Molecular marker closely linked with wheat spikelet number QTL and application thereof

Abstract

The invention discloses a molecular marker closely linked with wheat spikelet number QTL and application of the molecular marker, and belongs to the technical field of molecular biology and crop genetic breeding. The invention discloses the wheat spikelet number QTL QSNS.sicau-D12-3B for the first time, the wheat spikelet number QTL QSNS.sicau-D12-3B is located on a long arm of a wheat 3B chromosome, and the wheat spikelet number can be remarkably increased. The invention further discloses a molecular marker KASP-3B-1 closely linked with the wheat spikelet number QTL, the molecular marker can be used for rapidly screening plants with the wheat spikelet number QTL, then molecular assisted breeding of high-yield wheat is conveniently carried out, the breeding working efficiency is improved, and a theoretical basis and technical support are provided for genetic analysis research of the wheat spikelet number.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and crop genetic breeding, and in particular to a molecular marker tightly linked to a wheat spikelet number QTL and an application thereof. Background Art

[0002] Wheat (Triticum aestivum L.) yield is primarily determined by the number of spikelets per unit area, the number of grains per spike, and 1000-grain weight, with grain number per spike being the key factor. Spikelet number, a crucial component of grain number, is a complex quantitative trait formed by the differentiation of the spike meristem and is regulated by both genetic and environmental factors (such as temperature and nitrogen nutrition). Elucidating the genetic mechanisms of spikelet number and identifying its major genetic loci are crucial for uncovering yield formation mechanisms and exploring high-yield breeding strategies. To date, researchers have identified spikelet number QTLs on all 21 wheat chromosomes using biparental populations, including recombinant inbred lines, backcrosses, and doubled haploids. For example, Zhai Huijie et al. used a recombinant inbred line population to identify a major QTL controlling spikelet number on chromosome 1B, which explained 30.75% of the phenotypic variation. A SNP marker, Kukri_c11327_977, tightly linked to SNS, was identified on chromosome 1A. In addition, several genes associated with spikelet number have been reported, such as the homology-based cloned genes trs1 / WFZP-A, VRN-A3 / FT-A1, Q, TaTB1-4A, PPD-A1, and WAPO1 through map-based cloning. Although numerous QTLs / genes associated with spikelet number have been reported in wheat, the identification of major and stable QTLs for spikelet number across multiple environments remains limited. Therefore, identifying stable major genetic loci controlling spikelet number is crucial for elucidating the genetic basis of wheat yield traits and improving wheat yield.

[0003] Traditional wheat breeding methods are time-consuming, costly, and have low returns. Molecular marker-assisted breeding does not rely on phenotypic selection and is not affected by factors such as the environment and gene interactions. Instead, it directly selects the genotype, thus greatly improving breeding efficiency. Single Nucleotide Polymorphism (SNP) refers to DNA sequence polymorphism caused by changes such as conversion, transversion, insertion, and deletion at a specific nucleotide position in the DNA within the genome. The technology uses known sequence information to compare and search for SNP sites, and then uses the discovered variant sites to design specific primers to perform PCR amplification on genomic DNA or cDNA to obtain specific polymorphic products based on the SNP sites. Finally, the polymorphism of the products is analyzed using electrophoresis technology. The advantages of SNP markers are that they are numerous and widely distributed; they are unevenly distributed in single genes and the entire genome; and the SNP allele frequency is easy to estimate. Competitive Allele Specific PCR (KASP) is a low-cost, high-throughput genotyping technology developed by LGC (Laboratory of the Government Chemist) (http: / / www.lgcgenomics.com). It accurately types single-nucleotide polymorphisms (SNPs) and indels (InDel) by specifically matching primer terminal bases. It has been widely used in molecular marker-assisted selection of food crops such as rice, wheat, and soybeans.

[0004] While previous research has extensively investigated the mapping of QTLs for wheat spikelet number, there are currently few tightly linked molecular markers associated with spikelet number that can be used in marker-assisted selection breeding. Therefore, identifying QTLs or genes for spikelet number and leveraging modern molecular biology techniques to increase spikelet number, and thus grain number per spike, ultimately leading to the breeding of high-yield, high-quality wheat varieties, is of great significance in wheat breeding. Summary of the Invention

[0005] The purpose of the present invention is to provide a molecular marker tightly linked to the wheat spikelet number QTL and its application to solve the problems existing in the above-mentioned prior art. The present invention discloses for the first time the wheat spikelet number QTL QSNS.sicau-D12-3B, which is located on the long arm of wheat chromosome 3B and can significantly increase the number of wheat spikelets. The present invention also discloses a molecular marker KASP-3B-1 tightly linked to the wheat spikelet number QTL. This molecular marker can be used to quickly screen plants with the wheat spikelet number QTL, thereby facilitating molecular-assisted breeding of high-yield wheat, improving breeding efficiency, and providing a theoretical basis and technical support for genetic analysis research on wheat spikelet number.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a KASP-3B-1 molecular marker tightly linked to the wheat spikelet number QTL QSNS.sicau-D12-3B, wherein the KASP-3B-1 molecular marker and the wheat spikelet number QTL QSNS.sicau-D12-3B are co-localized on the long arm of wheat chromosome 3B;

[0008] The molecular marker is a nucleotide sequence as shown in SEQ ID NO.34; the nucleotide sequence shown in SEQ ID NO.34 has a C / T mutation at the 20th base; the number of wheat spikelets with the mutation base site being T is significantly greater than the number of wheat spikelets with the mutation base site being C.

[0009] Furthermore, the physical position of the wheat spikelet number QTL QSNS.sicau-D12-3B in the Chinese Spring RefSeqv2.1 genome version is 619673594-625487641bp.

[0010] The present invention also provides applications of the KASP-3B-1 molecular marker, including any of the following applications A1-A5:

[0011] A1. Detection of wheat spikelet number QTL QSNS.sicau-D12-3B;

[0012] A2. Early identification or selection of wheat varieties with a large number of spikelets;

[0013] A3. Wheat molecular genetics and breeding;

[0014] A4. Improvement of wheat germplasm resources;

[0015] A5. Genetic analysis and fine mapping of genes related to wheat spikelet number.

[0016] The present invention also provides a KASP primer set targeting the above-mentioned KASP-3B-1 molecular marker, wherein the KASP primer set includes primers shown as SEQ ID NOs. 25-27.

[0017] Furthermore, the two primers shown in SEQ ID NO. 25 and SEQ ID NO. 26 are modified with different fluorescent groups at their 5' ends or modified with different fluorescent groups at their 3' ends.

[0018] The present invention also provides the use of the KASP primer set in any one of the following items B1-B4:

[0019] B1. Detection of wheat spikelet number QTL QSNS.sicau-D12-3B;

[0020] B2. Early identification or selection of wheat varieties with a large number of spikelets;

[0021] B3. Preparation of reagents, kits or chips for detecting wheat spikelet number QTL QSNS.sicau-D12-3B;

[0022] B4. Prepare reagents, kits or chips for early identification or screening of wheat varieties with a large number of spikelets.

[0023] The present invention also provides a kit for detecting wheat spikelet number QTL QSNS.sicau-D12-3B, wherein the kit comprises the KASP primer set.

[0024] The present invention also provides a method for detecting wheat spikelet number QTL QSNS.sicau-D12-3B, comprising the following steps:

[0025] Using the genomic DNA of the plant to be tested as a template, perform fluorescence quantitative PCR amplification using the above-mentioned KASP primer set or the above-mentioned kit, and make judgments based on the fluorescence reading results;

[0026] The plants in which the fluorescent group labeled by SEQ ID NO. 25 was read were identified as plants without the wheat spikelet number QTL QSNS.sicau-D12-3B, and their genotype was CC. The plants in which the fluorescent group labeled by SEQ ID NO. 26 was read were identified as plants containing the wheat spikelet number QTL QSNS.sicau-D12-3B, and their genotype was TT.

[0027] The spikelet number of plants with genotype TT was significantly higher than that of plants with genotype CC.

[0028] Optionally, the reaction system of the fluorescent quantitative PCR is: 5 μL Master Mix, 5 ng template DNA, 1.4 μL mixed primers, and ddH2O added to a total volume of 10 μL;

[0029] The mixed primer is obtained by mixing 10 ng / μL of the primer shown in SEQ ID NO.25, 10 ng / μL of the primer shown in SEQ ID NO.26, 10 ng / μL of the primer shown in SEQ ID NO.27 and ddH2O in a volume ratio of 6:6:12:23.

[0030] Optionally, the fluorescent quantitative PCR reaction program is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 63°C for 60 s, for a total of 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 55°C for 1 min, for a total of 26 cycles.

[0031] The present invention discloses the following technical effects:

[0032] The present invention discloses for the first time the spikelet number QTL QSNS.sicau-D12-3B from wheat '03D501', which is located on the long arm of wheat chromosome 3B and has the effect of significantly increasing the number of wheat spikelets. This QTL has a high utilization value in wheat yield (regulating the number of spikelets) breeding. The present invention also discloses a molecular marker KASP-3B-1 located on wheat chromosome 3B and linked to the number of wheat spikelets. It is a co-dominant marker. This molecular marker is a flanking marker of the spikelet number QTL QSNS.sicau-D12-3B on the long arm of wheat chromosome 3B, has a high degree of linkage, and exhibits co-segregation marker characteristics. This molecular marker can be used to detect the spikelet number QTL on wheat chromosome 3B, quickly screen plants with this site, and then facilitate molecular-assisted breeding of high-yield wheat, improve breeding efficiency, and provide a theoretical basis and technical support for genetic analysis research on wheat spikelet number. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 This is the location map of the wheat spikelet number QTL QSNS.sicau-D12-3B on wheat chromosome 3B;

[0035] Figure 2 The fluorescence readings of the recombinant inbred line population of '03D501' × '2011' of the present invention detected using the molecular marker KASP-3B-1; HEX (blue, '03D501') fluorescence indicates plants with a large number of spikelets, FAM (orange, '2011') fluorescence indicates plants with a small number of spikelets, and black fluorescence indicates a blank control.

[0036] Figure 3 The fluorescence readings of the F5 strain of '03D501' × 'Liangmai No. 4' of the present invention were detected using the molecular marker KASP-3B-1; HEX (blue, '03D501') fluorescence indicates plants with a large number of spikelets, FAM (orange, '2011') fluorescence indicates plants with a small number of spikelets, and black fluorescence indicates the blank control.

[0037] Figure 4The spikelet number phenotypic test results of the F5 strain of '03D501'×'Liangmai No. 4' of the present invention are shown; TT refers to a plant population with the genotype TT, and CC refers to a plant population with the genotype CC. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] The wheat varieties used in the following examples were all from the germplasm resource bank of the Wheat Research Institute of Sichuan Agricultural University and are available to the public here.

[0044] The technical ideas of the present invention are as follows:

[0045] The present invention uses the high-spikelet wheat line 03D501 as the female parent and the wheat line 2011 with fewer spikelets as the male parent to produce F1 hybrids. Individual F1 plants are self-pollinated to obtain F2 hybrids. The F2 hybrids are propagated using the single-seed method up to the F7 generation, resulting in a recombinant inbred line containing 163 individual plants, constituting a genetic mapping population (abbreviated as D12). The spikelet number of the recombinant inbred line population was investigated and identified. DNA was extracted from the parents 03D501 and 2011, as well as from the recombinant inbred line population. Based on phenotypic data from the D12 population from 2021 to 2022, a pool of extreme strains was constructed by screening and analysis was performed using a wheat 60K SNP array. The specific screening process is as follows: (1) Sort the spikelet number phenotypic values ​​of each strain in the three environmental points of 2021-2022 respectively, screen out the strains in the top 50 and bottom 50 in each environmental point, and count the strain numbers of at least three environmental points in the top 50 or bottom 50; (2) Combine the spikelet number phenotypic data of the three environmental points, calculate the average value and sort, and screen out the top 50 and bottom 50 strains. Finally, take the intersection of the above two screening conditions to obtain 25 extreme few spikelet strains and 26 extreme many spikelet strains. Subsequently, the two F7 generation extreme mixed pools selected above were subjected to 60KSNP chip genotyping at Chengdu Tiancheng Future Technology Co., Ltd.

[0046] A genetic map was constructed using JoinMap 4.0 based on 60K SNP chip data. Combined with population spikelet number phenotypic data, the Inclusive Composite Interval Mapping-ADD (ICIM-ADD) method in QTL IciMapping 4.1 was used. With a threshold LOD ≥ 2.5, the BLUP (best linear unbiased prediction) values ​​for spikelet number at seven ecological sites from 2021 to 2024 were used to detect QTLs for spikelet number. A stably expressed major QTL for wheat spikelet number, QSNS.sicau-D12-3B, was located on the long arm of chromosome 3B. To further densify the map and identify molecular markers tightly linked to the spikelet number QTL, QSNS.sicau-D12-3B, flanking markers were located using 60K SNP array data and SNP markers located within the interval were screened. Furthermore, SNP loci with polymorphism between the parents were identified, leading to the development of highly efficient and tightly linked KASP molecular markers. A total of 11 pairs of KASP primers were designed, ultimately resulting in the identification of marker KASP-3B-1, which is tightly linked to the spikelet number QTL, QSNS.sicau-D12-3B. The present study discovered that the wheat spikelet number QTL, QSNS.sicau-D12-3B, is located on the long arm of wheat chromosome 3B, with a physical position of 619,673,594–625,487,641 bp in the Chinese Spring RefSeq v2.1 genome.

[0047] Example 1

[0048] Determination of wheat spikelet number QTL QSNS.sicau-D12-3B and its molecular marker KASP-3B-1:

[0049] (1) Phenotypic identification of spikelet number in recombinant inbred line population: The spikelet number of the recombinant inbred line population was analyzed and identified at the waxy stage of wheat. The marginal effect was eliminated and five single plants with the same growth were collected. The number of spikelets in the main ear was calculated and the average value was obtained to represent the spikelet number of the line.

[0050] (2) 60K SNP liquid phase chip analysis

[0051] a) DNA extraction

[0052] DNA from the parents '03D501', '2011' and the recombinant inbred line population was extracted using the CTAB method.

[0053] b) Genotype analysis

[0054] The quality of the extracted DNA was tested using an ultra-micro spectrophotometer (Thermo Fisher Scientific, USA). Qualified samples were sent to the company for genotyping analysis. Genotyping of the parental and recombinant inbred line populations was performed using a 16K SNP chip from Chengdu Tiancheng Future Company (http: / / www.molbreeding.com).

[0055] c) Construction of linkage map

[0056] A genetic map was constructed using JoinMap 4.0 based on 60K SNP chip data. Combined with population spikelet number phenotypic data, the Inclusive Composite Interval Mapping-ADD (ICIM-ADD) method in QTL IciMapping 4.1 was used. With a threshold LOD ≥ 2.5, the spikelet number QTL was detected using the best linear unbiased prediction (BLUP) values ​​of seven ecological sites from 2021 to 2024 for spikelet number. The major QTL for wheat spikelet number, QSNS.sicau-D12-3B, was located, and the genetic distance between the position of QSNS.sicau-D12-3B and the molecular markers was calculated.

[0057] d) Comparison of spikelet number loci and acquisition of tightly linked molecular markers

[0058] By compiling the chromosomal location information of loci or genes reported in previous studies, we compared these loci with the physical interval of QSNS.sicau-D12-3B in this example. Previously identified QTLs for spikelet number include QSpn.WJ.3B.1, QSpn.WY.3B.1, QSpn.abrii-3B.1, QSpn.abrii-3B.2, QSpn.abrii-3B.3, and QSns.sau-AM-3B.2. Physical interval alignment revealed no overlap between QSNS.sicau-D12-3B and previously reported spikelet number loci, indicating that QSNS.sicau-D12-3B, identified in this example, is a new key locus for regulating wheat spikelet number. The physical location of this QTL in the Chinese Spring RefSeq v2.1 genome is 619673521–625487833 bp.

[0059] To further confirm its uniqueness through fine mapping and functional validation. Based on the sequence information of flanking markers, 11 pairs of KASP primers were designed using DNAMAN (see Table 1), and genotyping was performed in the parents '03D501' and '2011' to obtain polymorphic sites. Finally, it was determined that the marker KASP-3B-1 (C / T) was closely linked to the spikelet number QTL QSNS.sicau-D12-3B. Among the 11 pairs of flanking marker KASP primers designed, one molecular marker, KASP-3B-1, was finally obtained. It was closely linked to the spikelet number QTL QSNS.sicau-D12-3B. The co-localization map of this molecular marker and QSNS.sicau-D12-3B is shown in the figure below. Figure 1 51 samples were randomly selected from the recombinant inbred line population for genotyping. The typing results are shown in Figure 2 As shown, this molecular marker was well typed in 51 randomly selected inbred lines.

[0060] Table 1 Primer sequences of molecular marker KASP

[0061]

[0062]

[0063]

[0064] The molecular marker KASP-3B-1 is located at the 20th base of the sequence shown in SEQ ID NO. 34 ("R" represents C or T), and there is a C / T mutation.

[0065] SEQ ID NO.34:

[0066] GTCTACTCAGCAGCCTCAAR.

[0067] Example 2

[0068] Application of KASP molecular marker KASP-3B-1 in identifying QTL QSNS.sicau-D12-3B controlling spikelet number:

[0069] (1) '03D501' is a line with many spikelets, and 'Liangmai No. 4' is a variety with a good plant type but few spikelets. A segregating population F5 was constructed with '03D501' as the female parent and 'Liangmai No. 4' as the male parent, and 51 lines were randomly selected from the progeny lines.

[0070] (2) The 51 strains obtained were tested for KASP-3B-1 markers. The specific method was as follows:

[0071] The genomic DNA of 51 strains was extracted and used as a template. PCR amplification was performed using a specific primer pair for the molecular marker KASP-3B-1 (SEQ ID NO. 25-27) and fluorescence reading was performed.

[0072] The PCR amplification system was as follows: 5 μL Master Mix; 1.4 μL of mixed primers (primers SEQ ID NO. 25, SEQ ID NO. 26, and SEQ ID NO. 27 were all prepared at a concentration of 10 ng / μL, with 60 μL, 60 μL, and 120 μL of each, respectively, added with 230 μL of ddH2O and mixed for use as the mixed primers); 5 ng of template DNA; double-distilled water was added to a total volume of 10 μL; and at least three independent blank controls were set up in which double-distilled water was used instead of the DNA template.

[0073] The PCR amplification procedure was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 63°C for 60 s, for a total of 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 55°C for 1 min, for a total of 26 cycles; and fluorescence reading at 37°C for 1 min.

[0074] Fluorescence reading results are as follows Figure 3 As shown in the figure, the genotype of the plant with FAM (orange) fluorescence is consistent with that of 'Liangmai No. 4', which is CC; the genotype of the plant with HEX (blue) fluorescence is consistent with that of '03D501', which is TT. The spikelet number phenotype of these plants was further investigated, and the results were as follows: Figure 4 As shown, the average spikelet number of plants with a CC genotype was 20.00, while the average spikelet number of plants with a TT genotype was 23.71. Plants with a TT genotype had significantly higher spikelet numbers than those with a CC genotype. This result is consistent with expectations, indicating that the spikelet number QTL QSNS.sicau-D12-3B of the present invention does significantly increase spikelet number. The molecular marker KASP-3B-1 of the present invention can be used to identify the wheat spikelet number QTL QSNS.sicau-D12-3B and wheat spikelet number.

[0075] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. The KASP-3B-1 molecular marker tightly linked to the wheat spikelet number QTL QSNS.sicau-D12-3B is characterized by: The KASP-3B-1 molecular marker and the wheat spikelet number QTL QSNS.sicau-D12-3B are co-localized on the long arm of wheat chromosome 3B; The molecular marker is a nucleotide sequence as shown in SEQ ID NO.34; the nucleotide sequence shown in SEQ ID NO.34 has a C / T mutation at the 20th base; the number of wheat spikelets with the mutation base site being T is significantly greater than the number of wheat spikelets with the mutation base site being C.

2. The KASP-3B-1 molecular marker according to claim 1, characterized in that The physical position of the wheat spikelet number QTLQSNS.sicau-D12-3B in the Chinese Spring RefSeqv2.1 genome version is 619673594-625487641bp.

3. A use of the KASP-3B-1 molecular marker according to claim 1 or 2, characterized in that: This includes any of the following applications: A1. Detection of wheat spikelet number QTL QSNS.sicau-D12-3B; A2. Early identification or selection of wheat varieties with a large number of spikelets; A3. Wheat molecular genetics and breeding; A4. Improvement of wheat germplasm resources; A5. Genetic analysis and fine mapping of genes related to wheat spikelet number.

4. A KASP primer set for the KASP-3B-1 molecular marker according to claim 1 or 2, characterized in that: The KASP primer set includes primers shown as SEQ ID NOs. 25-27.

5. The KASP primer set according to claim 4, characterized in that The two primers shown in SEQ ID NO. 25 and SEQ ID NO. 26 are modified with different fluorescent groups at their 5' ends or modified with different fluorescent groups at their 3' ends.

6. Use of the KASP primer set according to claim 4 or 5 in any one of the following items B1-B4: B1. Detection of wheat spikelet number QTL QSNS.sicau-D12-3B; B2. Early identification or selection of wheat varieties with a large number of spikelets; B3. Preparation of reagents, kits or chips for detecting wheat spikelet number QTL QSNS.sicau-D12-3B; B4. Prepare reagents, kits or chips for early identification or screening of wheat varieties with a large number of spikelets.

7. A kit for detecting wheat spikelet number QTL QSNS.sicau-D12-3B, characterized in that: The kit comprises the KASP primer set according to claim 4 or 5.

8. A method for detecting wheat spikelet number QTL QSNS.sicau-D12-3B, characterized in that: The steps include: Using the genomic DNA of the plant to be tested as a template, performing fluorescent quantitative PCR amplification using the KASP primer set of claim 4 or 5 or the kit of claim 7, and making a determination based on the fluorescence reading result; The plants in which the fluorescent group labeled by SEQ ID NO. 25 was read were identified as plants without the wheat spikelet number QTL QSNS.sicau-D12-3B, and their genotype was CC. The plants in which the fluorescent group labeled by SEQ ID NO. 26 was read were identified as plants containing the wheat spikelet number QTL QSNS.sicau-D12-3B, and their genotype was TT. The spikelet number of plants with genotype TT was significantly higher than that of plants with genotype CC.

9. The method according to claim 8, characterized in that The reaction system of the fluorescent quantitative PCR is: 5 μL Master Mix, 5 ng template DNA, 1.4 μL mixed primers, and ddH2O added to a total volume of 10 μL; The mixed primer is obtained by mixing 10 ng / μL of the primer shown in SEQ ID NO.25, 10 ng / μL of the primer shown in SEQ ID NO.26, 10 ng / μL of the primer shown in SEQ ID NO.27 and ddH2O in a volume ratio of 6:6:12:

23.

10. The method according to claim 8, characterized in that The fluorescence quantitative PCR reaction procedure was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 63°C for 60 s, for a total of 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 55°C for 1 min, for a total of 26 cycles.

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