A molecular marker closely linked to wheat spikelet number QTL and its application

Abstract

This invention discloses a molecular marker closely linked to a wheat spikelet number QTL and its application, belonging to the fields of molecular biology and crop genetics and breeding technology. This invention discloses for the first time the wheat spikelet number QTL QSNS.sicau-D12-3B, located on the long arm of wheat chromosome 3B, which can significantly increase the number of wheat spikelets. This invention also discloses the molecular marker KASP-3B-1, which is closely linked to the wheat spikelet number QTL. This molecular marker can be used to rapidly screen plants possessing the wheat spikelet number QTL, thereby facilitating molecular-assisted breeding of high-yielding wheat, improving breeding efficiency, and providing a theoretical basis and technical support for genetic analysis of wheat spikelet number.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and crop genetics and breeding technology, and in particular to a molecular marker closely linked to the QTL for wheat spikelet number and its application. Background Technology

[0002] Wheat (Triticum aestivum L.) yield is mainly composed of spike number per unit area, grain number per spike, and thousand-grain weight, with grain number per spike being the key factor. Spikelet number, as an important component of grain number per spike, is a complex quantitative trait formed by the differentiation of spike meristems and is jointly regulated by genetic and environmental factors (such as temperature and nitrogen nutrition). Elucidating the genetic mechanism of spikelet number and identifying its major genetic loci is of great significance for revealing the yield formation mechanism and exploring high-yield breeding pathways. To date, researchers have identified spikelet number QTLs on all 21 chromosomes of wheat using recombinant inbred lines, backcrosses, and double haploid populations. For example, Zhai Huijie et al. used a recombinant inbred line population to detect a major QTL controlling spikelet number located on chromosome 1B, which explained 30.75% of the phenotypic variation. The SNP marker Kukri_c11327_977, closely linked to the SNS, was found on chromosome 1A. In addition, several genes associated with spikelet number have been reported, such as the homology-based clones trs1 / WFZP-A, VRN-A3 / FT-A1, Q, TaTB1-4A, PPD-A1, and WAPO1, which was cloned using map-based methods. Although many QTLs / genes associated with spikelet number have been reported in wheat, the number of major and stable spikelet number QTLs identified under various environmental conditions remains limited. Therefore, identifying stable major genetic loci controlling spikelet number is of great significance 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, however, does not rely on phenotypic selection and is unaffected by environmental and gene-gene interactions; it directly selects for genotypes, thus significantly improving breeding efficiency. Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by changes such as transitions, transversions, insertions, and deletions at specific nucleotide positions within the genome. The technique involves using known sequence information to locate SNP sites, then designing specific primers to amplify genomic DNA or cDNA using PCR, obtaining specific polymorphic products based on the SNP sites. Finally, electrophoresis is used to analyze the polymorphism of the products. The advantages of SNP markers are their large number, wide distribution, uneven distribution within individual genes and the entire genome, and easily estimable SNP allele frequencies. Kompetitive Allele Specific PCR (KASP) is a novel, low-cost, high-throughput genotyping technology developed by LGC (Laboratory of the Government Chemist) (http: / / www.lgcgenomics.com). It uses specific matching of primer terminal bases to perform precise bicelestem typing of SNPs and InDel sites, and has been widely used in marker-assisted selection of food crops such as rice, wheat, and soybeans.

[0004] While previous studies have extensively investigated QTL mapping for spikelet number in wheat, few tightly linked molecular markers are currently available that are relevant to spikelet number and can be used for practical marker-assisted selection breeding. Therefore, obtaining QTLs or genes related to spikelet number and utilizing modern molecular biology techniques to increase spikelet number, thereby increasing grain number per spike, and ultimately achieving the goal of breeding high-yielding and high-quality new wheat varieties, is of great significance in wheat breeding. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a KASP-3B-1 molecular marker that is closely 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-located 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 mutated base site T is significantly greater than the number of wheat spikelets with the mutated base site C.

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

[0010] This invention also provides applications of the above-mentioned KASP-3B-1 molecular marker, including any one of the following applications: A1-A5

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

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

[0013] A3. Wheat molecular genetics breeding;

[0014] A4. Improvement of wheat germplasm resources;

[0015] A5. Perform genetic analysis and fine mapping of genes related to the number of wheat spikelets.

[0016] The present invention also provides a KASP primer set for the above-mentioned KASP-3B-1 molecular marker, the KASP primer set comprising primers as shown in SEQ ID NO.25-27.

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

[0018] This invention also provides the application of the above-described KASP primer set in any of the following B1-B4:

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

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

[0021] B3. Prepare 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 contains the above-mentioned KASP primer set.

[0024] This invention also provides a method for detecting the 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, the above-mentioned KASP primer set or the above-mentioned kit was used for real-time PCR amplification, and the results were judged based on the fluorescence readings.

[0026] Plants that showed the fluorescent group labeled by SEQ ID NO.25 were identified as plants without the wheat spikelet number QTL QSNS.sicau-D12-3B, with the genotype CC; plants that showed the fluorescent group labeled by SEQ ID NO.26 were identified as plants with the wheat spikelet number QTL QSNS.sicau-D12-3B, with the genotype TT.

[0027] Plants with genotype TT had a significantly higher number of spikelets than plants with genotype CC.

[0028] Optionally, the reaction system for the real-time 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 primers were prepared by mixing 10 ng / μL primers shown in SEQ ID NO.25, 10 ng / μL primers shown in SEQ ID NO.26, and 10 ng / μL primers shown in SEQ ID NO.27 with ddH2O in a volume ratio of 6:6:12:23.

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

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

[0032] This invention discloses for the first time a spikelet number QTL, QSNS.sicau-D12-3B, from wheat '03D501', located on the long arm of wheat chromosome 3B, which significantly increases the number of spikelets in wheat. This QTL has high utilization value in wheat yield (spikelet number regulation) breeding. This invention also discloses a co-dominant molecular marker, KASP-3B-1, located on wheat chromosome 3B and linked to wheat spikelet number. This marker is a flanking marker of the spikelet number QTL QSNS.sicau-D12-3B on the long arm of wheat chromosome 3B, exhibiting high linkage and co-segregating marker characteristics. This molecular marker can be used to detect spikelet number QTLs on wheat chromosome 3B, rapidly screen plants with this locus, thereby facilitating molecular-assisted breeding of high-yielding wheat, improving breeding efficiency, and providing a theoretical basis and technical support for genetic analysis of wheat spikelet number. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a mapping 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 '03D501'×'2011' of this invention were detected using the molecular marker KASP-3B-1; among them, 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;

[0036] Figure 3 The fluorescence readings of the F5 line of '03D501' × 'Liangmai 4' of this invention were detected using the molecular marker KASP-3B-1; among them, HEX (blue, '03D501') fluorescence indicates plants with more spikelets, FAM (orange, '2011') fluorescence indicates plants with fewer spikelets, and black fluorescence is the blank control;

[0037] Figure 4The results of spikelet number phenotypic detection of the F5 line of '03D501' × 'Liangmai 4' of this invention are shown; where TT represents the plant population with genotype TT and CC represents the plant population with genotype CC. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0043] The wheat varieties used in the following examples are all from the germplasm resource bank of the Wheat Research Institute of Sichuan Agricultural University, which can be obtained from here.

[0044] The technical concept of this invention is as follows:

[0045] This invention utilizes the multi-spikelet wheat cultivar 03D501 as the female parent and the wheat cultivar 2011 with fewer spikelets as the male parent for hybridization to obtain the F1 hybrid. F1 plants are then self-pollinated to obtain the F2 hybrid. The F2 hybrids are then propagated using the single-seed method until the F7 generation, resulting in a recombinant inbred line containing 163 individual plants, forming a genetic mapping population (D12). The spikelet count of the recombinant inbred line population was investigated and identified. DNA was extracted from the parental lines 03D501 and 2011, as well as the recombinant inbred line population. Based on the phenotypic data of the D12 population from 2021 to 2022, extreme lines were screened to construct a mixed pool, and analysis was performed using a wheat 60K SNP chip. The specific screening process is as follows: (1) The spikelet number phenotypic values ​​of each line in the three environmental points of 2021-2022 were sorted, and the lines in the top 50 and bottom 50 in each environmental point were screened out. The line numbers of the lines in the top 50 or bottom 50 in at least three environmental points were counted. (2) The average value of the spikelet number phenotypic data of the three environmental points was calculated and sorted to screen out the top 50 and bottom 50 lines. Finally, the intersection of the above two screening conditions was taken to obtain 25 extreme spikelet-less lines and 26 extreme spikelet-multiple lines. Subsequently, the two F7 extreme mixed pools selected above were genotyped using a 60K SNP chip at Chengdu Tiancheng Future Technology Co., Ltd.

[0046] Genetic maps were constructed using JoinMap 4.0 based on 60K SNP microarray data. Combined with spikelet number phenotypic data of the population, the Inclusive Composite Interval Mapping-ADD (ICIM-ADD) method in QTL IciMapping 4.1 was used to detect spikelet number QTLs by setting a threshold LOD ≥ 2.5. Seven ecopoints from four years (2021-2024) and the BLUP (best linear unbiased prediction) values ​​of spikelet number at these seven ecopoints were used to identify the stable major-effect QTL for wheat spikelet number, QSNS.sicau-D12-3B, on the long arm of chromosome 3B. To further refine the genome map and obtain molecular markers tightly linked to the spikelet number QTL QSNS.sicau-D12-3B, flanking markers were located using 60K SNP microarray data, and SNP markers within the interval were screened. Further exploration of polymorphic SNP sites between parents was conducted to develop a highly efficient and tightly linked KASP molecular marker. A total of 11 KASP primer pairs were designed, and the marker KASP-3B-1 was ultimately found to be tightly linked to the spikelet number QTL QSNS.sicau-D12-3B. This invention reveals that the wheat spikelet number QTL QSNS.sicau-D12-3B is located on the long arm of wheat chromosome 3B, with a physical location of 619673594-625487641 bp in the Chinese Spring RefSeqv2.1 genome version.

[0047] Example 1

[0048] Obtaining the 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 number of spikelets in the recombinant inbred line population was analyzed and identified at the waxy maturity stage of wheat. Marginal effects were excluded. Five single plants with the same growth were collected, the number of spikelets in the main spike was calculated, and the average value was obtained to represent the number of spikelets in the line.

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

[0051] a) DNA extraction

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

[0053] b) Genotype analysis

[0054] The extracted DNA was quality tested using an ultra-micro spectrophotometer (Thermo Fisher Scientific, made in the USA). After passing the quality test, the samples were sent to the company for genotyping analysis. Genotyping analysis of the parents 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 maps

[0056] Based on 60K SNP microarray data, a genetic map was constructed using JoinMap 4.0. Combining the spikelet number phenotypic data of the population, the Inclusive Composite Interval Mapping-ADD (ICIM-ADD) method in QTL IciMapping 4.1 was used. With a LOD threshold ≥ 2.5, spikelet number QTLs were detected using seven ecopoints from 2021-2024 and the BLUP (best linear unbiased prediction) values ​​of spikelet number at those seven ecopoints. The major-effect QTL for wheat spikelet number, QSNS.sicau-D12-3B, was located, and the location of QSNS.sicau-D12-3B and the genetic distance between the molecular markers were 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, these loci were compared with the physical region of QSNS.sicau-D12-3B in this embodiment. The spikelet number QTLs identified in previous studies are as follows: QSpn.WJ.3B.1, QSpn.WY.3B.1, QSpn.abrii-3B.1, QSpn.abrii-3B.2, QSpn.abrii-3B.3, QSns.sau-AM-3B.2, etc. Through physical region comparison, it was found that QSNS.sicau-D12-3B does not overlap with any previously reported spikelet number loci, indicating that QSNS.sicau-D12-3B screened in this embodiment is a new key locus regulating wheat spikelet number. The physical location of this QTL in the Chinese Spring RefSeqv2.1 genome version is 619673521-625487833bp.

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

[0060] Table 1. KASP primer sequences for molecular markers

[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” indicates C or T), and a C / T mutation exists.

[0065] SEQ ID NO.34:

[0066] GTCTACTCAGCAGCCTCAAR.

[0067] Example 2

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

[0069] (1) '03D501' is a multi-spikelet line, while 'Liangmai 4' is a variety with good plant type but few spikelets. Using '03D501' as the female parent and 'Liangmai 4' as the male parent, a segregating population F5 was constructed, and 51 lines were randomly selected from the progeny lines.

[0070] (2) KASP-3B-1 marker detection was performed on the 51 obtained strains. The specific method was as follows:

[0071] Genomic DNA was extracted from 51 strains; it was used as a template for PCR amplification using a specific primer pair of molecular marker KASP-3B-1 (SEQ ID NO.25-27) and fluorescence readings were performed.

[0072] The PCR amplification system described above consisted of: 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 prepared at a concentration of 10 ng / μL, with 60 μL, 60 μL, and 120 μL respectively, and 230 μL of ddH2O added before use as the mixed primers); 5 ng template DNA; double-distilled water to a total volume of 10 μL; and at least three independent blank controls were set up, using double-distilled water instead of DNA template.

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

[0074] Fluorescence readings as follows Figure 3 As shown, the genotype of plants exhibiting FAM (orange) fluorescence is consistent with that of 'Liangmai 4', with a genotype of CC; the genotype of plants exhibiting HEX (blue) fluorescence is consistent with that of '03D501', with a genotype of TT. Further investigation into the spikelet number phenotype of these plants yielded the following results: Figure 4 As shown, the average number of spikelets in plants with the CC genotype was 20.00, while the average number of spikelets in plants with the TT genotype was 23.71. The number of spikelets in plants with the TT genotype was significantly higher than that in plants with the CC genotype. This result is consistent with expectations, indicating that the spikelet number QTL QSNS.sicau-D12-3B of this invention does indeed significantly increase the number of spikelets. The molecular marker KASP-3B-1 of this invention can be used to identify the wheat spikelet number QTL QSNS.sicau-D12-3B and the total number of wheat spikelets.

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

Claims

1. A KASP-3B-1 molecular marker tightly linked to the wheat spikelet number QTL QSNS.sicau-D12-3B, characterized in that, The KASP-3B-1 molecular marker is co-located with the wheat spikelet number QTL QSNS.sicau-D12-3B on the long arm of the wheat 3B chromosome; The molecular marker is a nucleotide sequence as shown in SEQ ID NO. 34; the nucleotide sequence as shown in SEQ ID NO. 34 has a C / T mutation at the 20th base; the wheat spikelet number of the wheat plant with the mutant base site T is significantly more than the wheat spikelet number of the wheat plant with the mutant base site C; The wheat spikelet number QTL QSNS.sicau-D12-3B is located at the physical position of 619673594-625487641 bp in the Chinese Spring RefSeqv2.1 genome version.

2. A KASP primer set for the KASP-3B-1 molecular marker of claim 1, characterized in that, The KASP primer set comprises primers as shown in SEQ ID NO. 25-27.

3. The KASP primer set of claim 2, wherein, The 5' ends of the two primers as shown in SEQ ID NO. 25 and SEQ ID NO. 26 are modified with different fluorescent groups, or the 3' ends are modified with different fluorescent groups.

4. The KASP primer set of claim 2 or 3 is used in any one of B1-B4: B1. detecting the KASP-3B-1 molecular marker of claim 1; B2. early identification or screening of wheat varieties with more spikelets; B3. preparing reagents, kits or chips for detecting the KASP-3B-1 molecular marker of claim 1; B4. preparing reagents, kits or chips for early identification or screening of wheat varieties with more spikelets.

5. A kit for detecting the KASP-3B-1 molecular marker of claim 1, characterized in that, The kit comprises the KASP primer set of claim 2 or 3.

6. A method of detecting the KASP-3B-1 molecular marker of claim 1, characterized in that, comprising the following steps: using the KASP primer set of claim 2 or 3 or the kit of claim 5 to perform fluorescent quantitative PCR amplification with the genomic DNA of the plant to be tested as a template, and determining according to the fluorescence reading results; the plant reading the fluorescent group labeled by SEQ ID NO. 25 is identified as a plant not containing the wheat spikelet number QTL QSNS.sicau-D12-3B, and the KASP-3B-1 molecular marker genotype is CC; the plant reading the fluorescent group labeled by SEQ ID NO. 26 is identified as a plant containing the wheat spikelet number QTL QSNS.sicau-D12-3B, and the KASP-3B-1 molecular marker genotype is TT; the plant with genotype TT has significantly more spikelets than the plant with genotype CC.

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

23.

8. The method of claim 6, wherein, The fluorescent quantitative PCR reaction procedure is: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 63℃ recombination / elongation for 60 s, totally 10 cycles; 94℃ denaturation for 20 s, 55℃ recombination / elongation for 1 min, totally 26 cycles.

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