Molecular analysis of high-copy insertion sites in maize

Abstract

High-copy transposon mutagenesis is an effective tool for creating gene disruptions in maize. In order to molecularly define transposon-induced disruptions on a genome-wide scale, we optimized TAIL-PCR to amplify genomic DNA flanking maize Robertson's Mutator insertions. Sample sequencing from 43 Mutator stocks and the W22 inbred line identified 676 non-redundant insertions, and only a small fraction of the flanking sequences showed significant similarity to maize repetitive sequences. We further designed and tested 79 arbitrary primers to identify 12 primers that amplify all Mutator insertions within a DNA sample at 3.1-fold redundancy. Importantly, the products are of sufficient size to use as substrates or probes for hybridization-based identification of gene disruptions. Our adaptation simplifies previously published TAIL-PCR protocols and should be transferable to other high-copy insertional mutagens.

Figure 1

(A) MuTAIL-PCR products from the leaf mutant, ectopic auricle (eco). Products were amplified from a pool of eco homozygous plants using the AD1, AD2, AD3, AD10, AD20 and W4 arbitrary primers (lanes 1–6). The AD10 and AD20 primers frequently gave few or no products (lanes 4–5). Lane 7 is the 1 kbp size standard (Invitrogen). (B) Size selection of pooled MuTAIL products from two UniformMu Mu-inactive mutants used for sample sequencing experiments. Lanes 2 and 4 show total pooled products amplified from two embryo defective mutants using AD1, AD2, AD3, AD10 and W4. Lanes 3 and 5 are the same pools after sephacryl-400 size selection. Lane 1 is the 1 kbp size standard.

Figure 2

Average number of non-redundant sequences found in the MuTAIL-PCR microlibraries. White bars indicate average numbers of sequences that were shared between two or more genotypes within the set of 676 non-redundant sequences. Gray bars show the mean number of unique sequences in each microlibrary. The microlibraries were grouped by genetic background and Mu activity state (see Materials and Methods for details). Error bars are standard errors for each class of sequences.

Figure 3

Genomic DNA gel blots hybridized with Mu element specific probes. Genomic DNA that was used as the template for the MuTAIL sequencing libraries was digested with HindIII (A–D) or XhoI (E). Lanes 1–11 are the template DNA samples for libraries W22, 00-133, 00-136, 00-137, 99F-235, 99F-240, 99F-245, a1-mum2, eco, 96-603 and 99F-263B. The DNA gel blots were probed with (A) MuDR-, (B) Mu1-, (C) Mu3-, (D) Mu7- and (E) Mu8-specific probes.

Figure 4

DNA gel blots probed with cloned MuTAIL-PCR products. (A) DNA gel blot of HindIII digested genomic DNA extracted from W22 (lane 1) and F₂ progeny of the 99F-232 UniformMu parental pool (lanes 2–13). The blot is probed with the insert from MuTAIL clone 99F-232-G09. (B) DNA gel blot of SstI digested genomic DNA extracted from W22 (lane 1) and F₂ progeny of 99F-232 (lanes 2–13). The blot is probed with the insert of MuTAIL clone 99F-232-A07. Size standards are marked in kbp and polymorphic bands are denoted with arrows.

Figure 5

Ten-locus hybridization assay. DNA gel blots of MuTAIL-PCR products amplified from each of 10 DNA templates containing known Mu-tagged loci were probed with their respective gene sequences. All signals were scored after an overnight exposure. Lanes A–F (left of the vertical black line) are MuTAIL products using the TIR4 nested Mu primer and the Arabidopsis arbitrary primers AD1, AD2, AD3, AD10, AD20 and W4, respectively. Lanes G–R (right of the vertical black line) are MuTAIL products using the TIR8 nested Mu primer and the optimized maize arbitrary primers cggc1, SAD11, SW41, BAD5, CG2, CST1, CTG1, GAG3, geeky1, AG1, BAD8 and NC1, respectively.

Figure 6

Comparison of Mu-TIR sequences amplified with the TIR4 and TIR8 nested primers. (A) Maximum likelihood phylogenetic tree of Mu-TIR sequences recovered with the TIR4 primer. The tree is based on the nine non-redundant 33 bp terminal ends of the 5′ (L) and 3′ (R) Mu-TIRs found in DDBJ/EMBL/GenBank. The number of MuTAIL sequences that clustered with each TIR class are shown next to the tree. (B) Distribution of Mu-TIR sequences from products amplified with the TIR8 nested primer. The maximum likelihood phylogenetic tree is based on the six non- redundant 29 bp terminal ends of the Mu-TIRs found in DDBJ/EMBL/GenBank. (C) Bar graph showing the relative frequency of Mu-TIR classes cloned from products amplified with TIR4 (white) and TIR8 (gray) nested primers. The TIR4 clusters were grouped according to the terminal 29 bp of the specific TIR sequences amplified.