Primer, method and application for detecting polymorphism in the promoter region of wheat TaPpd-2D gene

Abstract

The present invention relates to a kind of primer, method and application for detecting polymorphism of wheat TaPpd 2D gene promoter region, utilizing the causal relationship between TaPpd 2D gene second genetic variation and spike length, design primer of the present invention, can complete the detection of two genetic variations simultaneously in the same PCR reaction system, detect 2511bp and 134bp bands in wheat genomic DNA to be detected, i.e. there is 2089bp nucleotide sequence and 24bp deletion, then wheat material to be detected spike length is longer, such as detecting 422bp and 158bp bands in wheat genomic DNA to be detected, indicating that there is 2089bp deletion and 24bp insertion in the material, and the material has shorter spike length. The method of the present invention has the characteristics of accurate result, fast and simple, is suitable for the selection of breeding different generations, can shorten the breeding cycle, accelerate breeding selection speed, reduce screening cost, simple to operate, low cost, high in flux.

Description

Technical Field

[0001] The present invention relates to a primer, a method and an application for detecting polymorphism in a promoter region of a wheat TaPpd-2D gene, and belongs to the field of molecular biology. Background Art

[0002] The foundation of plant breeding is the selection of genetic variation. Currently, plant breeding primarily relies on empirical field selection (conventional breeding), supplemented by molecular marker-assisted selection. Conventional breeding presents numerous practical challenges, including the inability to select for low-effect alleles, difficulty estimating gene-gene interactions, the inability to simultaneously select for genes that interact with the environment, the inability to perform phenotypic selection in the heterozygous state in early generations, and the inability to accommodate multi-trait selection.

[0003] A molecular marker is a specific, heritable DNA fragment that reflects differences in nucleotide sequences between individual organisms. Molecular markers are primarily used for a few traits, such as plant disease resistance and quality traits, and their effectiveness is generally good. The genetic mechanism of yield traits is complex, with low gene contribution to traits, abundant allelic variation but difficulty in evaluation, and a large localization interval. Linked markers are sensitive to genetic background and difficult to apply within the context of universal breeding resources, resulting in low breeding selection efficiency. In recent years, the development and utilization of functional molecular markers (hereinafter referred to as functional markers) have achieved good results in overcoming these deficiencies. Functional molecular markers are molecular markers located on genes that can detect specific genetic variations on genes that cause phenotypic variation. They co-segregate with genes, eliminating the need to consider the influence of genetic background during molecular marker selection. This offers irreplaceable advantages in plant molecular marker-assisted selection.

[0004] Wheat is the world's second-largest staple food and my country's third-largest. Continuously developing new wheat varieties is crucial for ensuring independent and reliable seed sources and ensuring national food security. The superior traits of a variety are the result of the interaction between functional genes and the environment. Therefore, discovering functional genes, utilizing superior allelic variants to improve crops, enhancing the technological content of breeding, and mastering key core genetic resources are not only scientific research tasks but also crucial to the food security of hundreds of millions of Chinese people.

[0005] The ear is a crucial photosynthetic organ in the later stages of wheat growth and development, and it also determines the total amount of dry matter it can hold. Large ears provide space for a greater number of grains per ear and larger grains. Large-ear varieties generally have larger flag leaves, more grains per ear, a higher 1000-grain weight, and sturdy stems. Large-ear varieties offer advantages such as high photosynthetic efficiency and a plant shape that facilitates ventilation. Therefore, ear length is an important high-yield trait in wheat breeding, especially since the base number of ears per mu in current breeding is relatively stable. Therefore, increasing the number of grains per ear and 1000-grain weight is the focus of current high-yield breeding. Large-ear varieties, in particular, are prone to achieving super-high yields when planted on high-fertility soils.

[0006] There are many genetic studies related to ear length, most of which are still at the preliminary positioning stage. A large number of molecular markers linked to it have been detected. These loci are almost distributed on all 21 chromosomes of wheat, among which the most QTLs are located on homologous groups 2, 4 and 7.

[0007] Using a population of recombinant inbred lines constructed from Yumai 8679 and Jing 411, stable QTLs controlling spike length were mapped on chromosomes 1B, 2B, 2D, 5A, and 7B, accounting for 4.88% to 7.96% of the phenotypic variation. QTL analysis using recombinant inbred lines constructed from hard white wheat WCB414 and hard red wheat HRSW revealed six QTLs controlling spike length, located on chromosomes 1A, 2B, 2D, 4A, and 4B, explaining 5.1% to 17.8% of the phenotypic variation. QTL mapping for spike length using recombinant inbred lines constructed from wheat lines '20828' and Chuannong 16 identified five novel QTLs (QSl.sau.2D.1, QSl.sau.2D.2, QSl.sau.4B.1, QSl.sau.4B.2, and QSl.sau.5B). Due to the limitations of the accuracy of initial positioning, the genetic distance between these molecular markers and traits is relatively far, which is not conducive to further breeding utilization.

[0008] To further obtain markers more closely linked to target traits, current research focuses on fine-mapping of target loci. Fine-mapping of a few loci has been completed, with the loci located within smaller genetic intervals. The QTLs on chromosome 2D in the Chinese local variety Wangshuibai were finely mapped, and both genes were identified as semi-dominant. Using an F2 population derived from a near-isogenic line, the two genes were fine-mapped to 0.9 cM and 0.2 cM, respectively. Although these loci have been further accurately mapped, no functional genes have been cloned, and the developed molecular markers remain linkage markers, lacking the advantages of functional markers.

[0009] Ppd encodes a pseudo-response regulator, homologous to Arabidopsis PRR37, involved in plant rhythmic regulation and photoperiod response. In wheat, this gene is closely associated with flowering traits under both long-day and short-day conditions. Ppd's partial homologs, TaPpd-2A and TaPpd-2B, in the wheat A and B subgenomes are less well-studied, and both copy number variation and sequence variation may be involved in photoperiod regulation. TaPpd-2D on the D subgenome exhibits extensive allelic variation, with five potential key sequence variants identified to date: 1) a 2089-bp deletion in the 5' noncoding region, resulting in abnormal gene expression; 2) a transposon insertion detected in the first intron; 3) a 5-bp deletion in exon 7, resulting in a frameshift mutation; 4) a single nucleotide polymorphism in exon 7, altering a single amino acid in the CCT domain; and 5) a 16-bp deletion in exon 7, altering the amino acid composition of the CCT domain at the carbon terminus. The 2089-bp deletion / insertion in the first region is generally believed to have the greatest impact on plant responses to photoperiod. Summary of the Invention

[0010] The present invention provides a primer, method and application for detecting polymorphisms in the promoter sequence region of the wheat TaPpd-2D gene. The two sequence polymorphisms are completely co-segregated with the ear length, which can realize direct selection of ear length in breeding.

[0011] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0012] A primer for detecting polymorphism in the promoter region of wheat TaPpd-2D gene, characterized in that the nucleotide sequence of the primer is as follows:

[0013] TaPPD2D24bpIndelF:

[0014] 5'-CACTCCGTTTTTATTTACTTCCCAGATCAG-3'

[0015] TaPPD2D24bpIndelR:

[0016] 5'-GTGAATCTATGCTATTGCTTAGGTATTGTAGATG-3'

[0017] TaPPD2D-MF1: 5'-CTCCTCTAAATTATGAAATCCCGACT-3'

[0018] TaPPD2D-MR1: 5'-CAACGCCCAAATTTAGTACCTCC-3'.

[0019] The present invention also provides a method for detecting polymorphism in the promoter region of the wheat TaPpd-2D gene, comprising the following steps:

[0020] (1) Design primers as follows:

[0021] TaPPD2D24bpIndelF:

[0022] 5'-CACTCCGTTTTTATTTACTTCCCAGATCAG-3'

[0023] TaPPD2D24bpIndelR:

[0024] 5'-GTGAATCTATGCTATTGCTTAGGTATTGTAGATG-3'

[0025] TaPPD2D-MF1: 5'-CTCCTCTAAATTATGAAATCCCGACT-3'

[0026] TaPPD2D-MR1: 5'-CAACGCCCAAATTTAGTACCTCC-3';

[0027] (2) PCR reaction system is:

[0028]

[0029] Among them, the DNA template is common wheat DNA,

[0030] (3) PCR reaction;

[0031] (4) Gel electrophoresis detection.

[0032] Furthermore, preferably, the Tks Gflex DNA Polymerase is a Taq enzyme, and the Taq enzyme must have the ability to amplify a 2-kb length sequence.

[0033] Furthermore, preferably, the PCR reaction is specifically as follows: pre-denaturation at 94°C for 1 minute; then 35 cycles, each cycle of denaturation at 98°C for 10 seconds, annealing at 60°C for 15 seconds, and extension at 68°C for 1 minute; and finally extension at 68°C for 5 minutes.

[0034] Further, preferably: the PCR reaction is specifically as follows: the gel electrophoresis detection is specifically 3% agarose gel electrophoresis for detection, 140V voltage electrophoresis for 30 minutes, and band display under ultraviolet light. If the 2089bp deletion and the 24bp deletion are not detected in the test results, the wheat is a long-spike type; if the 2089bp deletion is detected and the 24bp deletion is not detected, the wheat is a short-spike type; if the above two types are not detected, it is impossible to determine whether it is long-spike wheat or short-spike wheat.

[0035] The primers of the present invention are used in assisting the screening of wheat materials with longer spike length.

[0036] Furthermore, preferably, the wheat is common hexaploid wheat.

[0037] Beneficial effects of the present invention:

[0038] The present invention utilizes the causal relationship between two genetic variations of the TaPpd-2D gene and the ear length to design primers of the present invention to detect the absence of the TaPpd-2D gene. If bands of 2511bp and 134bp are detected in the wheat genomic DNA to be tested, that is, there is a nucleotide sequence of 2089bp and there is a 24bp deletion, then the wheat material to be tested has a longer ear length. If bands of 422bp and 158bp are detected in the wheat genomic DNA to be tested, it indicates that there is a 2089bp deletion and a 24bp insertion in the material, and the material has a shorter ear length.

[0039] The method of the present invention has the characteristics of accurate, rapid and simple results, is suitable for the selection of different generations of breeding, can shorten the breeding cycle, accelerate the breeding selection speed, reduce the screening cost, and has the advantages of simple operation, low cost and medium to high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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 or the description of the prior art. 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.

[0041] Figure 1 Schematic diagram of the sequence variation of TaPpd-2D gene between Yanda 1817 (YD) and Beinong 6 (BN);

[0042] Figure 2 This is the result map of QTL mapping for ear length;

[0043] Figure 3 Schematic diagram of TaPpd-2D molecular marker detection results. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions in this embodiment with reference to the accompanying drawings. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0045] The gene tested is TaPpd-2D, whose gene ID is TraesCS2D02G079600 in the IWGSC database (https: / / www.wheatgenome.org / ). The genetic effect of the key gene variant on spike length was determined after analysis of a high-generation population constructed from the offspring of the Chinese local wheat Yanda 1817 and Beinong No. 6. The key variant was discovered after amplifying the TaPpd-2D gene sequence in the Chinese local wheat Yanda 1817 and Beinong No. 6 and performing sequence comparison.

[0046] The TaPpd-2D gene sequences amplified from Yanda 1817 and Beinong 6 were 10068 bp and 7995 bp, respectively. The sequences are shown in Sequence Table 1 and Sequence Table 2, respectively. The TaPpd-2D gene from Yanda 1817 was used as the reference sequence. The gene consists of 10 exons and 9 introns. The deoxyribonucleotides from positions 1 to 3102 are the 5' upstream sequence of the gene, the deoxyribonucleotides from positions 3103 to 3250 are the first exon, the deoxyribonucleotides from positions 3251 to 3338 are the first intron, the deoxyribonucleotides from positions 3339 to 3616 are the second exon, and the deoxyribonucleotides from positions 3606 to 3608 are the first intron. The amino acid composition is ATG, which is the start codon of the gene, the deoxyribonucleotides from positions 3617 to 3723 are the second intron, the deoxyribonucleotides from positions 3724 to 3914 are the third exon, the deoxyribonucleotides from positions 3915 to 4021 are the third intron, the deoxyribonucleotides from positions 4022 to 4186 are the fourth exon, the deoxyribonucleotides from positions 4187 to 4294 are the fourth intron, and the deoxyribonucleotides from positions 4295 to 4428 are The fifth exon, the deoxyribonucleotides from positions 4429 to 4623 are the fifth intron, the deoxyribonucleotides from positions 4624 to 4779 are the sixth exon, the deoxyribonucleotides from positions 4780 to 5224 are the sixth intron, the deoxyribonucleotides from positions 5225 to 5408 are the seventh exon, the deoxyribonucleotides from positions 5409 to 5514 are the seventh intron, the deoxyribonucleotides from positions 5515 to 5932 are the eighth exon, and the deoxyribonucleotides from positions 5933 to 5937 are the eighth exon. The deoxyribonucleotides from positions 6015 to 6672 are the eighth intron, the deoxyribonucleotides from positions 6673 to 6782 are the ninth intron, and the deoxyribonucleotides from positions 6783 to 7322 are the tenth exon. The deoxyribonucleotides from positions 6882 to 6884 form a TAG, which is the termination codon of the gene. The deoxyribonucleotides from positions 7323 to 10068 are the 3' downstream sequence of the gene.

[0047] The gene sequence identity of TaPpd-2D between Beinong No. 6 and Yanda 1817 was 76.66%, with the following five sequence variations: 1) an insertion of 24 nucleotides after deoxyribonucleotide position 902, located in the 5' upstream region of the gene; 2) a complete deletion of the 2089 bp nucleotide sequence from position 1597 to 3685, resulting in the deletion of positions 1597 to 3102 corresponding to the 5' upstream region, positions 3103 to 3250 corresponding to the first exon, positions 3251 to 3338 corresponding to the first intron, positions 3339 to 3616 corresponding to the second exon, and positions 3360 to 3617 corresponding to the second intron. All deoxyribonucleotides from position 3617 to 3685 are missing, including the start codon; 3) a deoxyribonucleotide conversion occurs at position 5305, from thymine deoxyribonucleotide (T) to cytosine deoxyribonucleotide (C). This mutation is located in exon 7, and the triplet codon changes from CCT to CCC, but does not cause amino acid changes; 4) 8 of the 22 consecutive cytosine deoxyribonucleotides starting from position 8282 are deleted; 5) a deoxyribonucleotide transversion occurs at position 8590, from thymine deoxyribonucleotide (T) to adenine deoxyribonucleotide (A). The fourth and fifth mutations are both located in the 3' downstream sequence (see Appendix). Figure 1 ).

[0048] The purpose of the present invention is to detect the sequence variations at the first and second positions mentioned above. If the detection results show that a 24bp nucleotide sequence is inserted at the first position and a 2089bp nucleotide sequence exists at the second position, it indicates that the wheat is a candidate long-spike wheat.

[0049] The present invention detects the aforementioned mutations by configuring a PCR reaction system for polymerase chain reaction (PCR). The PCR reaction requires adding appropriate amounts of dNTPs, ddH2O, a tag enzyme, a reaction buffer corresponding to the tag enzyme, and a DNA template of the wheat material to be tested to an EP tube. Four primers are then added to the reaction. The tag enzyme used in the reaction must be capable of amplifying fragments larger than 2 kb.

[0050] The genomic DNA of the hexaploid wheat was amplified and tested in agarose gel electrophoresis with a concentration of 3%. A marker covering the range of 100-2500bp was used for comparison. The electrophoresis was performed at 140V for 30 minutes and the bands were displayed under ultraviolet light. The wheat materials with 134bp and 2511bp bands did not contain the 24bp nucleotide sequence in the first mutation, but contained the 2089bp nucleotide sequence in the second mutation. The wheat materials with 158bp and 422bp bands contained the 24bp nucleotide sequence in the first mutation, but lacked the 2089bp nucleotide sequence in the second mutation.

[0051] Another object of the present invention is to provide a method for genetically improving wheat spike length using molecular marker-assisted selection. The method provided by the present invention utilizes the causal relationship between two genetic variations in the promoter region of the TaPpd-2D gene and spike length. For example, if bands of 2511 bp and 134 bp are detected in the wheat genomic DNA to be tested, indicating the presence of a 2089 bp nucleotide sequence and a 23 bp deletion, the wheat material to be tested has a longer spike length. If bands of 422 bp and 158 bp are detected in the wheat genomic DNA to be tested, indicating the presence of a 2089 bp deletion and a 24 bp insertion, the material has a shorter spike length. This method is accurate, rapid, and simple, and is suitable for selection of different breeding generations. It can shorten the breeding cycle, accelerate breeding selection, reduce screening costs, and has the advantages of simple operation, low cost, and medium to high throughput.

[0052] Example 1: Discovery of genetic variation in the TaPpd-2D gene

[0053] The present invention uses Beinong No. 6 and Yanda 1817 as genetic materials, and conducts ear length phenotypic identification of the offspring recombinant inbred lines in 2012-2013. The long-ear family BY45 was screened out, and the ear length phenotype was 8.60±0.75cm. The ear length phenotype of the short-ear family BY210 was 9.63±0.46cm. BY45 was used as the recurrent parent and BY210 as the donor parent. After 5 backcrosses and two generations of self-pollination, a BC5F3 population containing 46 individual plants was obtained. It was found that Q21-10 (ear length 11.64±0.43cm in 2020) and Q11-8 (ear length 9.94±0.22cm in 2020), Q21-10 and Q11-8 were used to configure the secondary F2 population, DNA was extracted from 150 F2 plants and the ear length phenotype was examined. 30 extremely long-eared and 30 extremely short-eared plants were selected, and equal amounts of DNA were mixed. Genotyping was performed using a 660K wheat SNP chip, and 33% of the polymorphic SNPs were found to be located on chromosome 2D. Then, 10 polymorphic molecular markers on chromosome 2D were used to genotype the 150 plants and a genetic linkage map was constructed ( Figure 2 ), combined with the results of spike length phenotype identification, QTL positioning was performed, and the QTL was located between the molecular markers AX-109836946 / SNP48 / 2DSNP9 / Indel4 and 2DSNP41 / AX-111956072, with a genetic distance of 1.84 cM, corresponding to a minimum physical interval of 795.34-kb (SNP48-AX-111956072). The molecular markers SNP48 and AX-111956072 were used to detect recombinant plants in the F2 generation population containing 1920 plants, and four molecular markers were further developed. The identified genotypes were combined with the field spike length phenotypes to locate the QTL site between 2DIndel17 and 2DSNP59 (see Appendix). Figure 1 ), with an LOD value of 22.19, which could explain 53.46% of the phenotypic variation. There was only one gene in the corresponding interval, namely TaPpd-2D.

[0054] Primers were designed to amplify the full length of the TaPpd-2D gene in the parents, Yanda 1817 and Beinong 6. After sequence comparison, five sequence variations were found between the parents: 1) an insertion of 24 nucleotides after deoxyribonucleotide position 902, located in the 5' upstream region of the gene; 2) a complete deletion of the 2089 bp nucleotide sequence from position 1597 to 3685, resulting in the deletion of positions 1597 to 3102 corresponding to the 5' upstream region, positions 3103 to 3250 corresponding to the first exon, positions 3251 to 3338 corresponding to the first intron, positions 3339 to 3616 corresponding to the second exon, and positions 3360 to 3616 corresponding to the second intron. The corresponding deoxyribonucleotides from positions 3617 to 3685 are completely deleted, including the start codon. 3) A deoxyribonucleotide transition occurs at position 5305, from thymine (T) to cytosine (C). This mutation is located in exon 7, and the triplet codon changes from CCT to CCC, but does not result in an amino acid change. 4) Eight of the 22 consecutive cytosine deoxyribonucleotides starting at position 8282 are deleted. 5) A deoxyribonucleotide transversion occurs at position 8590, from thymine (T) to adenine (A). The fourth and fifth mutations are both located in the 3' downstream sequence.

[0055] Example 2 Establishment of the relationship between TaPpd-2D and spike length

[0056] Molecular markers were developed for the first and second mutations, including four primers: TaPPD24bpIndelF, TaPPD24bpIndelR, TaPPD-MF1, and TaPPD-MR1. The primer sequences are:

[0057] TaPPD24bpIndel:

[0058] 5'-CACTCCGTTTTTATTTACTTCCCAGATCAG-3';

[0059] TaPPD24bpIndelR:

[0060] 5'-GTGAATCTATGCTATTGCTTAGGTATTGTAGATG-3'; TaPPD-MF1: 5'-CTCCTCTAAATTATGAAATCCCGACT-3' and TaPPD-MR1: 5'-CAACGCCCAAATTTAGTACCTCC-3'.

[0061] The molecular marker was used to identify the recombinant strain in Example 1. The identification PCR reaction system was:

[0062]

[0063] The PCR reaction program is: 94℃ initial denaturation for 1 minute; then 35 cycles, each cycle of denaturation at 98℃ for 10 seconds, annealing at 60℃ for 15 seconds, and extension at 68℃ for 1 minute; and finally extension at 68℃ for 5 minutes. After completion, the reaction was detected by electrophoresis on a 3% agarose gel at 140V for 30 minutes. The bands were visualized under UV light. The results are as follows: Figure 3 As shown, Figure 3 Middle well 1: 1Kb DNA marker, 2-5: short-spike wheat materials; 6-9: long-spike wheat materials; 10: 50bp DNA marker.

[0064] If 134bp and 2511bp bands are detected in the wheat to be tested, it means that the wheat material does not contain the 24bp nucleotide sequence in the first mutation, but contains the 2089bp nucleotide sequence in the second mutation, and is recorded as genotype AA; the wheat material in which 158bp and 422bp bands are detected contains the 24bp nucleotide sequence in the first mutation, but lacks the 2089bp nucleotide sequence in the second mutation, and is recorded as genotype BB, Appendix 1.

[0065] Table 1 Relationship between TaPpd-2D molecular marker detection results and spike length phenotype results

[0066]

[0067] Combined with the genotype identification results of the recombinant plants using other molecular markers in Example 1, a one-way analysis of variance showed that only the TaPpd-2D allele had a causal relationship with the spike length phenotype. The wheat material TaPpd-2D gene was identified as a long spike type because it did not contain the 24bp nucleotide sequence in the first mutation, but contained the 2089bp nucleotide sequence in the second mutation (i.e., 134bp and 2511bp bands were identified).

[0068] In this example, a 2089 bp deletion and a 24 bp insertion were found to co-segregate in the tested materials, but this does not mean that this phenomenon also exists in other wheat materials.

[0069] Although the embodiments of the present invention have been described above, it will be apparent to those skilled in the art that modifications and substitutions made without departing from the principles and spirit of the present invention are intended to fall within the scope of protection claimed by the present invention.

Claims

1. A method for determining wheat spike type by detecting polymorphism in the promoter region of the wheat TaPpd-2D gene, characterized in that: The following steps are involved: (1) Design and synthesize primers: TaPPD2D24bpIndelF: 5'-CACTCCGTTTTTATTTACTTCCCAGATCAG-3' TaPPD2D24bpIndelR: 5'-GTGAATCTATGCTATTGCTTAGGTATTGTAGATG-3' TaPPD2D-MF1: 5'-CTCCTCTAAATTATGAAATCCCGACT-3' TaPPD2D-MR1: 5'-CAACGCCCAAATTTAGTACCTCC-3'; (2) Perform PCR reaction. The PCR reaction system is: ； The DNA templates were DNA from the recombinant inbred lines of Yanda 1817 and Beinong 6; (3) Gel electrophoresis detection: the gel electrophoresis detection is specifically performed using 3% agarose gel electrophoresis, 140V voltage for 30 minutes, and band display under ultraviolet light. If the band sizes in the lanes are 2511bp and 134bp, the wheat is a long-spike type; if the band sizes are 422bp and 158bp, the wheat is a short-spike type. If the above two types are not detected, it is impossible to determine whether it is long-spike wheat or short-spike wheat.

2. The method according to claim 1, wherein: The Tks Gflex DNA Polymerase is a Taq enzyme, which needs to be able to amplify a 2-kb length sequence.

3. The method according to claim 1, wherein: The PCR reaction was specifically as follows: pre-denaturation at 94°C for 1 minute; then 35 cycles, each cycle consisting of denaturation at 98°C for 10 seconds, annealing at 60°C for 15 seconds, and extension at 68°C for 1 minute; and finally extension at 68°C for 5 minutes.

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