Development and application of a set of core primers for SSR based on whole genome sequencing and re-sequencing of core collection of Glycine max

Abstract

The application discloses a SSR core primer group based on whole genome sequencing and core germplasm resequencing of Glycine max and application thereof. Specifically disclosed is the SSR core primer group, which comprises at least 5 pairs of primers (SEQ ID No. 1-460) in 230 pairs of primers. The application establishes a method for developing the whole genome SSR marker of Glycine max, which is comprehensive, accurate and reliable, and solves the problems of the current Glycine max SSR marker, such as small quantity, low polymorphism and low genome coverage. The SSR core primer group can maximize the reflection of the genetic diversity of Glycine max germplasm resources, has the characteristics of high polymorphism, uniform distribution, stable amplification, good reproducibility, clear site, clear and easy-to-distinguish statistical electrophoresis bands and the like, can accurately perform kinship analysis and diversity analysis, and is helpful to the researches such as genetic map construction, gene positioning, variety identification and variety DNA fingerprint construction, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically involving an SSR core primer set developed based on whole-genome sequencing and core germplasm resequencing of soybean and its application. Background Technology

[0002] Red beans (Vigna angularis), belonging to the genus Vigna L. of the tribe Phaseoleae in the family Leguminosae, are also known as adzuki beans, red beans, and adzuki beans. Originating in China, red beans have a long cultivation history of over 2000 years and are one of China's six major edible beans. They are a traditional specialty crop in my country and an important crop in the country's modern agricultural restructuring. China has the largest red bean planting area and output in the world. Rich in protein, vitamins, and minerals, red beans are widely used in making red bean buns, desserts, red bean rice, ice cream, and soy milk, holding an important position in the food consumption of Asian populations and in the international trade of edible beans. Red beans are also considered a food with medicinal value, possessing properties such as dispelling dampness, detoxifying, and lowering blood sugar, as well as weight loss and fat reduction health benefits. Mung bean is a diploid species (2n=22) with a genome of only 539Mb and a short growth period, making it an ideal species for genetic research in leguminous crops. Furthermore, mung bean is tolerant of poor soil and shade, has wide adaptability, and strong nitrogen-fixing and soil-enriching capabilities, making it suitable for intercropping, relay cropping, and crop rotation with gramineous crops. Therefore, strengthening research on mung bean genetics and breeding, and conducting research on mung bean variety identification, fingerprinting, and genetic diversity are of significant practical importance.

[0003] Molecular markers are heritable and detectable specific DNA fragments that reflect certain differences in the genomes of individuals or populations; they are also called DNA markers. Molecular markers are widely distributed in the genomes of eukaryotes. Simple sequence repeats (SSRs), also known as microsatellites, are DNA sequences that are repeated multiple times in tandem in the genome, with units of 1-6 nucleotides, and are generally less than 200 bp in length. Due to the different number of repeats or the different degrees of repetition, the length of SSRs is highly variable, thus giving rise to SSR markers. Although the locations of SSRs in the genome are not exactly the same, the sequences at both ends are mostly conserved single-copy sequences. Therefore, primer pairs can be designed using microsatellite regions, and polyacrylamide gel electrophoresis can be used to display the polymorphism of SSR sites among different individuals using PCR technology. SSR marker technology has the following advantages: (1) a large number of markers, high polymorphism, and a large amount of information; (2) no tissue specificity, unrelated to the degree of growth and development, and the sampling is not limited by development and seasonality; (3) it can clearly identify alleles; (4) the markers are evenly distributed throughout the genome; (5) selection is neutral and does not affect the expression of the target trait; (6) the detection method is simple and fast; (7) the cost is low; (8) it is stable and has good reproducibility; and (9) it is co-dominant. At present, this technology is widely used in plant genetic diversity research, variety identification, genetic map construction, molecular marker-assisted selection, QTL mapping, comparative genomics research and other fields. Compared with crops such as wheat, rice and corn, the number of existing soybean SSR markers is very small, and there are still problems such as low polymorphism and low genome coverage, which cannot meet the needs of soybean research. The large-scale development of SSR markers covering the whole genome is still the current work of soybean breeding research and has important application value. Summary of the Invention

[0004] One objective of this invention is to provide an SSR core primer set developed based on whole-genome sequencing and core germplasm resequencing of soybeans, and its applications. The technical problems to be solved are not limited to the described technical topics; other technical topics not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To achieve the above objectives, the present invention first provides an SSR core primer set developed based on the whole genome sequence of soybean, the SSR core primer set including at least 5 pairs of 230 primer pairs, the nucleotide sequences of the 230 primer pairs are shown as SEQ ID No.1-SEQ ID No.460.

[0006] Further, the at least 5 pairs may be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220 pairs.

[0007] Furthermore, the nucleotide sequences of the primers targeting chromosome 1 (VaC1) of Bean are shown in SEQ ID No. 1-SEQ ID No. 50; the nucleotide sequences of the primers targeting chromosome 2 (VaC2) of Bean are shown in SEQ ID No. 51-SEQ ID No. 98; the nucleotide sequences of the primers targeting chromosome 3 (VaC3) of Bean are shown in SEQ ID No. 99-SEQ ID No. 156; the nucleotide sequences of the primers targeting chromosome 4 (VaC4) of Bean are shown in SEQ ID No. 157-SEQ ID No. 198; the nucleotide sequences of the primers targeting chromosome 5 (VaC5) of Bean are shown in SEQ ID No. 199-SEQ ID No. 240; the nucleotide sequences of the primers targeting chromosome 6 (VaC6) of Bean are shown in SEQ ID No. 241-SEQ ID No. 268; the nucleotide sequences of the primers targeting chromosome 7 (VaC7) of Bean are shown in SEQ ID No. 269-SEQ ID No. 314; and the nucleotide sequences of the primers targeting chromosome 8 (VaC8) of Bean are shown in SEQ ID No. 50-SEQ ID No. 98. The nucleotide sequences of primers for chromosome 9 (VaC9) of lentil are shown in SEQ ID No. 315-SEQ ID No. 352; the nucleotide sequences of primers for chromosome 10 (VaC10) of lentil are shown in SEQ ID No. 383-SEQ ID No. 434; and the nucleotide sequences of primers for chromosome 11 (VaC11) of lentil are shown in SEQ ID No. 435-SEQ ID No. 460.

[0008] Furthermore, the SSR core primer set was developed based on whole-genome sequencing and core germplasm resequencing sequences of soybean.

[0009] The present invention also provides a kit that may contain the SSR core primer set.

[0010] The present invention also provides a DNA chip, which may contain the SSR core primer set.

[0011] The present invention also provides any of the following applications of the SSR core primer set, and / or the kit, and / or the DNA chip:

[0012] A1) Application in the analysis of genetic diversity in soybean;

[0013] A2) Application in the analysis of soybean variety diversity;

[0014] A3) Application in the analysis of kinship in soybean;

[0015] A4) Application in mung bean variety identification;

[0016] A5) Application in constructing genetic maps or DNA fingerprint maps of soybeans;

[0017] A6) Application in the improvement of mung bean germplasm resources;

[0018] A7) Application in soybean gene localization or functional gene mining;

[0019] Application of A8 in marker-assisted breeding of soybean.

[0020] Furthermore, A1) can be used in the analysis of genetic diversity of wild adzuki beans, adzuki beans and adzuki bean closely related species.

[0021] Furthermore, A4) can be applied in the diversity analysis of cultivated mung beans and mung bean closely related species.

[0022] The present invention also provides a method for screening the SSR core primer set, the method comprising the following steps:

[0023] B1) Perform genome resequencing on soybean germplasm resources;

[0024] B2) Using the whole genome sequence of Xiaodou Jingnong 6 (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_001190045.1, https: / / www.ncbi.nlm.nih.gov / assembly / GCA_001723775.1) as a reference genome, a whole genome scan of the SSR loci of the reference genome was performed to obtain SSR markers;

[0025] B3) Screening for diversity among germplasm resources using the SSR markers;

[0026] B4) Primers were designed for the SSR markers selected in B3) to obtain the core SSR primer set.

[0027] In the above method, the screening criteria described in B3) can be: the diversity ratio among the core germplasm materials of mung bean is more than 25%, the number of dinucleotide repeats is ≥11, and redundant SSR sites in the genome are removed.

[0028] In the above method, the primer design parameters described in B4) may include: primer annealing temperature of 60-65℃, primer length of 22-25bp, primer located 45-150bp away from the SSR site to ensure primer specificity, and amplification product length of 80-300bp.

[0029] The mung bean germplasm resources described in B1) can be core mung bean germplasm materials that can represent the genetic diversity of all mung bean germplasm resources.

[0030] Furthermore, the mung bean germplasm resources mentioned in B1) can be the 322 core mung bean germplasm materials in Table 1.

[0031] Furthermore, B2) also includes the step of screening the obtained SSR markers and flanking sequences to remove repetitive sequences, and obtaining non-repetitive SSR markers for the Jingnong 6 soybean genome after removing repetitive sequences.

[0032] The SSR marker mentioned in B3) can be the non-repetitive SSR marker of the Jingnong 6 soybean genome.

[0033] Furthermore, B3) can be the non-repetitive SSR marker of the Jingnong 6 soybean genome obtained in step B2) as a reference sequence to screen for polymorphic sites among the core soybean germplasm materials.

[0034] In one embodiment of the present invention, B3) may include the following steps:

[0035] (1) Using the non-repetitive SSR markers of the obtained Jingnong 6 soybean genome as reference sequences, we screened for polymorphic sites among the core soybean germplasm materials. The screening criteria were: the diversity ratio among the 322 core soybean germplasm materials was more than 25%.

[0036] (2) For the SSR marker sites obtained in step (1), use the SSR hunter software to search for SSR sequences. The criteria for searching for SSR sequences are: the number of dinucleotide repeats is ≥11, and redundant SSR sites in the genome are removed.

[0037] (3) Screening of SSR sites for the searched SSR sequences. The screening criteria is: develop one SSR marker within a physical distance range of 500K-1000K in the genome.

[0038] The present invention also provides a method for analyzing the genetic diversity or kinship of soybean using the SSR core primer set. The method may include: using soybean genomic DNA as a template, performing PCR amplification using the SSR core primer set to obtain PCR amplification products, performing electrophoresis detection on the PCR amplification products, and performing soybean genetic diversity or kinship analysis based on the electrophoresis detection results.

[0039] Furthermore, the genetic diversity analysis or kinship analysis of soybeans based on the electrophoresis results includes statistical analysis of the electrophoresis results, recording "1" for a band at the same electrophoretic mobility position and "0" for no band. Cluster analysis is then performed to construct a kinship tree diagram, and the genetic diversity analysis or kinship analysis of soybeans is conducted based on the statistical results.

[0040] Furthermore, the cluster analysis can be performed using NTSYS-PC Ver.2.10e software based on the UPGMA method.

[0041] This invention also provides a method for identifying mung bean varieties by performing fingerprint analysis using the SSR core primer set. The method may include: using genomic DNA of the mung bean to be tested and a standard mung bean variety as templates, respectively, performing PCR amplification using the SSR core primer set; detecting the PCR amplification products by polyacrylamide gel electrophoresis to obtain the fingerprint patterns of the mung bean to be tested and the standard mung bean variety; and identifying the mung bean variety based on the fingerprint pattern comparison analysis results.

[0042] In the above method, the PCR amplification reaction system can be 15 μL, containing 0.1 μmol / L upstream primer, 0.1 μmol / L downstream primer, 1 mmol / L dNTPs, and Mg. 2+ 1.5 mmol / L, Taq polymerase 1 U, soybean genomic DNA 50 ng; PCR amplification program can be: 94℃ 2 min; 94℃ 30 s, 55℃ 30 s, 72℃ 30 s, 35 cycles; 72℃ 5 min.

[0043] Furthermore, in the above method, the electrophoresis detection can be performed by detecting the PCR amplification products using 7% denaturing polyacrylamide gel electrophoresis.

[0044] Furthermore, the electrophoresis detection includes the following steps: loading 1 μL of PCR product, using a 100bp Ladder DNA marker as the molecular weight standard, electrophoresis at a constant voltage of 60W for 1 hour, and silver staining for development.

[0045] The soybeans to be tested in this article may be wild soybeans, soybeans grown by farmers, local soybean varieties, commercial soybean varieties, or cultivated soybeans.

[0046] Furthermore, the method for identifying mung bean varieties can be to use core primers (two pairs randomly selected from each chromosome VaC1-VaC11) to perform PCR fingerprinting on the DNA of the standard mung bean variety and the test sample. When the fingerprint patterns of the standard sample and the test sample are inconsistent, the test sample and the standard mung bean variety are different varieties.

[0047] This invention develops SSR markers and their core primer sets based on whole-genome sequencing and core germplasm resequencing of soybean. The SSR core primer set includes at least 5 pairs of 230 primer pairs, the nucleotide sequences of which are shown in SEQ ID No. 1-SEQ ID No. 460 (Table 2). This invention establishes a method for screening (developing) whole-genome SSR marker sites in soybean. This method has the advantages of being comprehensive, complete, accurate, and reliable, solving the problems of limited number of existing soybean SSR markers, low polymorphism, and low genome coverage. This invention designs primers, performs PCR amplification, and detects the SSR markers using polyacrylamide gel electrophoresis, and applies the SSR markers to identify the kinship of cultivated soybean. Experiments show that the primer combinations disclosed in this invention can reflect the genetic diversity of soybean germplasm resources to the greatest extent, exhibiting high polymorphism, uniform distribution, stable amplification, good repeatability, clear loci, clear and easily identifiable electrophoretic bands, and ease of statistical analysis. It can cover the entire genome and has significant practical value. The primer combinations of this invention can be applied to wild mung beans, cultivated mung beans, mung bean farm varieties, bred mung beans, and mung bean closely related species. They can perform kinship analysis and diversity analysis with maximum accuracy, and help to accurately and quickly carry out research such as genetic map construction, gene mapping, variety identification, and variety DNA fingerprinting. This lays the foundation for molecular marker-assisted breeding of mung beans and has broad application prospects. Attached Figure Description

[0048] Figure 1 This is a cluster diagram of 14 soybean samples based on the core SSR data of soybeans. Detailed Implementation

[0049] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0051] Example 1: Obtaining the whole genome SSR markers and core primer set of soybean

[0052] This embodiment developed a whole-genome SSR marker and its core primer set for soybean based on whole-genome sequencing and core germplasm resequencing. The specific steps are as follows:

[0053] 1. The genomes of 322 core germplasm materials of mung bean were resequencing. The basic information of the 322 core germplasm materials of mung bean is shown in Table 1. The mung bean varieties in Table 1 are from the College of Plant Science and Technology of Beijing University of Agriculture.

[0054] 2. Using the whole genome sequence of Xiaodou Jingnong 6 (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_001190045.1, https: / / www.ncbi.nlm.nih.gov / assembly / GCA_001723775.1) as a reference genome, a whole genome scan of SSR loci was performed to obtain SSR markers.

[0055] 3. Screen the SSR markers and flanking sequences obtained in step 2, remove repetitive sequences, and obtain the non-repetitive SSR markers of the Jingnong 6 soybean genome. Further, use the obtained non-repetitive SSR markers of the Jingnong 6 soybean genome as reference sequences to screen for polymorphic sites among the core soybean germplasm materials. The screening criteria are: the diversity ratio among the 322 core soybean germplasm materials is more than 25%.

[0056] 4. For the SSR marker sites obtained in step 3, use the SSR hunter software to search for SSR sequences. The criteria for searching for SSR sequences are: the number of dinucleotide repeats ≥ 11, and redundant SSR sites in the genome are removed.

[0057] 5. Screen the SSR sites of the searched SSR sequences. The screening criteria is: develop one SSR marker within a physical distance range of 500K-1000K in the genome.

[0058] 6. Design primers for the obtained SSR markers. Primer design parameters are as follows: primer annealing temperature 60-65℃, primer length 22-25bp, primers located 45-150bp flanking the SSR site to ensure primer specificity, and amplification product length 80-300bp.

[0059] Ultimately, 230 SSR markers covering the entire soybean genome were obtained, and a core primer set (230 primer pairs) for the entire soybean genome SSR was designed. The sequences of the 230 primer pairs (as shown in the sequence listing SEQ ID No. 1-SEQ ID No. 460) are shown in Table 2.

[0060] Table 1. Basic Information of 322 Core Germplasm Materials of Mung Bean

[0061]

[0062]

[0063]

[0064]

[0065] Table 2. Sequences of 230 primer pairs (SSR core primer set)

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] In Table 2, “SEQ ID No.” represents the serial number.

[0077] Example 2: Application of whole-genome SSR markers and core primer set of soybean

[0078] The diversity analysis of soybean germplasm resources was performed using the SSR core primer set developed and designed based on soybean whole-genome sequencing and core germplasm resequencing in Example 1. The specific steps are as follows:

[0079] 1. Select 14 representative materials from soybean germplasm resources (GM97, GM309, GM868, GM437, Yuhong No.1, B2621, Jingnong No.6, Jingnong No.21, Jingnong No.23, CCA004, Zhongnayansha 47, AG109, KCA004, and Nonglin No.3).

[0080] 2. Genomic DNA was extracted from soybeans using a modified CTAB method. The specific steps are as follows:

[0081] (1) 0.5g of fresh leaves of mung bean germplasm resources were frozen and ground in liquid nitrogen to obtain sample powder. 800μL of CTAB buffer (CTAB 2%, NaCl 1.4mol / L, EDTA (pH 8.0) 20mmol / L, Tris-HCl (pH 8.0) 100mmol / L, 0.2% mercaptoethanol) preheated at 65℃ was added and incubated at 65℃ for 1h.

[0082] (2) Add 600 μL of chloroform, mix by inverting for 5-10 minutes, and centrifuge at 12,000 rpm for 5 minutes.

[0083] (3) Take the supernatant and add 0.7 times the volume of isopropanol pre-cooled at 4℃. Let it stand at -20℃ for 30 min, centrifuge at 12,000 rpm for 5 min, carefully discard the supernatant, and obtain the centrifuged DNA precipitate.

[0084] (4) Air dry the centrifuged DNA, add 100 μL of TE buffer (Tris-HCl 10 mM / L, EDTA 1 mM / L, pH=8.0), incubate at 55℃ until completely dissolved, and store at 4℃ for later use.

[0085] 3. The SSR core primer set obtained in Example 1 (eight randomly selected primer pairs from Table 2: VaC1S139_6800, VaC2S105_14351, VaC3S21_1100, VaC4S24_8000, VaC5S619_9080, VaC6S174_10824, VaC7S276_9557, VaC8S11_7048) was used to amplify the extracted soybean genomic DNA by PCR. The PCR reaction system contained: 0.1 μmol / L upstream primer, 0.1 μmol / L downstream primer, 1 mmol / L dNTPs, and Mg... 2+ 1.5 mmol / L, 1 U Taq polymerase, 50 ng soybean genomic DNA; PCR amplification program: 94℃ for 2 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min.

[0086] Specifically, the reaction system for the PCR amplification is 15 μL.

[0087] 4. PCR products were detected by 7% denaturing polyacrylamide gel electrophoresis. 1 μL of PCR product was loaded and electrophoresed at 60 W for 1 hour using a 100 bp Ladder DNA marker as the molecular weight standard. Silver staining was then performed for development.

[0088] 5. Photographic statistical analysis of electrophoresis results: if there is a band at the same electrophoretic mobility position, record it as "1"; if there is no band, record it as "0".

[0089] 6. Use NTSYS-PC Ver.2.10e software to perform cluster analysis based on the UPGMA method and construct a kinship dendrogram. Figure 1 The kinship relationship between different mung bean varieties can be determined by using a kinship tree diagram.

[0090] The method for identifying mung bean varieties involves using the DNA of standard mung bean varieties and test samples to perform PCR fingerprinting using core primers (two pairs randomly selected from each chromosome VaC1-VaC11). When the fingerprint patterns of the standard sample and the test sample are inconsistent, the test sample is a different variety from the standard mung bean variety.

[0091] The results showed that Chinese local varieties and varieties bred using Chinese mung bean resources, such as GM97, GM309, GM868, GM437, Yuhong No. 1, and B2621, clustered into one group; Japanese-bred varieties, Korean local varieties, and varieties bred using Japanese varieties as parents, such as Jingnong No. 6, Jingnong No. 21, Jingnong No. 23, CCA004, Zhongnayansha 47, AG109, KCA004, and Nonglin No. 3, clustered into another group.

[0092] It is evident that the genetic similarity coefficients of different mung bean varieties are all less than 0.24, which can distinguish all varieties. This indicates that by selecting a limited number of core primers from 230 pairs of core primers, the kinship of mung bean resources can be effectively identified.

[0093] In summary, the whole-genome SSR markers and core primer set of the present invention can comprehensively reflect the genetic information of soybeans, have good polymorphism and stability, and can be used for genetic diversity analysis, kinship analysis and variety identification of soybean resources.

[0094] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A primer set, characterized in that, The primer set is any one of the following: C1) a primer set consisting of 230 pairs of primers, the nucleotide sequences of the 230 pairs of primers being shown as SEQ ID No. 1-SEQ ID No. 460; C2) a primer set consisting of 8 pairs of primers, the nucleotide sequences of the 8 pairs of primers being shown as SEQ ID No. 3, 4, 51, 52, 119, 120, 167, 168, 233, 234, 243, 244, 291, 292, 315, 316.

2. A kit characterized in that, The kit comprises the primer set of claim 1.

3. A DNA chip characterized by The DNA chip comprises the primer set of claim 1.

4. Use of the primer set of claim 1, or the kit of claim 2, or the DNA chip of claim 3, which is any one of the following: A1) use in analysis of diversity of varieties of adzuki bean; A2) use in identification of varieties of adzuki bean.

5. A method for analyzing the diversity of varieties of Glycine max using the primer set according to claim 1, characterized by, The method comprises: using the primer set of claim 1 to perform PCR amplification with the genomic DNA of the adzuki bean to be tested as a template, obtaining the PCR amplification product, performing electrophoresis detection on the PCR amplification product, and performing analysis of diversity of varieties of adzuki bean according to the result of electrophoresis detection.

6. A method for identifying a variety of azuki bean by fingerprinting the variety of azuki bean using the primer set according to claim 1, characterized by, The method comprises: using the primer set of claim 1 to perform PCR amplification with the genomic DNA of the adzuki bean to be tested and the standard adzuki bean variety as templates, respectively, performing polyacrylamide gel electrophoresis detection on the PCR amplification product, obtaining the fingerprint of the adzuki bean to be tested and the fingerprint of the standard adzuki bean variety, and performing identification of varieties of adzuki bean according to the result of comparison and analysis of the fingerprints.

7. The method according to claim 5 or 6, characterized in that, The reaction system of PCR amplification was 15 μL, containing 0.1 μmol / L of upstream primer, 0.1 μmol / L of downstream primer, 1 mmol / L of dNTPs, 1.5 mmol / L of Mg 2+ 1.5 mmol / L, 1 U of Taq polymerase, 50 ng of genomic DNA of Glycine soja; the PCR amplification procedure was as follows: 94℃ for 2 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min.

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