Multiplex PCR (Polymerase Chain Reaction) detection primer for multiple disease-resistant genes of wheat and application thereof

Abstract

The invention relates to the technical field of crop molecular detection, in particular to a multiplex PCR (polymerase chain reaction) detection primer for multiple disease-resistant genes of wheat and application of the multiplex PCR detection primer. The invention discloses a multiplex PCR primer group for detecting or assisting in detecting to-be-detected wheat disease resistance related genes. The primer group is used for simultaneously distinguishing whether to-be-detected wheat contains stripe rust, leaf rust, powdery mildew and gibberellic disease related resistance genes or not. The invention also discloses a method for simultaneously detecting whether wheat contains stripe rust resistance related genes, leaf rust resistance related genes, powdery mildew resistance related genes and gibberellic disease resistance related genes or not. The method comprises a method for carrying out multiplex PCR (Polymerase Chain Reaction) and sequencing genotype analysis by utilizing the screened primer group. 27 single nucleotide polymorphic sites and multiple PCR primer groups related to the disease resistance of wheat stripe rust, brown leaf rust, gibberellic disease and powdery mildew are screened for the first time, the corresponding detection method is developed, 20 wheat disease resistance related functional genes are involved in single PCR detection, and the method has the advantages of being efficient and accurate in detection.

Description

Technical Field

[0001] The present invention relates to the technical field of crop molecular detection, and in particular to a multiplex PCR detection primer for multiple disease-resistant genes in wheat and an application thereof. Background Art

[0002] Wheat is an important food crop in the world. The main diseases in my country's wheat production include stripe rust, leaf rust, powdery mildew, and ergot. Although a variety of chemical agents can be used to effectively prevent and control wheat stripe rust, leaf rust and other diseases in production, the most economical, effective and safe means of preventing and controlling them is to select, identify and utilize new resistance sources to breed disease-resistant varieties.

[0003] Current molecular-assisted breeding and genetic engineering technologies have made it possible to aggregate multiple disease-resistant genes into the same variety, but how to quickly and accurately detect whether a certain wheat variety has all the target disease-resistant genes is a new requirement in current wheat breeding research.

[0004] Although multiplex PCR technology can amplify multiple target loci in a single system, the wheat genome is large and complex, making it difficult to develop highly polymorphic, co-dominant molecular markers that are tightly linked to the target trait. Furthermore, when multiplex PCR is used to detect multiple target genes, multiple primer pairs in the same PCR system may fail to amplify the target bands due to sequence interference or reduce the accuracy of the test results due to low primer specificity. The more target traits there are, the more difficult it is to detect multiple traits in aggregate using multiplex PCR. This has led to existing multiplex PCR technology focusing on detecting a single disease-resistance gene or a limited number of multiple disease-resistance genes in wheat disease-resistance gene detection. Therefore, finding molecular markers tightly linked to target disease-resistance genes, developing highly specific wheat multiplex PCR primers, establishing an efficient wheat multiplex PCR system, and developing a method for rapid and accurate detection of multiple disease-resistance genes in wheat are of great significance to the field of molecular-assisted wheat breeding. Summary of the Invention

[0005] To address the current difficulty in rapidly and effectively detecting multiple disease-resistance genes in wheat, the first objective of the present invention is to identify molecular markers closely linked to target disease-resistance genes and screen multiplex PCR primer sets that can specifically and efficiently amplify the target genes, thereby enabling simultaneous detection of multiple disease-resistance genes in wheat. A second objective of the present invention is to provide a method for simultaneously detecting genes associated with resistance to stripe rust, leaf rust, powdery mildew, and scab, enabling rapid and accurate simultaneous detection of multiple disease-resistance traits in wheat varieties.

[0006] In order to achieve the first object of the present invention, the present invention provides a multiplex PCR primer set, which adopts the following technical solution:

[0007] A multiplex PCR primer set for detecting or assisting in detecting disease resistance-related genes in the wheat to be tested, wherein the primer set is used to simultaneously distinguish whether the wheat to be tested contains resistance genes related to stripe rust, leaf rust, powdery mildew, and ergot. The sequences of the primer set are shown in SEQ ID No: 1 to SEQ ID No: 54.

[0008] Implementations may include any or all of the following features.

[0009] In one embodiment, all primer sets are placed in the same PCR reaction system for amplification.

[0010] In one embodiment, the stripe rust resistance-related genes include: Yr5, YrSP, Yr7, Yr15, YrAS2388R / Yr28, Yr27, Yru1, Yr18 / Lr34 / Pm38, Lr67 / Yr46 / Pm46; the leaf rust resistance-related genes include: Lr1, Lr10, Lr13, Lr21, Lr22a, Lr42; the powdery mildew resistance-related genes include: Pm1a, Pm3, Pm5e, Pm21; the ergot resistance-related genes include: Fnb1.

[0011] In one embodiment, the present invention further provides a reagent for detecting or assisting in detecting a disease resistance-related gene in wheat to be detected, wherein the reagent comprises any one of the primer sets described above.

[0012] In order to achieve the second purpose of the present invention, the present invention provides a method for simultaneously detecting whether wheat contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and ergot resistance-related genes, including the following technical solutions.

[0013] The method for simultaneously detecting whether wheat contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and scab resistance-related genes comprises the following steps:

[0014] Find genes and molecular marker loci that are closely linked to the target disease resistance trait, and design and screen multiplex PCR primer sets that specifically amplify the target disease resistance gene;

[0015] Using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using any of the primer sets described above to obtain a PCR product;

[0016] Determine whether the wheat to be tested contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and scab resistance-related genes based on the PCR products.

[0017] In one embodiment, the PCR amplification is a multiplex PCR amplification.

[0018] In one embodiment, the method for simultaneously detecting whether wheat contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and scab resistance-related genes comprises the following steps:

[0019] S1, performing a first-round PCR amplification using any of the primer sets described above to obtain a first-round PCR amplification product;

[0020] S2, after purifying the first-round PCR amplification product, adding universal primers containing sequencing adapters to perform a second-round PCR amplification to obtain the second-round PCR amplification product;

[0021] S3, constructing a DNA library and sequencing the products of the second round of PCR amplification;

[0022] S4, performing genotype analysis on the sequencing results to determine whether the wheat to be tested contains disease resistance-related genes.

[0023] In one embodiment, performing genotyping analysis on the sequencing results includes the following steps:

[0024] S41, using data quality control software to optimize the adapter sequences and low-quality bases in the sequencing data to obtain optimized sequences;

[0025] S42, the reference sequence is a short sequence comprising the target sites and flanking sequences of the stripe rust resistance-related gene, leaf rust resistance-related gene, powdery mildew resistance-related gene, and scab resistance-related gene;

[0026] S43, analyzing the genotype of the target site using bioinformatics analysis software.

[0027] In one embodiment, the sequencing adapter is an Illumina sequencing adapter, and the universal primer comprises an i5 / i7 adapter sequence, a sequencing primer binding site, and index sequences of different samples.

[0028] In one embodiment, the second-round PCR amplification products are mixed into a product pool, and the second-round PCR amplification products in the product pool are purified before constructing a DNA library and sequencing.

[0029] In summary, the present invention provides a multiplex PCR detection primer for multiple disease resistance genes in wheat and its application, which has the following beneficial effects:

[0030] First, the present invention first screened out a group of genes closely linked to stripe rust resistance, leaf rust resistance, powdery mildew resistance, and ergot resistance from wheat genomic DNA, and screened out multiple single nucleotide polymorphism sites (SNPs) linked to these genes as detection markers. Based on the single nucleotide polymorphism sites, candidate primers were designed and screened. After experimental verification, a multiplex PCR primer set was finally obtained that can efficiently and specifically amplify 27 single nucleotide polymorphism sites of 4 important disease resistance traits and 20 functional genes of wheat, which has important application value in the simultaneous detection of multiple traits in wheat.

[0031] Second, through the multiplex PCR primer set and detection method of the present invention, the simultaneous detection and analysis of 20 disease-resistance-related genes in the wheat genome can be completed at one time, realizing multiple detection of samples by high-throughput sequencing. It has the advantages of being fast and accurate, and is suitable for genotype detection of large-scale populations, providing an effective method reference for the simultaneous detection of multiple traits in wheat molecular marker-assisted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the technical roadmap of the method of the present invention.

[0033] Figure 2 Graph showing the agarose gel electrophoresis results of 42 pairs of candidate primers in Example 1. DETAILED DESCRIPTION

[0034] The following will further illustrate the embodiments of the present invention with reference to the accompanying drawings and specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. It should be noted that many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present invention is not limited to the specific embodiments disclosed below. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained through commercial channels. If there are no special instructions, the test methods used are all conventional methods.

[0035] In current multiplex PCR systems for detecting multiple disease resistance traits in wheat, the more target traits to be detected, the more interference there is between the multiple sequences, making it more difficult to screen multiple molecular marker sites and suitable specific primers. This results in a limited number of target disease resistance traits that can be detected in a single multiplex PCR system. This invention, for the first time, has developed a multiplex PCR primer set and detection method capable of simultaneously detecting 27 SNP molecular marker sites associated with wheat resistance to stripe rust, leaf rust, powdery mildew, and scab. A single PCR assay involves a total of 20 wheat disease resistance functional genes, demonstrating efficient and accurate detection, making it suitable for genotyping large populations and possessing significant application value in the simultaneous detection of multiple traits in wheat.

[0036] Technical roadmap reference for the method of the present invention Figure 1 , the following will be combined Figure 1 The method of the present invention is described in detail with reference to the accompanying drawings and specific embodiments.

[0037] Example 1 Design and verification of multiplex PCR primer sets

[0038] 1. Collect SNP / InDels sites in the wheat genome

[0039] We searched the NCBI database and related literature for cloned genes related to wheat disease resistance to collect key SNP / InDel loci for genes associated with resistance to stripe rust, leaf rust, powdery mildew, and scab. Basic information on the cloned genes is shown in Table 1.

[0040] Table 1 Basic information of cloned genes and loci

[0041]

[0042]

[0043] 2. Multiplex PCR primer design and initial screening

[0044] For all collected single nucleotide polymorphism (SNP) sites, 2-3 pairs of candidate primers were designed in the flanking regions of the SNP sites using Primer3 software. The amplification specificity was evaluated by e-PCR software (v2.3.12), and the primer dimers were evaluated through the website https: / / www.detaibio.com. All candidate primers were preliminarily screened.

[0045] 3. Primer verification and screening

[0046] Twenty-seven single-gene lines or varieties containing known related genes, including Parula, Chinese Spring, and Yr7, were selected as test materials to verify the feasibility of PCR amplification using the designed primers. These varieties or test materials were sourced from the Huazhong Agricultural University Germplasm Resource Bank (see Table 2 for a list). The designed primers were synthesized by a biotechnology company.

[0047] Table 2 Sample material list

[0048]

[0049] The specific steps for primer verification and screening are as follows:

[0050] The genomic DNA of the above 27 samples was extracted and diluted to 50-100 ng / μL, and used as DNA template for PCR amplification.

[0051] Specifically, the PCR system is as follows: DNA template 50-100ng; 10μL 2ⅹTaq Master Mix (Novozyme); upstream primer (10μM) 1μL; downstream primer (10μM) 1μL; ddH2O to 20 (10μM) 1μL. According to the PCR reaction procedure: 95℃ pre-denaturation for 5min, (95℃ denaturation for 30s; set the temperature according to the primer Tm value, annealing for 30s; 72℃ extension for 1min) 30-35 cycles; 72℃ extension for 5min, 25℃, 1min, PCR amplification is performed to obtain PCR amplification products. Each pair of primers randomly selects 4 of the PCR amplification products of 27 test materials for electrophoresis to observe the PCR amplification bands. In this embodiment, agarose gel electrophoresis is used for electrophoresis, and the electrophoresis results are as follows: Figure 2 As shown, lane 13 is DNA Maker.

[0052] Figure 1 For the target disease-resistance gene, all primers with amplified bands can be used as candidate multiplex PCR detection primers.

[0053] Example 2 Multiplex PCR detection of sample genotypes

[0054] 1. Genomic DNA sample quality testing

[0055] To ensure the quality of subsequent library construction, the genomic DNA samples were tested using the following method. Library construction was performed after the genomic DNA samples passed the test. The specific testing steps are as follows:

[0056] (1) Agarose gel electrophoresis showed that the main band of genomic DNA was intact and clear, without degradation and RNA contamination.

[0057] (2) The OD260 / 280 ratio detected by Nanodrop was between 1.8 and 2.2, and there was no protein or visible impurities contamination.

[0058] (3) The detection concentration of Qubit 3.0 is greater than 20ng / μL, and the total amount is greater than 2ug.

[0059] 2. Library construction and sequencing

[0060] According to the experimental purpose and site information, the primers verified and screened in Example 1 were mixed to form a multiplex PCR primer set, wherein the primers were mixed at equal concentrations and in equal moles.

[0061] Using the genomic DNA of 27 wheat materials, including Parula and Chinese Spring, that passed the above quality inspection as templates, multiplex PCR was performed using the KAPA KK5802 kit to amplify the target sites. The specific steps are as follows:

[0062] S1, first round of PCR amplification.

[0063] PCR system: DNA 50ng; 4×KAPA2G Fast Multiplex Mix 5μL; Pool-primer 2μL; ddH2O to 20μL.

[0064] PCR reaction program: pre-denaturation at 95°C for 5 min, (denaturation at 95°C for 15 s; annealing at 60°C for 30 s; extension at 72°C for 2 min) 25-30 cycles; extension at 72°C for 10 min, and 25°C for 1 min.

[0065] Among them, Pool-primer is a mixed primer of the multiplex PCR primer set, and the rest are reagents in the KAPA KK5802 kit.

[0066] S2, second round of PCR amplification.

[0067] PCR system: 1 μL of first-round PCR product; 5 μL of 4×KAPA2G Fast Multiplex Mix; 5 μL of Nova-index; 0.5 μL each of the left and right primers of the adapter; ddH2O to 20 μL.

[0068] PCR reaction program: pre-denaturation at 95°C for 2 min, (denaturation at 95°C for 15 s; annealing at 60°C for 30 s; extension at 72°C for 2 min) 25-30 cycles; extension at 72°C for 10 min, and 25°C for 1 min.

[0069] Among them, the left and right primers of the adapter are universal primers for sequencing adapters, the sequencing adapter is an Illumina sequencing adapter, and the universal primer contains an i5 / i7 adapter sequence, a sequencing primer binding site, and index sequences of different samples.

[0070] S3, DNA library construction and sequencing.

[0071] The products of the multiplex PCR reactions of all species were mixed (Pooling), purified with AMPure XP Beads, and the library was constructed using conventional library construction methods in the art.

[0072] In some embodiments of the present invention, a second-generation sequencing library is constructed and sequenced on a second-generation sequencing platform (eg, Roche / 454FLX, Illumina / Solexa, GenomeAnalyzer, Applied Biosystems SOLID system).

[0073] After the library is constructed, its quality is tested using conventional methods in the field, such as qPCR to measure the library concentration. Only when the test results meet the requirements can the next-generation sequencing be performed.

[0074] S4, Genotype data analysis.

[0075] Fastp software was used to remove the adapter sequences contained in the reads series and the low-quality bases in the reads sequence to obtain clean reads sequences.

[0076] The clean reads were aligned to the reference sequence using the MEM algorithm of the BWA software. The reference sequence used here is a short sequence containing the target site and flanking sequences.

[0077] Use the python script to analyze the genotype of the target site and obtain the genotype analysis results.

[0078] The read sequence is the entire sequence data obtained from sequencing. The process from read sequence to clean read sequence is the sequencing data quality control process, which can be performed using fastp software or other data quality control software that can perform the same function. Alignment of clean read sequences with reference sequences and genotyping of target loci can also be performed using other bioinformatics data analysis software that can perform the same function.

[0079] All primers screened in Example 1 were subjected to various combinations and multiplex PCR, sequencing, and genotyping analysis according to the methods of this example. Ultimately, 27 specific primer pairs were selected. These 27 specific primer pairs, as a multiplex PCR primer set, showed the best amplification efficiency for 27 SNPs in 20 cloned genes associated with wheat head blight, leaf rust, powdery mildew, and stripe rust. Basic information for the 27 selected specific primer pairs is shown in Table 3.

[0080] Table 3 Basic information of 27 multiplex PCR primer sets

[0081]

[0082]

[0083]

[0084] Note: When a gene involves two or more pairs of primers, the primer names in the table represent the order of the SNP sites of the gene in Table 1. For example, gene Lr10 involves two pairs of primers, Lr10-1-F / Lr10-1-R and Lr10-3-F / Lr10-3-R. Lr10-1-F / Lr10-1-R corresponds to the 151C>T site that ranks first in the Lr10 gene in Table 1, and Lr10-3-F / Lr10-3-R2 corresponds to the 3461G>A site that ranks third in the Lr10 gene in Table 1.

[0085] 3. Test result data

[0086] The 27 screened primer pairs were used as the multiplex PCR primer set, and the 27 materials in Table 2 were selected as the test materials. The test materials were tested according to the above-mentioned method 1-2 of this example. The obtained genotype analysis results are shown in Table 4.

[0087]

[0088]

[0089] As can be seen from the results in Table 4, each primer pair was able to obtain a detection result (0 / 0 or 0 / 1), demonstrating that the multiplex PCR system and detection method of the present invention can simultaneously capture 27 SNP loci associated with wheat disease resistance traits, with minimal interference between multiple loci or primers. When the multiplex PCR system and detection method of the present invention are used to test a wheat variety, the presence of the target gene can be determined by interpreting the genotype results.

[0090] In addition, the detection method of the present invention can simultaneously identify 27 single nucleotide polymorphism sites of 20 genes related to wheat stripe rust, leaf rust, ergot, and powdery mildew resistance through single-tube operation, providing an efficient technical solution for molecular marker-assisted selection (MAS) breeding, reducing detection costs, and has important application value in wheat breeding.

[0091] Finally, it should be noted that the above-described embodiments are only specific implementation methods of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the above-described embodiments, those skilled in the art should understand that any person skilled in the art can modify or easily conceive of changes to the technical solutions described in the above-described embodiments within the technical scope disclosed by the present invention, or replace some of the technical features therein with equivalents. Such modifications, changes, or replacements do not deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A multiplex PCR primer set for detecting or assisting in detecting a disease resistance-related gene in wheat, characterized in that: The primer set is used to simultaneously distinguish whether the wheat to be tested contains resistance genes related to stripe rust, leaf rust, powdery mildew, and head blight. The sequences of the primer set are shown in SEQ ID No: 1 to SEQ ID No:

54.

2. The multiplex PCR primer set for detecting or assisting in detecting a disease resistance-related gene in wheat according to claim 1, wherein: All primer sets were placed in the same PCR reaction system for amplification.

3. The multiplex PCR primer set for detecting or assisting in detecting genes related to wheat traits to be tested according to claim 1, wherein: The stripe rust resistance-related genes include: Yr5, YrSP, Yr7, Yr15, YrAS2388R / Yr28, Yr27, Yru1, Yr18 / Lr34 / Pm38, Lr67 / Yr46 / Pm46; the leaf rust resistance-related genes include: Lr1, Lr10, Lr13, Lr21, Lr22a, Lr42; the powdery mildew resistance-related genes include: Pm1a, Pm3, Pm5e, Pm21; the ergot resistance-related genes include: Fnb1.

4. A reagent for detecting or assisting in detecting a disease resistance-related gene in wheat, characterized in that: The reagent comprises the primer set according to any one of claims 1 to 3.

5. A method for simultaneously detecting whether wheat contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and scab resistance-related genes, characterized in that: The following steps are involved: Find genes and molecular marker loci that are closely linked to the target disease resistance trait, and design and screen multiplex PCR primer sets that specifically amplify the target disease resistance gene; Using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using the primer set according to any one of claims 1 to 3 to obtain a PCR product; Determine whether the wheat to be tested contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and scab resistance-related genes based on the PCR products.

6. The method for simultaneously detecting whether wheat contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and scab resistance-related genes as claimed in claim 5, characterized in that: The PCR amplification is multiplex PCR amplification.

7. The method for simultaneously detecting whether wheat contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and scab resistance-related genes according to claim 6, characterized in that: The following steps are involved: S1, performing a first-round PCR amplification using the primer set according to any one of claims 1 to 3 to obtain a first-round PCR amplification product; S2, after purifying the first-round PCR amplification product, adding universal primers containing sequencing adapters to perform a second-round PCR amplification to obtain the second-round PCR amplification product; S3, constructing a DNA library and sequencing the products of the second round of PCR amplification; S4, performing genotype analysis on the sequencing results to determine whether the wheat to be tested contains disease resistance-related genes.

8. The method for simultaneously detecting whether wheat contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and scab resistance-related genes according to claim 7, characterized in that: The genotype analysis of the sequencing results includes the following steps: S41, using data quality control software to optimize the adapter sequences and low-quality bases in the sequencing data to obtain optimized sequences; S42, aligning the optimized sequence to a reference sequence, wherein the reference sequence is a short sequence comprising the target sites and flanking sequences of the stripe rust resistance-related gene, the leaf rust resistance-related gene, the powdery mildew resistance-related gene, and the scab resistance-related gene; S43, analyzing the genotype of the target site using bioinformatics analysis software.

9. The method for simultaneously detecting whether wheat contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and scab resistance-related genes as claimed in claim 7, characterized in that: The sequencing adapter is an Illumina sequencing adapter, and the universal primer contains an i5 / i7 adapter sequence, a sequencing primer binding site, and index sequences of different samples.

10. The method for simultaneously detecting whether wheat contains stripe rust resistance-related genes, leaf rust resistance-related genes, powdery mildew resistance-related genes, and scab resistance-related genes according to claim 7, characterized in that: The second-round PCR amplification products are mixed into a product pool, and the second-round PCR amplification products in the product pool are purified and then used to construct a DNA library and sequence.

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