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Characterization of an Ac transposon system based on apt1-m1 (Ac) on the long arm of maize chromosome 9

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Abstract

Activator/Dissociation (Ac/Ds) transposable elements have been used in maize insertional mutagenesis as a complement to Mutator (Mu). In this study, to further improve the efficiency of the Ac/Ds mutagenesis system, we adopted apt1-m1 (Ac) on the long arm of chromosome 9 (9L) as a donor Ac to create an Ac insertion library. This system is based on the negative selection pressure against the donor Ac, and it was highly efficient for isolating new transposition events. We obtained 9,625 transposition events from 1083 F1 ears with an average transposition rate of 8.66 % (rates ranged from 1.11 to 29.73 %). We also adopted a modified PCR-based genome walking strategy to improve the efficiency of the new method for isolating transposon-flanking sequences. This method is more efficient than the Southern-based method that was used in previous studies. A validation step was developed to distinguish transposon tags derived from newly transposed Ac or Ds elements. Using this PCR-based method, we isolated 67 inheritable flanking sequences from the apt1-m1 (Ac) transposition library; of these, 51 were confirmed as tr-Ac-flanking sequences and 11 were tr-Ds-flanking sequences. Similar to other Ac donors from different loci, the apt1-m1 (Ac) system also exhibited a preference for short distance transposition. In this study, we have further improved the Ac mutagenesis system in maize for gene isolation and functional genomics studies.

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Acknowledgments

This work was supported by National Natural Science Foundation of China (30900900), The Ministry of Agriculture (2009ZX08009-111B), Ministry of Science and Technology of China (2012AA10A305 and 2009CB118400), Shanghai Science and Technology Committee No. 11DZ2272100.

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Authors and Affiliations

  1. School of Life Sciences, Shanghai Key Laboratory of Bio-energy Crop, Shanghai University, 333 Nanchen Road, Shanghai, 200444, People’s Republic of China

    Fei Wang, Pengfei Li, Yuanping Tang, Jun Fan, Dabin Xu, Shengming Guo, Zhengkai Xu & Rentao Song

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  1. Fei Wang
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  2. Pengfei Li
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  3. Yuanping Tang
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  4. Jun Fan
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  5. Dabin Xu
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  6. Shengming Guo
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  7. Zhengkai Xu
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  8. Rentao Song
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Corresponding author

Correspondence to Rentao Song.

Additional information

Fei Wang and Pengfei Li contributed equally to this work.

Nucleotide sequence data reported are available in the GenBank database under the accession numbers JS187029—JS187092.

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10709_2012_9685_MOESM1_ESM.jpg

Supplemental Figure 1. Amplification of Shu00071 flanking sequence. Genomic DNA was digested by EcoRV and ligated to adaptors. For each group, lanes 1-5 were Shu00071-1, Shu00071-2, the W22 reporter line, the donor Ac line and a blank control, respectively. Shu00071-1 and Shu00071-2 were independent lines containing the same tr-Acs. M: 100 bp DNA marker (Invitrogen). A: Amplification of the Ac 3′ end flanking sequence in Shu00071. The primer Ac4131 was used as the first round Ac end-specific primer. The Ac end-specific primers used in the second round of PCR were Ac4217, Ac4411, Ac4442, Ac4490, Ac 4528 and Ac 4547 for groups 1, 2, 3, 4, 5 and 6, respectively. The white arrow indicates the expected band that was present in Shu00071-1 and Shu00071-2 and absent from the parental lines. B: Amplification of the Ac 5′ end flanking sequence in Shu00071. The primer Ac317 was used as the first round Ac end-specific primer. The Ac end-specific primers used in the second round of PCR were Ac281, Ac215, Ac169, Ac86, Ac53 and Ac22 for groups 1, 2, 3, 4, 5 and 6, respectively. The white arrow indicates the expected band that was present in Shu00071-1 and Shu00071-2 and absent from the parental lines. (JPEG 184 kb)

10709_2012_9685_MOESM2_ESM.jpg

Supplemental Figure 2. Amplification of the Ac flanking sequences of the identified tr- Ac s. Lanes 1 and 2 were two independent lines of Shu00071 (positive control). Lanes 3 and 4 were independent lines of Shu00114, and lanes 5 and 6 were independent lines of Shu00132. Lane 7, W22 reporter line; lane 8, Ac donor line; lane 9, blank control; lane 10, 100 bp DNA marker (Invitrogen) (lane 10). A: Amplification of the Ac 5′ flanking sequence using Ac317 and Ac169 as the first and second round Ac end-specific primers, respectively. The white arrow indicates the expected bands. B: Amplification of Ac 3′ flanking sequence using Ac4131 and Ac4217 as the first and second round Ac end-specific primers, respectively. The white arrow indicates the expected bands. (JPEG 136 kb)

10709_2012_9685_MOESM3_ESM.jpg

Supplemental Figure 3. The distribution of tr - Acs and tr - Dss. The numbers on the left sides of each chromosome indicate the physical location of each tr-Ac in Mb. The tr-Acs are indicated in red, and the tr-Dss are indicated in blue. (JPEG 239 kb)

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Wang, F., Li, P., Tang, Y. et al. Characterization of an Ac transposon system based on apt1-m1 (Ac) on the long arm of maize chromosome 9. Genetica 140, 337–347 (2012). https://doi.org/10.1007/s10709-012-9685-2

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