Primer combinations and methods for isolating opie transposon insertion site genomic sequences
By designing specific primer combinations and adapter primers, combined with PCR amplification and NGS sequencing, the problems of low efficiency and high cost in existing maize genome Opie transposon detection technologies have been solved, achieving high-throughput and low-cost Opie transposon site detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SANYA NATIONAL INSTITUTE OF SOUTHERN BREEDING CHINESE ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2023-12-18
- Publication Date
- 2026-07-14
AI Technical Summary
Existing technologies for detecting Opie transposon insertion sites in the maize genome suffer from low marker counts, high costs, and low efficiency, making it difficult to achieve high-throughput and low-cost high-density genotyping.
Specific primer combinations were designed, including Opie1-inner, Opie1-outer, and Opie2-inner, and adapter primers were combined. The genomic sequence of the Opie transposon insertion site was isolated and enriched by PCR amplification and NGS sequencing. The DNA fragments were processed by NlaIII digestion and T4 DNA ligase, and the products were recovered using an Axygen gel recovery kit and Beckman magnetic beads.
It achieves efficient enrichment of almost all Opie transposon sites on the maize genome, reduces library preparation and sequencing costs, improves detection efficiency, and is suitable for high-throughput detection.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology, and specifically relates to a primer combination and method for isolating the genomic sequence of the Opie transposon insertion site. Background Technology
[0002] Transposons are widespread in plants and animals, playing a crucial role in species evolution, domestication, and the formation of new varieties. A jumping gene, or transposon, is a DNA sequence that can replicate independently in situ or break off, circularize, and insert into another site, regulating subsequent genes. For example, transposons have a wide range of applications. The Sleeping Beauty Transposon (SB-Tn) system—a gene therapy technique that avoids the drawbacks of viral gene transfer technology—can correct the gene defect causing sickle cell anemia (SCD) in the laboratory. Simultaneously, transposons are extensively used to create mutant libraries, generate artificial variations, and create new crop varieties. They are also used to create in vitro and in vivo DNA cutting tools for producing fragmented DNA.
[0003] Currently, retrotransposons with distinct sequence characteristics have been discovered in various plants, playing crucial roles in speciation, crop domestication, and other processes. Opie is the largest LTR transposon superfamily discovered in maize. Its length varies depending on the elements it contains, ranging from approximately 8000-11000 bp. The elements it contains include Gag, Integrase, Protease, Ribonuclease, and reverse transcriptase. A key characteristic of this transposon is its near-uniform distribution across the entire genome. The copy number of Opie is generally consistent across different maize varieties, exhibiting good coverage. The libraries generated by this method are high-density molecular markers or liquid-phase microarrays, making them highly efficient genotyping tools with significant application potential.
[0004] In agricultural breeding, applications such as resource material background analysis, breeding authenticity verification, whole-genome locus scanning of breeding populations, and screening for superior haplotypes can all be achieved using genomic sequence markers obtained by this method. However, existing detection methods, including SSR markers, indel markers, multiplex PCR, and kasp markers, suffer from low marker counts (generally less than 1000); while resequencing to determine random sequences results in low efficiency and high cost. These methods limit the development of this field, and there is an urgent need to establish a method with a marker count of 1000 or more. 4 It is a detection method with clearly defined capture sites, high throughput, and low cost.
[0005] The maize genome is 4Gb in size, containing 40,000 genes, with an average distribution of approximately one gene every 10-100kb. When we have 100,000 high-density markers, there will be an associated marker near each gene. Theoretically, 10 sets of markers in the "10,000" range can yield "100,000" markers. Calculations show that 10 different types of 10 4 By cumulatively stacking the copied transposons, a total of 10 can be achieved. 5 The total copy number reaches the ideal state of genomic marker site saturation. Therefore, each transposon in the "tens of thousands" range has valuable significance.
[0006] Opie is a type of high-copy transposon with a unique sequence and insertion site in the maize genome. It appears frequently in the coding region. Developing novel marker detection methods and technologies using opie has great potential for scientific research and industrial applications. Summary of the Invention
[0007] The purpose of this invention is to provide a primer combination and method for isolating the genomic sequence of the Opie transposon insertion site.
[0008] To achieve the objective of this invention, in a first aspect, this invention provides specific primers for detecting Opie transposon sites in the maize genome, the specific primers comprising Opie1-inner, Opie1-outer and Opie2-inner, the sequences of which are shown in SEQ ID NO:1-3, respectively.
[0009] According to the Opie transposon sequence published in the appendix of the 2021 scientific paper "De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes" (https: / / www.science.org / doi / full / 10.1126 / science.abg5289), its serial number is opie_AC185480_1137#L. TR、opie_AC187149_1780#LTR、opie_AC187207_1792#LTR、opie_AC188002_2029#LTR、opie_AC196469_5133#LT R. opie_AC197084_5382#LTR, opie_AC197201_5474#LTR, opie_AC197691_5727#LTR, opie_AC198173_5898#LTR, opie_AC198924_6206#LTR, opie_AC201793_7083#LTR, opie_AC202020_7258#LTR, opie_AC202033_7274#LTR, op ie_AC210610_10772#LTR, opie_AC211653_11340#LTR, opie_AC214122_12495#LTR, opie_AC217577_13524#LTR.
[0010] In a second aspect, the present invention provides primer combinations for isolating the genomic sequence of the Opie transposon insertion site, including specific primers with sequences as shown in SEQ ID NO:1-3 and adapter primers with sequences as shown in SEQ ID NO:6.
[0011] Thirdly, the present invention provides a kit for isolating the genomic sequence of the Opie transposon insertion site, comprising the above-mentioned primer combination and adapter 1 and adapter 2 with sequences as shown in SEQ ID NO:4 and 5, respectively.
[0012] Fourthly, the present invention provides the application of specific primers with sequences as shown in SEQ ID NO:1-3, combinations of the above primers, or kits in high-throughput detection of Opie transposon whole genome loci based on NGS sequencing.
[0013] Fifthly, the present invention provides a method for isolating the genomic sequence of the Opie transposon insertion site, comprising the following steps:
[0014] a. Extract maize genomic DNA, break the DNA with a DNA fragmentation enzyme, and obtain DNA fragments between 250bp and 5kb.
[0015] b. The DNA fragment obtained in step a is ligated with adapter 1 and adapter 2 using DNA ligase to obtain a DNA fragment with adapters, and then recovered using a kit (such as the Axygen Gel Recovery Kit AP-GX-250).
[0016] c. Perform PCR amplification on the recovered product from step b using Opie2-inner, Opie1-outer, Opie1-inner and adapter primers. Perform two rounds of PCR, first using Opie1-outer and adapter primers for 3-5 cycles, then adding Opie1-inner, Opie2-inner and adapter primers for 10-20 cycles.
[0017] d. The amplification product from step c is recovered, quantified, and sequenced using an NGS sequencer. The sequencing results are then compared with the maize genome sequence to obtain the corresponding loci.
[0018] Opie2-inner, Opie1-outer, Opie1-inner, adapter primers, adapter 1, and adapter 2 were derived from the aforementioned kit.
[0019] Furthermore, the cleavage enzyme mentioned in step a is a DNA enzyme that recognizes 4-6 sticky ends of the sequence, preferably an NlaIII and NspI iso-suppressive endonuclease to increase cleavage efficiency; the cleavage condition is incubation at 37°C for 1-5 hours.
[0020] Furthermore, the DNA ligase mentioned in step b is T4 DNA ligase, and the ligation conditions are incubation at 25°C for 1-3 hours.
[0021] Furthermore, in step b, the ratio of adapter 1, adapter 2, and DNA fragment is 10 pM: 10 pM: 50 ng.
[0022] Further, the amplification conditions in step c are as follows: 98℃ for 2 minutes, first amplify with Opie1-outer and adapter primers for 5 cycles, with conditions of 98℃ for 10 seconds, 62℃ for 20 seconds, and 72℃ for 1 minute; then add Opie1-inner, Opie2-inner and adapter primers for 20 cycles, with conditions of 98℃ for 10 seconds, 62℃ for 20 seconds, and 72℃ for 1 minute; finally, amplify at 72℃ for 8 minutes.
[0023] Furthermore, in step d, magnetic beads (such as Beckman's AMPure XP beads) are used to recover the amplification products.
[0024] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:
[0025] Currently, high copy number transposon sites are generally obtained through resequencing. Existing OPIE transposon site identification methods can only obtain whole-genome information through resequencing, which is non-specific and random. Sequence filtering then removes 99% of useless sequences, resulting in high costs and low efficiency for both library construction and sequencing. In contrast, the method provided by this invention can enrich almost all sites on the genome with extremely high efficiency. Furthermore, due to the tag sequences on the primers, this method helps to separate sites from large-scale data after mixed sequencing, making it suitable for high-throughput detection. Moreover, the mixed PCR and sequencing significantly reduces the cost of library construction and sequencing. The specific primer design ensures that the OPIE end length is a specific sequence of 150-350 bp during subsequent analysis, and also ensures that 150-350 bp flanking genome sequences are obtained during subsequent second-generation NGS sequencing, representing a clever and efficient novel design. Attached Figure Description
[0026] Figure 1 This invention provides a common sequence for the 3' end alignment analysis of different Opie family members in the maize genome.
[0027] Figure 2 This invention provides a common sequence for 5' end alignment analysis of different Opie family members in the maize genome.
[0028] Figure 3 This invention is based on the Illumina sequencing platform and the sequence structure of the sequencing sample, wherein the sequences labeled Opie1-inner and Opie2-inner are the 5' and 3' of the Opie transposon, respectively.
[0029] Figure 4 This is the quality control result of the Opie transposon NGS library construction for this invention.
[0030] Figure 5 This is the distribution of the Opie transposon in the maize DH-1 genome as measured in this invention.
[0031] Figure 6 This is the distribution of the Opie transposon of this invention in the maize K17 genome as measured in practice.
[0032] Figure 7 This is the distribution of the Opie transposon in the maize Zheng58 genome as measured in this invention. Detailed Implementation
[0033] This invention aims to provide a method for capturing more than 10 sites at the whole genome level. 5 Primers and methods with fixed capture sites, high throughput, high efficiency, and low cost.
[0034] The present invention also provides a method for detecting the insertion site of an Opie transposon.
[0035] The present invention adopts the following technical solution:
[0036] This invention provides specific primers for isolating genomic sequences of Opie transposon insertion sites. The specific primers include Opie1-inner, Opie1-outer, and Opie2-inner, wherein Opie1-inner, Opie1-outer, and Opie2-inner satisfy one of the following conditions: a, b, c, or d.
[0037] a. Conserved common sequences located flanking transposons in the genome sequence;
[0038] b. The distance from the inner base to the last base of the transposon is approximately 70-300 bp, and the distance from the outer base to the last base of the transposon is approximately 300-600 bp;
[0039] c. Carrying a 4-8 bp tag sequence to distinguish samples;
[0040] d. Contains anchor sequences for NGS deep sequencing.
[0041] The genome is a maize genome, Opie1-inner and Opie1-outer are sequences shown in SEQ ID NO:1-2, and Opie2-inner is a sequence shown in SEQ ID NO:3.
[0042] Furthermore, the specific primer combination is Opie1-inner, Opie1-outer, Opie2-inner, and adapter primer (SEQ ID NO:6).
[0043] This invention also provides a method for isolating the genomic sequence of Opie transposon insertion sites, the specific steps of which are as follows:
[0044] a. Take maize genomic DNA, break it with DNA-breaking enzyme to obtain the broken product, the broken DNA fragments are between 250bp and 5kb;
[0045] b. The fragmented product obtained in step a is ligated with synthetic adapter 1 and adapter 2 and DNA ligase to obtain DNA fragments with adapters.
[0046] c. Recover the product obtained in step b using a kit;
[0047] d. The recovered product from step c is subjected to PCR amplification using the Opie2-inner, Opie1-outer, and Opie1-inner and adapter PCR primers. To ensure sequence specificity, two rounds of PCR are designed. First, the outer and adapter are used for 3-5 cycles of amplification, and then the inner and adapter primers are added for 10-20 cycles of amplification.
[0048] e. The amplification product from step d is recovered, quantified, and sequenced using an NGS sequencer. The sequencing results are then compared with the genome sequence to obtain the corresponding loci.
[0049] In step a, the cleavage enzyme is NlaIII or other DNases that recognize 4-6 sticky ends of the sequence, and the cleavage conditions are 37°C for 1-5 hours.
[0050] In step b, connector 1 is adapter1, whose sequence is sequence 3 in the sequence list, and connector 2 is adapter2, whose sequence is sequence 4 in the sequence list.
[0051] The ligase used in step b is T4 DNA ligase, and the ligation conditions are 25℃ for 1-3 hours; the kit used in step c is the Axygen gel extraction kit AP-GX-250; the PCR amplification conditions in step d are: 98℃ pre-denaturation for 2 minutes, adding outer and adapter primers for the first 3-5 cycles, adding inner and adapter primers for the next 10-20 cycles (98℃ for 10 seconds, 62℃ for 20 seconds, 72℃ for 1 minute), and extension at 72℃ for 8 minutes.
[0052] The present invention also provides the application of the primers or the method described therein in isolating the genomic sequence of the Opie transposon insertion site.
[0053] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.
[0054] Example 1: Design and Testing of Opie-Specific Primers
[0055] Based on the Opie sequences published in the appendix of the 2021 scientific paper "De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes" (https: / / www.science.org / doi / full / 10.1126 / science.abg5289), a scan and alignment were performed on the maize B73 genome. Based on the common sequence characteristics of these Opies, the conserved sequences at the 3' and 5' ends were extracted. The alignment results are shown below. Figure 1 , Figure 2 Ultimately, NGS sequencing technology will be used to obtain all Opie loci at the whole genome level. Currently, NGS read lengths are generally 150 bp at one end and 300-700 bp for both ends, which are considered short read lengths. To ensure that adjacent genomic sequences larger than 100 bp can be obtained within this read length range, this invention designs 5' or 3' end primers on the Opie loci with a distance of approximately 70 bp from their respective ends, estimating the obtained genomic sequences to be between 70-700 bp. We designed Opie1-inner, Opie1-outer, and Opie2-inner specific primers for the 5' and 3' ends of the Opie loci, as shown in Table 1, to anchor the transposon end-specific sequences. In addition, corresponding adapter sequences Adaptor1 and Adaptor2, and an adapter PCR primer adapterprimer were also designed. The adaptor and primer match the anchor sequence and sequencing primer sequence used in NGS next-generation high-throughput sequencing. The primer can anneal with the same sequence of the adaptor to complete PCR amplification and obtain the product for sequencing.
[0056] This invention utilizes NlaIII (NEB, Cat. No. R0125L) to cleave genomic fragments and load specific adapters. NlaIII is a key feature of this invention; using this restriction site (4 bp) allows for the acquisition of numerous cleavage sites from the chromosome, facilitating the capture of Opie sequences. Highly specific adapter sequences, adapter1 and adapter2, were also designed. adapter1 and adapter2 were subjected to high-temperature denaturation followed by annealing at 95°C for 2 minutes. After mixing adapter1 and adapter2, the mixture was slowly cooled to room temperature.
[0057] The specific primers, adapters, and adapter primer sequences used for detecting Opie sites across the entire genome are shown in Table 1.
[0058] Table 1. Specific primers, adapters, and adapter primer sequences used for detecting the Opie site in the whole genome.
[0059]
[0060]
[0061] This invention is based on the Illumina sequencing platform and the sequence structure of the sequencing sample, wherein the sequences labeled Opie1-inner and Opie2-inner are the 5' and 3' of the Opie transposon, respectively. Figure 3 ).
[0062] Example 2: NGS sequencing method for enriching Opie sites in the maize genome
[0063] Select K17, Zheng58, and DH-1 corn samples and follow these steps:
[0064] (1) Take 50 ng of maize genomic DNA and break it with (NEB, Cat. No R0125L) at 37℃ for 5 hours to obtain the broken product. The DNA fragments obtained are between 250 bp and 5 kb.
[0065] (2) Add 10 pM each of Adaptor1 and Adaptor2 and 1 μl of T4 DNA ligase to the fragmentation product, react at 25°C for 3 hours to ligate, and obtain DNA fragments with adapters; in this step, the ratio of adapter to fragmentation product is 10 pM: 50 ng.
[0066] (3) The PCR product was recovered using a purification kit.
[0067] (4) Add 10 pM Opie1-outer, 10 pM adaptor primer and 1 μg of the recovered product from step (3) to the system and perform PCR. The amplification conditions are 98℃ pre-denaturation for 2 minutes, 5 cycles (98℃ for 10 seconds, 62℃ for 20 seconds, 72℃ for 1 minute), then add 5 pM Opie1inner and Opie2inner and continue PCR. The amplification conditions are 20 cycles (98℃ for 10 seconds, 62℃ for 20 seconds, 72℃ for 1 minute) and 72℃ extension for 8 minutes.
[0068] (5) The product was recovered by equal volume using Beckman's AMPure XP beads.
[0069] (6) NGS novaseq sequencing, library quality control results are shown in […]. Figure 4 .
[0070] (7) According to the sequencing analysis results, the number of effective sequences refers to the genome sequence containing the Opie sequence and the wing (the number of effective sequences on each chromosome and their proportions are shown in Table 2).
[0071] Table 2 shows the number of Opie loci on each chromosome of the genome of the three maize materials detected by the method of the present invention.
[0072]
[0073]
[0074] The distribution of Opie transposons in the maize DH-1 genome was measured as follows: Figure 5 The distribution of Opie transposons in the maize K17 genome was measured as follows: Figure 6 The distribution of Opie transposons in the maize Zheng 58 genome was measured as follows: Figure 7 .
[0075] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. Application of specific primers for detecting Opie transposon sites in the maize genome in high-throughput detection of whole-genome Opie transposon sites in maize based on NGS sequencing; The specific primers include Opie1-inner, Opie1-outer, and Opie2-inner, and their sequences are shown in SEQ ID NO:1-3, respectively.
2. Application of primer combinations for isolating genomic sequences of Opie transposon insertion sites in high-throughput detection of whole-genome Opie transposon sites in maize based on NGS sequencing; The primer combination includes specific primers for detecting Opie transposon sites in the maize genome and adapter primers with sequences as shown in SEQ ID NO:6; The specific primers include Opie1-inner, Opie1-outer, and Opie2-inner, and their sequences are shown in SEQ ID NO:1-3, respectively.
3. Application of the kit for isolating the genomic sequence of Opie transposon insertion sites in high-throughput detection of whole-genome Opie transposon sites in maize based on NGS sequencing; The kit includes a primer combination for isolating the genomic sequence of the Opie transposon insertion site, and adapter 1 and adapter 2 with sequences as shown in SEQ ID NO:4 and 5, respectively. The primer combination includes specific primers for detecting Opie transposon sites in the maize genome and adapter primers with sequences as shown in SEQ ID NO:6; The specific primers include Opie1-inner, Opie1-outer, and Opie2-inner, and their sequences are shown in SEQ ID NO:1-3, respectively.
4. A method for isolating the genomic sequence of the Opie transposon insertion site, characterized in that, Includes the following steps: a. Extract maize genomic DNA, break the DNA with a DNA-breaking enzyme, and obtain DNA fragments between 250 bp and 5 kb; b. The DNA fragment obtained in step a is ligated with adapter 1 and adapter 2 using DNA ligase to obtain a DNA fragment with adapters, which is then recovered using a kit. c. Perform PCR amplification on the recovered product from step b using Opie2-inner, Opie1-outer, Opie1-inner and adapter primers. Perform two rounds of PCR, first using Opie1-outer and adapter primers for 5 cycles, then adding Opie1-inner and Opie2-inner for 5 cycles. d. The amplification product from step c is recovered, quantified, and sequenced using an NGS sequencer. The sequencing results are then compared with the maize genome sequence to obtain the corresponding loci. Among them, Opie2-inner, Opie1-outer, Opie1-inner, adapter primer, adapter 1 and adapter 2 are from the kit used to isolate the genomic sequence of the Opie transposon insertion site; The kit for isolating the genomic sequence of the Opie transposon insertion site includes a primer combination for isolating the genomic sequence of the Opie transposon insertion site, and adapter 1 and adapter 2 with sequences as shown in SEQ ID NO:4 and 5, respectively. The primer combination includes specific primers for detecting Opie transposon sites in the maize genome and adapter primers with sequences as shown in SEQ ID NO:6; The specific primers include Opie1-inner, Opie1-outer, and Opie2-inner, and their sequences are shown in SEQ ID NO:1-3, respectively.
5. The method according to claim 4, characterized in that, The cleavage enzyme mentioned in step a is a DNA enzyme that recognizes 4-6 sticky ends of the sequence; the cleavage condition is incubation at 37°C for 1-5 hours.
6. The method according to claim 5, characterized in that, The interrupting enzyme mentioned in step a is an NlaIII restriction endonuclease.
7. The method according to claim 4, characterized in that, The DNA ligase mentioned in step b is T4 DNA ligase, and the ligation conditions are incubation at 25°C for 1-3 hours.
8. The method according to claim 4, characterized in that, In step b, the ratio of adapter 1, adapter 2, and DNA fragment is 10 pM: 10 pM: 50 ng.
9. The method according to any one of claims 4-8, characterized in that, In step d, magnetic beads are used to recover the amplification products.