Molecular marker combination capable of identifying germplasm type of pogostemon cablin and application of molecular marker combination

Abstract

The invention relates to a molecular marker combination capable of identifying germplasm types of pogostemon cablin and application of the molecular marker combination. The molecular marker combination comprises a first molecular marker, a second molecular marker, a third molecular marker and a fourth molecular marker; the first molecular marker is located at a chromosome 44138387bp of a nuclear genome A02, the second molecular marker is located at a chromosome 25567854bp of the nuclear genome A02, the third molecular marker is located at a chromosome 33983091bp of a nuclear genome A03, and the fourth molecular marker is located at a chromosome 42533373bp of a chromosome A05 of a nuclear genome A03. Compared with an early molecular identification method, pogostemon cablin of different germplasm can be rapidly and accurately identified through molecular marker identification, the developed molecular marker can be used for commercial application of pogostemon cablin germplasm type identification, and good economic benefits and good social value can be brought.

Description

Technical Field

[0001] The invention relates to the field of gene technology, and in particular to a molecular marker combination capable of identifying patchouli germplasm types and an application thereof. Background Art

[0002] Patchouli is a famous southern Chinese medicine. After nearly a thousand years of cultivation and domestication in my country, it has formed germplasm types represented by Shipai Patchouli, Zhaoqing Patchouli, Hainan Patchouli, and Zhanjiang Patchouli. Among them, Hainan Patchouli and Zhanjiang Patchouli are actually the same germplasm type; according to chemical composition, patchouli can be divided into "alcohol type" and "keto type". Zhaoqing Patchouli and Shipai Patchouli are "keto type" patchouli, and Hainan Patchouli and Zhanjiang Patchouli are "alcohol type" patchouli. Due to long-term seed propagation by cuttings, the problem of germplasm degeneration and mixing is becoming increasingly serious. In order to overcome the problem of germplasm degeneration, growers often blindly introduce patchouli germplasm from other production areas, which not only fails to improve the problem of patchouli germplasm degeneration, but also brings about the problem of patchouli germplasm mixing. Traditionally, it is believed that the medicinal value of mellow patchouli and ketone patchouli is different due to their different chemical composition. Therefore, the mixed patchouli germplasm not only affects the stability of the quality of patchouli medicinal materials, but also hinders the guarantee of the safety of patchouli medicinal materials in clinical application. At present, researchers have conducted research on the identification of patchouli germplasm through macro / micro phenotypic traits, chemical fingerprints, and conventional molecular markers, but a rapid and effective identification method has not yet been established.

[0003] Identification of patchouli by phenotypic traits relies heavily on the experience and subjective judgment of the identifier. Morphological identification is affected by the environment and season, and the workload is large but the accuracy is low. Although chemical fingerprint identification has achieved certain results in patchouli identification, there are still some challenges and limitations. For example, the sample processing process is relatively complex and easily affected by multiple factors. At the same time, this method requires professional chemical analysis technology and equipment support, and has high technical requirements for operators.

[0004] Early molecular identification methods such as amplified fragment length polymorphic (AFLP), random amplified polymorphic (RAPD), restriction fragment length polymorphism (RFLP) and other marker technologies have been applied to the identification of patchouli germplasm, but the identification results are still unsatisfactory. With the rapid development of high-throughput sequencing technology, molecular markers developed based on whole-genome genetic variations such as single nucleotide polymorphism (SNP) and insertion and deletion (InDel) have gradually played an important role in germplasm identification. However, up to now, there has been no research report on the use of whole-genome genetic variation to develop molecular markers to identify patchouli germplasm resources in my country. In view of this, the present invention provides a molecular marker combination and its application that can identify the type of patchouli germplasm. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a molecular marker combination and application thereof for identifying the types of patchouli germplasm, with the purpose of identifying different patchouli germplasms by using whole genome genetic variation.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, a molecular marker combination capable of identifying the germplasm type of Patchouli, the molecular marker combination comprising a first molecular marker, a second molecular marker, a third molecular marker and a fourth molecular marker;

[0008] The first molecular marker is located at 44138387bp of chromosome A02 of the nuclear genome, and the polymorphism is T or TTCT;

[0009] The second molecular marker is located at 25567854bp of chromosome A02 of the nuclear genome, and the polymorphism is T or TGGAATTAGTGAGT;

[0010] The third molecular marker is located at 33983091bp of chromosome A03 of the nuclear genome, and the polymorphism is A or AA;

[0011] The fourth molecular marker is located at 42533373bp of chromosome A05 of the nuclear genome, and the polymorphism is A or GTA.

[0012] Furthermore, when the inserted base at 44138387bp of chromosome A02 is TTCT, it is Zhanjiang patchouli-mellow type; when the inserted base at 25567854bp of chromosome A02 of the nuclear genome is TGGAATTAGTGAGT, it is keto type-Zhaoqing patchouli; when the inserted base at 33983091bp of chromosome A03 of the nuclear genome is AA, it is keto type-Shipai patchouli; when the inserted base at 42533373bp of chromosome A05 of the nuclear genome is GTA, it is intermediate type-high ethanol and ketone content patchouli.

[0013] In a second aspect, a primer for amplifying a molecular marker combination that can identify the germplasm type of Patchouli includes a primer set for amplifying the molecular marker combination.

[0014] Furthermore, the primer set of the first molecular marker is shown in SEQ ID No: 1-2;

[0015] The primer set for the second molecular marker is shown in SEQ ID No: 3-4;

[0016] The primer set of the third molecular marker is shown in SEQ ID No: 5-6;

[0017] The primer of the fourth molecular marker is shown as SEQ ID No:7.

[0018] In a third aspect, a kit for amplifying a molecular marker combination that can identify the type of patchouli germplasm comprises the primer set for amplifying a molecular marker combination that can identify the type of patchouli germplasm.

[0019] In a fourth aspect, an application of a molecular marker combination that can identify the germplasm type of Patchouli, the application of the molecular marker combination in the identification of the germplasm type of Patchouli; or the application of the molecular marker combination in assisted breeding of Patchouli.

[0020] In a fifth aspect, a primer set for amplifying a molecular marker combination that can identify the type of patchouli germplasm is used, characterized in that the primer set of the molecular marker combination is used in the identification of the patchouli germplasm type; or the primer set of the molecular marker combination is used in patchouli assisted breeding.

[0021] In a sixth aspect, a kit for amplifying a combination of molecular markers that can identify the germplasm type of Patchouli is used, and the kit is used in identifying the germplasm type of Patchouli; or the kit is used in assisted breeding of Patchouli.

[0022] In a seventh aspect, a method for identifying the germplasm type of Patchouli comprises the following steps:

[0023] Step 1: extracting the DNA of the sample patchouli as a DNA template;

[0024] Step 2: using the primer set or the kit to perform PCR amplification on the DNA template to obtain a PCR amplification product;

[0025] Step 3: Sequence the PCR amplification products and perform genotype analysis based on the sequencing results.

[0026] Furthermore, the reaction system for PCR amplification in step 2 is: 2X Pro Taq Master Mix (dye plus) 12.5 μl, ddH2O 8.5 μl, 10 μmol / L forward primer and reverse primer 1.0 μl, 10-30 ng / μL template DNA 2.0 μl;

[0027] The reaction program of PCR amplification in step 2 is: pre-denaturation at 94°C for 30s; denaturation at 94°C for 30s, annealing at 56°C for 30s, extension at 72°C for 1min, 35 cycles; extension at 72°C for 10min; and insulation at 4°C.

[0028] The beneficial effects of the present invention are as follows: the present invention uses four molecular markers of whole genome genetic variation to identify different patchouli germplasms, and compared with the early molecular identification method, the advantage is that patchouli of different germplasms can be quickly and accurately identified. In addition, the method is simple to operate, easy to operate in the laboratory, easy to promote and use, and has broad market application prospects. In addition, the technology can be effectively used for patchouli germplasm identification and protection, and the developed molecular markers can be used for commercial applications of patchouli germplasm type identification, which can bring good economic benefits and good social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The electrophoresis results of the fragment amplified by the first primer pair of the present invention are marker, Zhanjiang patchouli-mellow fragrance type, ketone fragrance type-Shipai patchouli, ketone fragrance type-Zhaoqing patchouli, intermediate type-alcohol and ketone content high patchouli from left to right;

[0030] Figure 2 The electrophoresis results of the fragment amplified by the second primer pair of the present invention are marker, Zhanjiang patchouli-mellow fragrance type, ketone fragrance type-Shipai patchouli, ketone fragrance type-Zhaoqing patchouli, intermediate type-alcohol and ketone content high patchouli from left to right;

[0031] Figure 3 The electrophoresis results of the fragment amplified by the third primer pair of the present invention are marker, Zhanjiang patchouli-mellow fragrance type, ketone fragrance type-Shipai patchouli, ketone fragrance type-Zhaoqing patchouli, intermediate type-alcohol and ketone content high patchouli from left to right;

[0032] Figure 4The electrophoresis results of the amplified fragments of the fourth primer pair of the present invention are marker, Zhanjiang patchouli-mellow fragrance type, ketone fragrance type-Shipai patchouli, ketone fragrance type-Zhaoqing patchouli, intermediate type-alcohol and ketone content high patchouli from left to right;

[0033] Figure 5 The results of the forward sequencing peak comparison of the PCR products of the first primer pair of the present invention are, from top to bottom, Zhanjiang patchouli-mellow fragrance type, ketone fragrance type-Shipai patchouli, ketone fragrance type-Zhaoqing patchouli, intermediate type-alcohol and ketone content high patchouli;

[0034] Figure 6 The reverse sequencing peak comparison result of the PCR product of the first primer pair of the present invention is shown in the figure, from top to bottom: Zhanjiang patchouli-mellow fragrance type, ketone fragrance type-Shipai patchouli, ketone fragrance type-Zhaoqing patchouli, intermediate type-alcohol and ketone content high patchouli;

[0035] Figure 7 The forward sequencing peak comparison result of the PCR product of the second primer pair of the present invention is shown in the figure, from top to bottom: Zhanjiang patchouli-mellow fragrance type, ketone fragrance type-Shipai patchouli, ketone fragrance type-Zhaoqing patchouli, intermediate type-alcohol and ketone content high patchouli;

[0036] Figure 8 The reverse sequencing peak comparison results of the PCR products of the second primer pair of the present invention are, from top to bottom, Zhanjiang patchouli-mellow fragrance type, ketone fragrance type-Shipai patchouli, ketone fragrance type-Zhaoqing patchouli, intermediate type-alcohol and ketone content high patchouli;

[0037] Fig. 9 The forward sequencing peak comparison result of the PCR product of the third primer pair of the present invention is shown in Figure 1, which is Zhanjiang Patchouli-mellow type, ketone type-Shipai Patchouli, ketone type-Zhaoqing Patchouli, and intermediate type-high alcohol and ketone content Patchouli from top to bottom;

[0038] Fig.10 The reverse sequencing peak comparison result of the PCR product of the third primer pair of the present invention is shown in the figure, from top to bottom: Zhanjiang patchouli-mellow fragrance type, ketone fragrance type-Shipai patchouli, ketone fragrance type-Zhaoqing patchouli, intermediate type-alcohol and ketone content high patchouli;

[0039] Fig.11 The forward sequencing peak comparison result of the PCR product of the fourth primer pair of the present invention is shown in Figure 1, which is Zhanjiang Patchouli-mellow type, ketone type-Shipai Patchouli, ketone type-Zhaoqing Patchouli, and intermediate type-high alcohol and ketone content Patchouli from top to bottom;

[0040] Fig.12 This is the forward primer sequencing peak diagram of sample A of the present invention;

[0041] Fig.13 This is the reverse primer sequencing peak diagram of sample A of the present invention;

[0042] Fig.14 This is the forward primer sequencing peak diagram of sample B of the present invention;

[0043] Fig.15 This is the reverse primer sequencing peak diagram of sample B of the present invention;

[0044] Fig.16 This is the forward primer sequencing peak diagram of sample C of the present invention;

[0045] Fig.17 This is the reverse primer sequencing peak diagram of sample C of the present invention;

[0046] Fig.18 This is the forward primer sequencing peak diagram of sample D of the present invention. DETAILED DESCRIPTION

[0047] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0048] Example

[0049] 1. Development of specific molecular markers for patchouli germplasm

[0050] The Shipai patchouli, Zhaoqing patchouli, Zhanjiang patchouli and patchouli with high alcohol and ketone content of the present invention were selected and bred by Professor Ouyang Puyue of Guangdong Food and Drug Vocational College, and were sampled in Liantang Town, Gaoyao District, Zhaoqing City, Guangdong Province, on March 18, 2023. Whole genome sequencing was performed on Shipai patchouli, Zhaoqing patchouli, Zhanjiang patchouli and patchouli with high alcohol and ketone content, and it was found that the 44138387 and 25567854 positions of chromosome A02, the 33983091 position of chromosome A03 and the 42533373 position of chromosome A05 showed germplasm-specific variation, and primers were designed to amplify the above candidate sites respectively.

[0051] Specifically, a molecular marker combination that can identify the germplasm type of patchouli includes a first molecular marker, a second molecular marker, a third molecular marker and a fourth molecular marker; the first molecular marker is located at 44138387bp on chromosome A02, and the polymorphism is T or TTCT; the second molecular marker is located at 25567854bp on chromosome A02 of the nuclear genome, and the polymorphism is T or TGGAATTAGTGAGT; the third molecular marker is located at 33983091bp on chromosome A03 of the nuclear genome, and the polymorphism is A or AA; the fourth molecular marker is located at 42533373bp on chromosome A05 of the nuclear genome, and the polymorphism is A or GTA.

[0052] When the inserted base at 44138387bp of chromosome A02 is TTCT, it is Zhanjiang patchouli-mellow type; when the inserted base at 25567854bp of chromosome A02 of the nuclear genome is TGGAATTAGTGAGT, it is keto type-Zhaoqing patchouli; when the inserted base at 33983091bp of chromosome A03 of the nuclear genome is AA, it is keto type-Shipai patchouli; when the inserted base at 42533373bp of chromosome A05 of the nuclear genome is GTA, it is intermediate type-high ethanol and ketone content patchouli.

[0053] 2. Primer design for molecular markers

[0054] The software used for primer design is Primer3, and the parameter settings are as follows:

[0055] PRIMER_PICK_LEFT_PRIMER=1,

[0056] PRIMER_PICK_INTERNAL_OLIGO=0,

[0057] PRIMER_PICK_RIGHT_PRIMER=1,

[0058] PRIMER_OPT_SIZE=20,

[0059] PRIMER_MIN_SIZE=18,

[0060] PRIMER_MAX_SIZE=22,

[0061] PRIMER_PRODUCT_SIZE_RANGE=80-150, PRIMER_EXPLAIN_FLAG=1.

[0062] The first primer pair of the first molecular marker (Zhanjiang Patchouli-mellow type):

[0063] Forward primer: 5'-AGGTGAGGGAAGGCATTGTT-3' (SEQ ID No: 1),

[0064] Reverse primer: 5'-TCAACCACCATCAACCACACT-3' (SEQ ID No: 2).

[0065] The second primer pair of the second molecular marker (ketone fragrance - Zhaoqing patchouli):

[0066] Forward primer: 5'-AGGAGCCTTTAGGTGTTGCT-3' (SEQ ID No: 3),

[0067] Reverse Primer 2: 5'-ACACTATGGAAAAGGGAGGCC-3' (SEQ ID No: 4).

[0068] The third primer pair of the third molecular marker (ketone fragrance-Shipai patchouli):

[0069] Forward primer: 5'-CCCTGAAAAGAAAAGGCTACC-3' (SEQ ID No: 5),

[0070] Reverse primer: 5'-CCCACCATAGTAAAAAAGTTGTAAG-3' (SEQ ID No: 6).

[0071] The fourth primer of the fourth molecular marker (intermediate-high alcohol and ketone content patchouli):

[0072] Primer: 5'-TGGTGCACTTGAACAATCCT-3' (SEQ ID No: 7).

[0073] 3. Validation of primer sets for molecular markers

[0074] A method for identifying the germplasm type of Patchouli comprises the following steps:

[0075] Step 1: Extract the DNA of the sample Patchouli as a DNA template, wherein the DNA is extracted using the Tiangen Plant Genome Kit (DP305) method to extract the patchouli sample DNA, the method is as shown in the instructions, and the purity and concentration of the DNA are detected using Nanodrop 2000c (Thermo Scientific, USA). The DNA samples with OD260 / 280 in the range of 1.8 to 2.0 are stored at -20°C for later use.

[0076] Step 2: Perform PCR amplification on the DNA template using the above primer set to obtain a PCR amplification product;

[0077] The PCR reaction system was: 2X Pro Taq Master Mix (dye plus) 12.5 μl, ddH2O 8.5 μl, 10 μmol / L forward primer and reverse primer 1.0 μl, 10-30 ng / μL template DNA 2.0 μl;

[0078] The PCR reaction program was as follows: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0079] Step 3: Sequence the PCR amplification products and perform genotype analysis based on the sequencing results.

[0080] (1) Electrophoresis detection

[0081] The PCR products were detected by 0.7% agarose gel electrophoresis at 80 V for 50 min. Figure 1 to Figure 4 As shown. The imaging results showed that for the patchouli samples of four different germplasms, the above four pairs of primers successfully amplified clear bands, but the band lengths of the PCR amplification products of the same primers did not differ significantly in the patchouli of the four germplasms. This result shows that on the gel imaging layer, we cannot effectively distinguish the Zhanjiang patchouli-mellow type, the ketone type-Zhaoqing patchouli, the ketone type-Shipai patchouli, and the intermediate type-high alcohol and ketone content patchouli.

[0082] (2) Sanger sequencing and sequence alignment

[0083] The PCR products and their corresponding forward and reverse primers were sent to Shanghai Bioengineering Co., Ltd. for bidirectional sequencing. The sequencing results of each sample included two sequences, forward and reverse. The sequencing results of the forward and reverse primers and the corresponding reference sequences were imported into SnapGene software for sequence alignment. The sequence alignment results are shown in Figure 2. Figure 5 to Figure 12 As shown. Comparing the T base at 44138387 of the nuclear genome A02 chromosome, when the base at this position is inserted into TTCT, it is Zhanjiang Patchouli-mellow aroma type; comparing the T base at 25567854 of the nuclear genome A02 chromosome, when the base at this position is inserted into TGGAATTAGTGAGT, it is ketone aroma type-Zhaoqing Patchouli; comparing the A base sequence at 33983091 of the nuclear genome A03 chromosome, when one more A is inserted into the base at this position, it is ketone aroma type-Shipai Patchouli; comparing the A base at 42533373 of the nuclear genome A05 chromosome, when the base at this position is inserted into GTA, it is intermediate type-high alcohol and ketone content Patchouli.

[0084] 4. Application of primer sets with four marker loci in practical identification of germplasm types of Patchouli

[0085] A single-blind experimental design was adopted, in which the designer numbered various germplasms, and then the testers were asked to test samples with different numbers. In order to verify the detection efficiency of the developed molecular markers, patchouli of different germplasm types were randomly numbered (A: ketone-flavored patchouli, B: Zhanjiang patchouli-mellow flavored patchouli, C: ketone-flavored patchouli, Zhaoqing patchouli, D: intermediate-high-alcohol-ketone content patchouli) (Table 1), and operators who did not know the germplasm type performed DNA extraction, PCR amplification, and first-generation sequencing. The identification steps were the same as those recorded in the above three parts. Primers were directly used for PCR amplification, and the conditions were sent for sequencing, and the germplasm was identified according to the sequence composition.

[0086] Table 1 Sample sources and numbers

[0087]

[0088] Depend on Figure 12-18 It can be seen that the identification results are consistent with the actual situation, and the germplasm types of all samples can be successfully detected.

[0089] In summary, the present invention uses four molecular markers of whole genome genetic variation to identify different patchouli germplasms. Compared with the early molecular identification method, the advantage is that it can quickly and accurately identify patchouli of different germplasms. In addition, the method is simple to operate, easy to operate in the laboratory, easy to promote and use, and has broad market application prospects. In addition, the technology can be effectively used for patchouli germplasm identification and protection, and the developed molecular markers can be used for commercial applications in patchouli germplasm type identification, which can bring good economic benefits and good social value.

[0090] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A molecular marker combination capable of identifying the germplasm type of Patchouli, characterized in that: The molecular marker combination includes a first molecular marker, a second molecular marker, a third molecular marker and a fourth molecular marker; The first molecular marker is located at 44138387bp of chromosome A02 of the nuclear genome, and the polymorphism is T or TTCT; The second molecular marker is located at 25567854bp of chromosome A02 of the nuclear genome, and the polymorphism is T or TGGAATTAGTGAGT; The third molecular marker is located at 33983091bp of chromosome A03 of the nuclear genome, and the polymorphism is A or AA; The fourth molecular marker is located at 42533373bp of chromosome A05 of the nuclear genome, and the polymorphism is A or GTA.

2. A molecular marker combination capable of identifying the germplasm type of Pogostemon cablin according to claim 1, characterized in that: When the inserted base at 44138387bp of chromosome A02 is TTCT, it is Zhanjiang patchouli-mellow type; when the inserted base at 25567854bp of chromosome A02 of the nuclear genome is TGGAATTAGTGAGT, it is keto type-Zhaoqing patchouli; when the inserted base at 33983091bp of chromosome A03 of the nuclear genome is AA, it is keto type-Shipai patchouli; when the inserted base at 42533373bp of chromosome A05 of the nuclear genome is GTA, it is intermediate type-high ethanol and ketone content patchouli.

3. A primer set for amplifying a molecular marker combination capable of identifying the germplasm type of Patchouli, characterized in that: Comprising a primer set for amplifying the molecular marker combination described in claim 1 or 2.

4. A primer set for amplifying a molecular marker combination capable of identifying the germplasm type of Pogostemon cablin according to claim 3, characterized in that: The primer set for the first molecular marker is shown in SEQ ID No: 1-2; The primer set for the second molecular marker is shown in SEQ ID No: 3-4; The primer set for the third molecular marker is shown in SEQ ID No: 5-6; The primer of the fourth molecular marker is shown as SEQ ID No:

7.

5. A kit for amplifying a combination of molecular markers that can identify the germplasm type of Patchouli, characterized in that: A primer set comprising a combination of molecular markers for amplifying and identifying the germplasm type of Patchouli as described in claim 3 or 4.

6. An application of a molecular marker combination for identifying the germplasm type of Patchouli, characterized in that: Use of the molecular marker combination described in claim 1 or 2 in identification of patchouli germplasm types; or use of the molecular marker combination described in claim 1 or 2 in patchouli assisted breeding.

7. An application of a primer set for amplifying a molecular marker combination capable of identifying the germplasm type of Patchouli, characterized in that: Use of the primer set described in claim 3 or 4 in identification of patchouli germplasm types; or use of the primer set described in claim 3 or 4 in patchouli assisted breeding.

8. Use of a kit for amplifying a combination of molecular markers capable of identifying the germplasm type of Patchouli, characterized in that: Use of the kit described in claim 5 in identification of patchouli germplasm types; or use of the kit described in claim 5 in patchouli assisted breeding.

9. A method for identifying the germplasm type of Patchouli, characterized in that: The steps include: Step 1: extracting the DNA of the sample patchouli as a DNA template; Step 2: performing PCR amplification on the DNA template using the primer set described in claim 3 or 4 or the kit described in claim 5 to obtain a PCR amplification product; Step 3: Sequence the PCR amplification products and perform genotype analysis based on the sequencing results.

10. A method for identifying the germplasm type of Patchouli according to claim 9, characterized in that: The reaction system for PCR amplification in step 2 is: 2X Pro Taq Master Mix (dye plus) 12.5 μl, ddH2O 8.5 μl, 10 μmol / L primer set 1.0 μl, 10-30 ng / μL DNA template 2.0 μl; The reaction program of PCR amplification in step 2 is: pre-denaturation at 94°C for 30s; denaturation at 94°C for 30s, annealing at 56°C for 30s, extension at 72°C for 1min, 35 cycles; extension at 72°C for 10min; and insulation at 4°C.

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