene Lr.ace-4A.

[0011] Further, b) comprises: detecting the PCR product system, and if there is no electrophoresis band, the wheat to be tested does not contain the wheat leaf rust resistance gene Lr.ace-4A.

[0012] Further, the detection method in b) comprises detecting the PCR product using gel electrophoresis.

[0013] Further, the PCR amplification procedure in a) is as follows: 94℃ for 3 min; 94℃ for 30 s, 54℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; and 72℃ for 5 min.

[0014] In order to achieve the above-mentioned purpose, according to a fourth aspect of the present application, there is provided an application of the functional molecular marker for detecting the wheat leaf rust resistance gene Lr.ace-4A, or the kit for detecting the wheat leaf rust resistance gene Lr.ace-4A, or the method for detecting the wheat leaf rust resistance gene Lr.ace-4A in the breeding of wheat with leaf rust resistance.

[0015] Beneficial technical effects: the technical scheme of the present application provides a functional molecular marker for detecting wheat leaf rust resistance gene Lr.ace-4A, which comprises an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2. The functional molecular marker is located inside the gene and is designed based on SNP (single nucleotide polymorphism). Using the functional molecular marker to perform PCR on the genomic DNA of the wheat to be tested can realize the detection of whether the wheat to be tested contains the wheat leaf rust resistance gene Lr.ace-4A by whether a PCR amplification product fragment is obtained, which is simple in operation, low in cost, can realize early molecular assisted selection of the leaf rust resistance gene Lr.ace-4A, and improves the efficiency of wheat disease breeding. BRIEF DESCRIPTION OF DRAWINGS

[0016] The drawings constituting a part of the specification of the present application are used to provide a further understanding of the present application, the illustrative embodiments of the present application and the description thereof serve to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0017] Figure 1 A schematic diagram according to embodiment 1 of the present application is shown, which shows the phenotype of the hard wheat disease-resistant parent PI 192051, the disease-susceptible parent Rusty and the F2 separation population generated by hybridization after being inoculated with leaf rust race PHQS for 10 days.

[0018] Figure 2 A structural schematic diagram of the leaf rust resistance gene Lr.ace-4A according to embodiment 2 of the present application is shown.

[0019] Figure 3 A sequence alignment diagram of the Lr.ace-4A protein according to embodiment 2 of the present application and the disease-susceptible homologous protein derived from other wheat at the position of the functional molecular marker is shown.

[0020] Figure 4 A PCR electrophoresis gel map of the molecular marker according to embodiment 3 of the present application amplified in the F2 separation population generated by hybridization of PI 192051 and Rusty is shown.

[0021] Figure 5 A PCR electrophoresis gel map of the molecular marker according to embodiment 3 of the present application amplified in part of tetraploid and hexaploid wheat varieties is shown. DETAILED DESCRIPTION

[0022] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0023] TERMS EXPLANATION:

[0024] Single nucleotide polymorphism (SNP): DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level.

[0025] As mentioned in the background, the detection of Lr.ace-4A gene in wheat in the prior art can only use linkage markers for molecular marker assisted selection, and there is a lack of molecular markers located in the gene, which cannot realize accurate and rapid detection. Therefore, the inventors attempt to develop a new functional molecular marker of wheat leaf rust resistance gene Lr.ace-4A in the present application, and on this basis, a series of protection schemes of the present application are proposed.

[0026] In the first typical embodiment of the present application, a functional molecular marker for detecting wheat leaf rust resistance gene Lr.ace-4A is provided, and the functional molecular marker comprises: an upstream primer (Lr30MAS-47F) having SEQ ID NO: 1 and a downstream primer (Lr30MAS-47R) having SEQ ID NO: 2.

[0027] SEQ ID NO: 1: CCTTATTGCCTAGATCCTTTG.

[0028] SEQ ID NO: 2: GCTTAAGGGAATCTGGACG.

[0029] The recombination rate near the Lr.ace-4A gene is very low, it is difficult to narrow down the target interval by recombination, and the gene is very conservative, the homologous sequence similarity in different wheat varieties is high, and there are certain difficulties in designing the gene internal probe. In the present application, the inventors developed the above-mentioned functional molecular marker, and used the functional molecular marker to perform PCR amplification on wheat genomic DNA. If the PCR product can be obtained, the detection method of the PCR product includes but is not limited to agarose gel electrophoresis, it indicates that the wheat to be detected contains Lr.ace-4A gene. On the contrary, if no PCR amplification product is detected, it indicates that the wheat to be detected does not contain Lr.ace-4A gene. The use of the above-mentioned functional molecular marker can simply and quickly realize the detection of whether the wheat contains this kind of leaf rust resistance gene. This kind of functional molecular marker can also be regarded as a primer set.

[0030] Linkage markers refer to molecular markers located near the target gene but not inside the gene. They are linked to the target gene, i.e. in the genetic process, they are highly likely to be inherited together with the target gene to the offspring. That is, linkage markers can only provide indirect information of the gene, and the detection accuracy is lower than that of the markers located inside the gene. Moreover, the distance between the linkage markers and the target gene can affect the accuracy of the markers, leading to misjudgment, and with the increase of recombination, the originally closely linked genes can gradually separate, affecting the effectiveness of the markers.

[0031] The above functional molecular markers provided in the present application are intragenic markers located inside the gene and directly related to the function of the gene. They are molecular markers designed based on single nucleotide polymorphisms (SNPs), and the detection of the target gene using such molecular markers has higher accuracy. In a second typical embodiment of the present application, a kit for detecting the wheat leaf rust resistance gene Lr.ace-4A is provided, which comprises the above functional molecular marker, and further comprises any one or more of the following: reagents for PCR amplification, reagents for DNA extraction, or reagents for PCR product analysis.

[0032] In the above kit, the reagents for PCR amplification include but are not limited to DNA polymerase, PCR amplification buffer and the like; the reagents 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 and the like; and the reagents for PCR product analysis include but are not limited to reagents used in methods such as gel electrophoresis, fluorescent probe capture or sequencing and the like.

[0033] In a third typical embodiment of the present application, a detection method for the wheat leaf rust resistance gene Lr.ace-4A is provided, which comprises: using the above functional molecular marker or the above kit to amplify the wheat genome, and detecting the amplified product.

[0034] Using the above functional molecular marker or the kit to amplify the genome of the wheat to be tested, based on the analysis of the amplified product, it can be determined whether the wheat to be tested contains the wheat leaf rust resistance gene Lr.ace-4A.

[0035] In a preferred embodiment, the detection method comprises: a) using the functional molecular marker to perform PCR amplification with the genomic DNA of the wheat to be tested as a template to obtain a PCR product system; b) detecting the PCR product system to determine whether it contains the PCR amplification product; if the PCR amplification product is present, the wheat to be tested contains the wheat leaf rust resistance gene Lr.ace-4A.

[0036] In a preferred embodiment, b) comprises: if the PCR amplification product with a length of 916 bp is contained in the PCR product system, the wheat to be tested contains the wheat leaf rust resistance gene Lr.ace-4A.

[0037] Preferably, the sequence of the PCR amplification product with a length of 916 bp is shown as SEQ ID NO: 3.

[0038] SEQ ID NO: 3:

[0039]

[0040]

[0041] In a preferred embodiment, b) comprises: detecting the PCR product system, if there is no PCR amplification product with a length of 916 bp, the wheat to be tested does not contain the wheat leaf rust resistance gene Lr.ace-4A.

[0042] In a preferred embodiment, b) comprises: detecting the PCR product system, if there is no electrophoresis band, the wheat to be tested does not contain the wheat leaf rust resistance gene Lr.ace-4A.

[0043] Using the above functional molecular marker of the present application to perform PCR amplification on the genome of the wheat to be tested, if the wheat to be tested does not contain the wheat leaf rust resistance gene Lr.ace-4A, there will be no new DNA fragments amplified in the PCR amplification system except for primer dimers, so there will be no electrophoresis bands in the electrophoresis detection, especially in the range of 100 bp-2000 bp. In a preferred embodiment, the detection method in b) comprises detecting the PCR product by gel electrophoresis.

[0044] It should be noted that the detection method in the present application uses the above functional molecular marker to perform PCR amplification on the genome DNA of the wheat to be tested, if a clear 916 bp electrophoresis band can be obtained in the gel electrophoresis, it means that the wheat to be tested contains the wheat leaf rust resistance gene Lr.ace-4A. Therefore, using the above detection method can clearly determine whether the wheat contains the Lr.ace-4A gene, without the need for more accurate analysis of the exact length of the electrophoresis band and the specific sequence of the product to achieve detection.

[0045] In a preferred embodiment, the detection method in b) comprises detecting the PCR product by gel electrophoresis.

[0046] If the sample lacks the wheat leaf rust resistance gene Lr.ace-4A, the PCR reaction will only produce primer dimers, and no additional specific DNA amplification products will be generated. Therefore, in the gel electrophoresis detection, no bands other than primer dimers will be observed, and in particular, no bands in the molecular weight range of 100 bp to 2000 bp should appear.

[0047] In a preferred embodiment of the present application, the PCR amplification results are directly observed using gel electrophoresis technology. Specifically, when the genomic DNA of the wheat sample to be tested is used as the PCR template and amplified using the above-mentioned molecular marker, if a clear and distinct band of 916 bp appears in the gel electrophoresis, it indicates that the wheat sample carries the leaf rust resistance gene Lr.ace-4A. The significant advantage of this method is that it allows direct determination of the presence or absence of the gene from the gel electrophoresis image, without the need for further length measurement of the electrophoresis band or sequence analysis of the amplification product, thereby greatly simplifying the detection process and improving the detection efficiency and accuracy.

[0048] In short, the detection method of the present application realizes rapid and accurate identification of the wheat leaf rust resistance gene Lr.ace-4A by using specific molecular markers combined with PCR and gel electrophoresis technology, without the need for complex post-processing analysis.

[0049] In a preferred embodiment, the PCR amplification procedure in a) is: 94°C for 3 min; 94°C for 30 s, 54°C for 30 s, 72°C for 30 s, for a total of 30 cycles; 72°C for 5 min.

[0050] Preferably, the PCR reaction system in a) includes: 100 ng of DNA template, 12.5 μL of 2x Rapid Taq MasterMix (Novozyme, P222), and 0.5 μL of 10 μm / μL primers.

[0051] Preferably, the method for extracting the genomic DNA of the wheat to be tested includes the CTAB method.

[0052] Those skilled in the art can flexibly select the existing genomic extraction methods, PCR methods, and PCR reagents in the prior art to perform the above-mentioned detection method.

[0053] In a fourth typical embodiment of the present application, the application of the functional molecular marker for detecting the wheat leaf rust resistance gene Lr.ace-4A, or the kit for detecting the wheat leaf rust resistance gene Lr.ace-4A, or the detection method for the wheat leaf rust resistance gene Lr.ace-4A in the breeding of wheat resistant to leaf rust is provided.

[0054] The beneficial effects of the present application will be further explained in detail below in connection with specific examples.

[0055] The experimental methods in the following examples are all conventional methods, and the reagents used are all commercially available, unless otherwise specified.

[0056] The wheat leaf genome extraction method used in the present application is the CTAB method, and the steps are as follows:

[0057] (1) Put an appropriate amount of leaves into a 2 mL round-bottom centrifuge tube, add steel balls, and freeze in liquid nitrogen. Use a grinder to grind the leaves into powder;

[0058] (2) Add 600 μL of CTAB extraction solution, heat in a 65°C water bath for 1 hour, and mix well twice during the process;

[0059] (3) Take out the centrifuge tube and cool it to room temperature. Add 600 μL of chloroform:isopropyl alcohol (24:1) mixture, shake well, stand for 15 minutes, and centrifuge at 12000 rpm for 15 minutes;

[0060] (4) Take 600 μL of supernatant, add 600 μL of pre-cooled isopropyl alcohol, mix gently, stand at 4°C for 5 minutes, and centrifuge at 12000 rpm for 15 minutes;

[0061] (5) Discard the supernatant, add 600 μL of 70% ethanol for washing, and centrifuge at 12000 rpm for 10 minutes;

[0062] (6) Discard the supernatant, air dry, add 200 μL of ddH2O to dissolve, and obtain the wheat leaf genomic DNA.

[0063] Those skilled in the art can also flexibly select other methods in the prior art to extract the wheat leaf genome.

[0064] Example 1 Leaf rust resistance identification at seedling stage

[0065] The disease-resistant parent durum wheat (T. durum) PI 192051 and the disease-susceptible parent Rusty, as well as the F2 separation population generated by crossing the two, were planted in a phytotron. The phytotron conditions were set as follows: photoperiod 16 hours, day 24°C, night 22°C, and humidity 80-90%. When the seedlings grew to the two-leaf-one-heart stage, fresh leaf rust spores were inoculated onto the seedlings by artificial spraying. After inoculation, the seedlings were treated in the dark for 24 hours. Leaf rust resistance identification was performed 10 days after inoculation.

[0066] For example, Figure 1As shown, after 10 days of inoculation, the families without the gene Lr.ace-4A and the families with the gene Lr.ace-4A showed different phenotypes against Puccinia triticina race PHQS. The families with the gene Lr.ace-4A showed immune or near-immune (R), and the families without the gene Lr.ace-4A showed moderate or high susceptible (S). The phenotypic data were used for subsequent analysis of the linkage relationship between the molecular marker and the gene Lr.ace-4A, and the map-based cloning of the gene.

[0067] Example 2 Development of functional molecular marker of leaf rust resistance gene Lr.ace-4A

[0068] In the inventors’ recent study, it was found that the full length of Lr.ace-4A gene is 4252 bp, which consists of 3 exons, encodes a protein containing 1175 amino acids, and the gene structure is as shown in Figure 2 (boxes represent exons, and solid lines represent introns). By analyzing the sequence polymorphism of Lr.ace-4A gene between the resistant parent PI 192051 and the susceptible variety, comparing the amino acid sequence translated from the gene with the published tetraploid and hexaploid wheat genomic data, and analyzing the resequencing or exon sequencing data of about 1500 tetraploid / hexaploid wheat (https: / / triticeaetoolbox.org / wheat / ; http: / / wheatgenomics.plantpath.ksu.edu / 1000EC / ; https: / / doi.org / 10.1038 / s41588-019-0393-z), it was found that the 533rd and 662nd amino acids are specific amino acids of Lr.ace-4A protein. Part of the comparison results are shown in Figure 3 Figure 3 The partial sequence of Lr.ace-4A protein shown in SEQ ID NO: 4, Figure 3 The partial sequence of CS shown in SEQ ID NO: 5, Figure 3 The partial sequence of DAS5-004027 shown in SEQ ID NO: 6, Figure 3 The partial sequence of DAS5-004579 shown in SEQ ID NO: 7, Figure 3 The partial sequence of DAS5-002342 shown in SEQ ID NO: 8, Figure 3 The partial sequence of Waskada shown in SEQ ID NO: 9, Figure 3 The partial sequence of PI 74108 shown in SEQ ID NO: 10, Figure 3 The partial sequence of PI 97616 shown in SEQ ID NO: 11.​

[0069] A functional molecular marker of Lr.ace-4A gene was designed at the position of the 533th and 662th amino acid by Primer5 software. The forward primer is shown as SEQ ID NO: 1, the reverse primer is shown as SEQ ID NO: 2, and this functional molecular marker is named as Lr30MAS-47F / R, and the sequence of the amplification product is shown as SEQ ID NO: 3.

[0070] SEQ ID NO: 1: CCTTATTGCCTAGATCCTTTG.

[0071] SEQ ID NO: 2: GCTTAAGGGAATCTGGACG.

[0072] SEQ ID NO: 3:

[0073]

[0074] SEQ ID NO: 4:

[0075]

[0076]

[0077] SEQ ID NO: 5:

[0078]

[0079] SEQ ID NO: 6:

[0080]

[0081] SEQ ID NO: 7:

[0082]

[0083] SEQ ID NO: 8:

[0084]

[0085] SEQ ID NO: 9:

[0086]

[0087] SEQ ID NO: 10:

[0088]

[0089] SEQ ID NO: 11:

[0090]

[0091] Application of Lr.ace-4A functional molecular marker

[0092] (1) Genotype and phenotype of 144 single plants of F2 segregation population of resistant parent PI 192051, susceptible parent Rusty and their hybrid were identified by using the functional molecular marker.

[0093] Specifically, the genomic DNA of the above wheat samples was extracted by CTAB method and PCR amplification was carried out according to the designed molecular marker Lr30MAS-47F / R, and the specific reaction system was as follows:

[0094]

[0095] ddH2O was added to 25 μL.

[0096] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 5 min, 4℃ preservation.

[0097] The amplification 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 colored with EB, and the photograph was taken by using BIO-RAD imager. Part of the results are shown in Figure 4 As shown in the table, F2 single plants 3, 4, 5, 6, 7, 13, 14, 15, 16 and 17 amplified the same band as PI 192051 (lane 1), indicating that they contained the gene Lr.ace-4A; while single plants 8, 9, 10, 11 and 12 had no PCR amplification product, which was the same as Rusty (lane 2), indicating that they did not contain the gene Lr.ace-4A.

[0098] The resistance of the parents and the above 144 F2 single plants to leaf rust was identified by using leaf rust race, and the results showed that the phenotype was completely consistent with the genotype.

[0099] (2) Genotype of 81 tetraploid wheat (T. dicoccon and T. durum) and 139 hexaploid wheat (Triticum aestivum) was identified by using the molecular marker Lr30MAS-47F / R.

[0100] Specifically, the genomic DNA of the above wheat samples was extracted by CTAB method and PCR amplification was carried out according to the designed molecular marker Lr30MAS-47F / R, and the specific reaction system was as follows:

[0101]

[0102] Add ddH2O to 25 μL.

[0103] PCR amplification reaction program: 94℃ pre-denaturation 3 min; 94℃ denaturation 30 s, 54℃ annealing 30 s, 72℃ extension 30 s, 30 cycles; 72℃ extension 5 min, 4℃ preservation. The amplification product is analyzed by 1.5% agarose gel electrophoresis, the electrophoresis buffer is 1×TAE, and the constant voltage of the electrophoresis instrument is 100 V. The gel is colored with EB, and a photograph is taken using a BIO-RAD imager.

[0104] The statistical results are shown in Table 1 and Table 2. The results show that one tetraploid wheat PI 192051 and two hexaploid wheat RL 6049 and PI 619381 can amplify a 916 bp target band. We perform full-length gene PCR amplification on RL 6049 and PI 619381, and the sequencing results prove that the sequence is completely identical to PI 192051, indicating that the three materials all contain the gene Lr.ace-4A. Other test materials do not amplify the target band, indicating that the measured materials do not contain the Lr.ace-4A gene (as shown in Table 1). Figure 5 An electrophoresis gel map showing the amplification of the molecular marker Lr30MAS-47F / R in part of tetraploid and hexaploid wheat varieties is shown, lanes 2-4 are PI 192051, RL 6049 and PI 619381 respectively, lanes 5-18 are hexaploid wheat Chinese Spring, Fielder, Kenong 9204, Dwarf 58, Ningchun 4, GSTR 425, PI 610750, Kern, Avocet-S, Bainong 207 and tetraploid wheat PI 101971, PI 154582, PI 164578 and PI 168673. Only PI 192051, RL 6049 and PI 619381 amplify a 916 bp target band, and other materials do not amplify a band, indicating that the molecular marker Lr30MAS-47F / R provided by the application can be used to efficiently and accurately identify whether the wheat material contains the leaf rust resistance gene Lr.ace-4A.

[0105] Table 1

[0106]

[0107] Table 2

[0108]

[0109] From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects: the present application provides a functional molecular marker for detecting wheat leaf rust resistance gene Lr.ace-4A, which comprises an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2. Using the upstream primer and the downstream primer, the genome 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 leaf rust resistance gene Lr.ace-4A, which has low detection difficulty, simple operation method, low cost, and good product specificity. The problem that it is difficult to accurately and quickly identify the leaf rust resistance gene Lr.ace-4A in wheat breeding is solved, and the present application has a wide application prospect and great economic value.

[0110] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting wheat leaf rust resistance genes Lr.ace-4A Functional molecular markers, characterized by, The functional molecular markers are: the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO:

2.

2. A method for detecting wheat leaf rust resistance genes Lr.ace-4A The reagent kit is characterized by, The kit includes the functional molecular marker of claim 1, and the kit further includes any one or more of the following: Reagents used for PCR amplification, reagents used for DNA extraction, or reagents used for PCR product analysis.

3. A wheat leaf rust resistance gene Lr.ace-4A The detection method is characterized by, The detection method includes: a) Using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using the functional molecular marker described in claim 1 or the kit described in claim 2 to obtain the PCR product system; b) Detect the PCR product system to determine whether it contains PCR amplification products; If the PCR product system contains a PCR amplification product of 916 bp in length, then the wheat to be tested contains the wheat leaf rust resistance gene. Lr.ace-4A .

4. The detection method according to claim 3, characterized in that, b) includes detecting the PCR products using gel electrophoresis.

5. The detection method according to claim 3, characterized in that, The PCR amplification program in a) is as follows: 94℃ for 3 min; 94℃ for 30 s, 54℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; 72℃ for 5 min.

6. The method for detecting wheat leaf rust resistance genes as described in claim 1 Lr.ace-4A Functional molecular markers, or the detection of wheat leaf rust resistance genes as described in claim 2 Lr.ace-4A The kit, or the wheat leaf rust resistance gene as described in any one of claims 3 to 5 Lr.ace-4A Application of detection methods in wheat breeding for resistance to leaf rust.

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