Key words

1 Introduction

Gene disruption is an important technique to investigate gene function. Targeted gene disruption via homologous recombination is applicable for a number of eukaryotes ranging from yeasts to animals. Because the frequency of homologous recombination is quite low in flowering plants, the success of gene targeting has been limited [1]. Recently, genome editing techniques such as TAL effector nuclease and CRISPR/Cas9 systems have been adapted for use in plants, making it easier to produce knockout mutants [2]. However, their application in large-scale chromosomal and genomic reconstructions has not yet been established [3]. In contrast, the maize transposon system using activator/dissociation (Ac/Ds), which was originally developed for insertional mutagenesis, is widely used to induce relatively large chromosomal deletions in combination with site-specific recombination systems (i.e., Cre/LoxP , Flp/Frt) that originated from non-plant organisms [4]. The Ac is a single-component system that carries the transposase (TPase) gene required for its own transposition [5]. A single-component system does not require genetic crossing for transposition. Because of the mobility of Ac, however, it is difficult to establish lines with a stable Ac position. To overcome this problem, the Ac/Ds two-component system was developed [6–9]. The Ac/Ds system comprises an Ac TPase gene derived from an autonomous Ac transposon, and a non-autonomous element, Ds, which is unable to transpose without the Ac TPase. To monitor transposition, it is preferable that Ds is inserted between a promoter and a resistance marker gene. If Ds transposition occurs, the gene will be activated, expressing resistance. The transposition can be fixed by crossing with a wild-type plant to remove the Ac TPase gene (Fig. 1). Although the Ac/Ds transposons themselves can induce chromosomal breakage and rearrangements [10], the combination with the Cre/LoxP system induces more efficient chromosomal rearrangements [11, 12] and can be used to generate artificial ring chromosomes [13]. However, because T-DNA insertion and Ds transposition occur mostly at random [14, 15], their inserted and transposed positions on chromosomes should be determined to estimate the scales of chromosomal reconstruction or the size and structure of artificial minichromosomes .

Fig. 1
figure 1

Schematic representation of Ds transposable element system (pDs-Lox, [12]). T-DNA insertions or Ds transpositions are selected by resistance to BASTA or hygromycin, respectively. Ds transposon is moved by a cut-and-paste process; thus, the Ds does not remain in its original location after being inserted into a new location

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Several methods have been developed to determine the genomic sequences flanking T-DNA or transposons . One of the common methods is inverse PCR [16]. Whereas standard PCR amplifies a DNA fragment between two inward primers, inverse PCR amplifies DNA sequences that are flanked with one end of a known DNA sequence. The individual restriction fragments are converted into circles by self-ligation, and the DNA can be used directly for PCR amplification with appropriate primer sets designed from the inserted DNA sequences. Some pretreatments are required before inverse PCR, such as restriction-enzyme digestion of genomic DNA followed by self-ligation. Another method to amplify unknown sequences adjacent to known DNA is thermal asymmetric interlaced (TAIL)-PCR [17, 18], which does not require any pretreatments. TAIL-PCR consists of two or three nested insertion-specific primers that anneal at relatively high temperatures during a series of reactions (Fig. 2), in combination with arbitrary degenerate (AD) primers that anneal at relatively low temperatures. AD primers are degenerate primers that anneal throughout the genome. The relative amplification efficiencies of specific products versus nonspecific products can be thermally controlled. From the primary to the tertiary reaction, the primers get closer to the edge of the inserted DNA (Fig. 3) (see Note 1 ). TAIL-PCR does not need special DNA manipulations before PCR, and the product specificity can be estimated by agarose gel electrophoresis. The TAIL-PCR reaction can be completed in only 1 day. Thus, this method is very effective for obtaining the genomic sequences flanking known DNA inserts such as T-DNA or Ds.

Fig. 2
figure 2

Sequences of 5′ and 3′ ends of Ds element. Grey arrows indicate inverted repeats. Ds element contains short inverted repeats at end, but internal sequence is identical

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Fig. 3
figure 3

Schematic representation of TAIL-PCR. One side of T-DNA or Ds element is shown

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Here, we provide a detailed TAIL-PCR method for isolating the flanking sequences of T-DNA or Ds transposable elements inserted into plant genomes, to predict chromosomal rearrangements.

2 Materials

2.1 DNA Extraction from Plants

  1. 1.

    Plant with T-DNA and/or a Ds transposable element. The site of Ds transposition via the Ac TPase can be determined after crossing to remove the Ac gene.

  2. 2.

    Plant DNA isolation kit (e.g., DNeasy Plant Mini Kit, Qiagen, Hilden, Germany).

  3. 3.

    Extraction buffer: 200 mM Tris–HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA, 0.5 % (w/v) SDS [19].

  4. 4.

    Disposable grinders or tooth picks.

  5. 5.

    Isopropanol.

  6. 6.

    Ethanol: 70 % (v/v).

  7. 7.

    TE buffer: 10 mM Tris–HCl (pH 8.0), 1 mM EDTA.

2.2 Thermal Asymmetric Interlaced PCR (TAIL-PCR)

  1. 1.

    Ex Taq polymerase (e.g., Takara Bio Inc., Kusatsu, Japan).

  2. 2.

    Specific primers (Table 1) (see Note 1 ).

    Table 1 Specific primers to amplify DNA adjacent to T-DNA or Ds in TAIL-PCR
    Full size table
  3. 3.

    AD primers:

    AD2-1: NGTCGASWGANAWGAA (N = A,G,C or T, S = C or G, W = A or T).

    AD17: TCNGSATWTGSWTGT (N = A,G,C or T, S = C or G, W = A or T).

  4. 4.

    Thermal cycler (e.g., Veriti® Thermal Cycler, Applied Biosystems, Foster City, CA, USA).

  5. 5.

    TAE buffer : 4.84 g Tris base, 1.14 ml acetic acid, 2 ml 0.5 M EDTA (pH 8.0), adjust to the volume to 1.0 l with ddH2O.

  6. 6.

    Agarose.

  7. 7.

    Agarose gel electrophoresis apparatus (e.g., Mupid System, Advance, Tokyo, Japan).

  8. 8.

    Agarose gel extraction kit (e.g., Wizard SV Gel and PCR Clean-up System, Promega, Madison, WI, USA).

3 Methods

3.1 DNA Extraction from Plants

Extract DNA from 50 mg leaf tissue with a plant DNA isolation kit according to the manufacturer’s protocol.

Alternatively, because TAIL-PCR is a robust method, rapid and crude techniques to extract plant genomic DNA, such as that described by Edwards et al. [19], can be used to obtain a large number of DNA samples.

  1. 1.

    Crush leaf tissue (3 mm × 3 mm) in 100 μl extraction buffer in a 1.5 ml tube using a disposable grinder or tooth pick.

  2. 2.

    Centrifuge the extract at top speed for 2 min and transfer supernatant to a new 1.5 ml tube.

  3. 3.

    Mix the supernatant with an equal volume of isopropanol. Centrifuge the mixture at top speed for 5 min, and then wash the pellet with 70 % (v/v) ethanol. Vacuum-dry the pellet and dissolve in 40 μl TE.

3.2 Thermal Asymmetric Interlaced PCR (TAIL-PCR)

  1. 1.

    Program the thermal cycler for primary TAIL-PCR as follows:

    • TAIL-PCR1:

    • 94 °C for 1 min

    • (94 °C for 1 min, 65 °C for 1 min, 68 °C for 3 min) × 5

    • 94 °C for 1 min, 30 °C for 1.5 min, 68 °C (ramp 10 %) for 3 min

    • (94 °C for 1 min, 65 °C for 1 min, 68 °C for 3 min, 94 °C for 1 min, 65 °C for 1 min, 68 °C for 3 min, 94 °C for 1 min, 44 °C for 1 min, 68 °C for 3 min) × 13

  2. 2.

    Prepare reaction mixture for primary TAIL-PCR as follows:

    • 0.5 μl Extracted genomic DNA

    • 6.9 μl ddH2O

    • 1.0 μl 10× Ex Taq buffer

    • 0.8 μl 2.5 mM dNTPs

    • 0.2 μl Specific primer 1 (e.g., LT6 for T-DNA of pDs-Lox, Ds1-I for 5′ Ds of pDs-Lox) (10 μM) (Table 1)

    • 0.5 μl AD primer (one of the AD primers) (100 μM)

    • 0.1 μl Ex Taq polymerase

  3. 3.

    Run program TAIL-PCR1. The program is completed in approximately 4–5 h.

  4. 4.

    Program for secondary TAIL-PCR as follows:

    • TAIL-PCR2:

    • 94 °C, 1 min

    • (94 °C for 1 min, 65 °C for 1 min, 68 °C for 3 min, 94 °C for 1 min, 65 °C for 1 min, 68 °C for 3 min, 94 °C for 1 min, 44 °C for 1 min, 68 °C for 3 min) × 13

  5. 5.

    Prepare reaction mixture for secondary TAIL-PCR as follows:

    • 0.5 μl 1/10 dilution of primary PCR product

    • 7.0 μl ddH2O

    • 1.0 μl 10× Ex Taq buffer

    • 0.8 μl 2.5 mM dNTPs

    • 0.2 μl Specific primer 2 (e.g., P745 for T-DNA of pDs-Lox, Ds1-II for 5′ Ds of pDs-Lox) (10 μM) (Table 1)

    • 0.4 μl AD primer (100 μM)

    • 0.1 μl Ex Taq polymerase

  6. 6.

    Run program TAIL-PCR2 . The program is completed in approximately 3.5–4 h.

  7. 7.

    Prepare reaction mixture for tertiary TAIL-PCR, if applicable; otherwise skip to step 9.

    • 0.5 μl 1/10 dilution of secondary PCR product

    • 7.0 μl ddH2O

    • 1 μl 10× Ex Taq buffer

    • 0.8 μl 2.5 mM dNTPs

    • 0.2 μl Specific primer 3 (e.g., Ds1-III for 5′ Ds of pDs-Lox) (10 μM) (Table 1)

    • 0.4 μl AD primer (100 μM)

    • 0.1 μl Ex Taq polymerase

  8. 8.

    Run program TAIL-PCR2.

  9. 9.

    Electrophorese 1 μl PCR product on a 1.2 % (w/v) agarose gel, stain with ethidium bromide, and visualize under ultraviolet light (Fig. 4).

    Fig. 4
    figure 4

    TAIL-PCR products. Agarose gel images of TAIL-PCR products from secondary (II) and tertiary (III) reactions. Lanes 1–5 show products from individual lines

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  10. 10.

    Extract all DNA fragments using an agarose gel extraction kit according to the manufacturer’s protocol (see Note 3 ).

  11. 11.

    Sequence DNA fragments with the specific primers used at the last step (e.g., P745 for T-DNA of pDs-Lox, Ds1-III for 5′ Ds of pDs-Lox) (Table 1).

  12. 12.

    Conduct DNA sequence analyses using the BLASTN (nucl query vs nucl db) program (BLAST : https://blast.ncbi.nlm.nih.gov/Blast.cgi) for the plant genome. If the band is correct, the sequences will begin with the T-DNA or Ds border sequence, followed by plant genome sequences. To confirm whether the authentic flanking sequences have been amplified, design primer sets around the boundary region (Primers A and B in Fig. 5).

    Fig. 5
    figure 5

    Confirmation of Ds insertion. DNA amplification will be observed when Primer A–Ds1-III, Primer B–Ds2-III, and Primer A–Primer B are used

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4 Notes

  1. 1.

    The border primers should be specific to the borders of the T-DNA or to the boundary sequence of the Ds transposable element. Because T-DNA integration occurs from the right border, the left-border side of the T-DNA is frequently truncated. Thus, successful TAIL-PCR amplification from the LB increases the probability of full-length T-DNA integration . A number of binary vectors have been developed from different Agrobacterium strains, with divergent border sequences. If your binary vectors are not commonly used, check the T-DNA sequences and design primer sets with Tm > 65 °C from the border sequences.

  2. 2.

    pBin19 derivatives [20] include pBI101, pBI121, pRok2 (Salk lines [21]), and others.

  3. 3.

    All amplified bands should be used for sequencing. When a single band appears, purify the PCR product using exonuclease and shrimp alkali phosphatase (e.g., ExoSAP-IT (Affymetrix, Inc, Cleveland, OH, USA)). However, in the case of Agrobacterium-mediated T-DNA transfer, multiple T-DNA insertions often occur (multiple copies at single locus and/or multiple loci). Some of them contain truncated T-DNA regions or binary vector backbone sequences . A single-locus insertion line can be selected based on segregation of resistance to antibiotics, and the number of insertions can be determined by Southern blot hybridization analysis.