Telomere-mediated chromosomal truncation in maize

Abstract

Direct repeats of Arabidopsis telomeric sequence were constructed to test telomere-mediated chromosomal truncation in maize. Two constructs with 2.6 kb of telomeric sequence were used to transform maize immature embryos by Agrobacterium-mediated transformation. One hundred seventy-six transgenic lines were recovered in which 231 transgene loci were revealed by a FISH analysis. To analyze chromosomal truncations that result in transgenes located near chromosomal termini, Southern hybridization analyses were performed. A pattern of smear in truncated lines was seen as compared with discrete bands for internal integrations, because telomeres in different cells are elongated differently by telomerase. When multiple restriction enzymes were used to map the transgene positions, the size of the smears shifted in accordance with the locations of restriction sites on the construct. This result demonstrated that the transgene was present at the end of the chromosome immediately before the integrated telomere sequence. Direct evidence for chromosomal truncation came from the results of FISH karyotyping, which revealed broken chromosomes with transgene signals at the ends. These results demonstrate that telomere-mediated chromosomal truncation operates in plant species. This technology will be useful for chromosomal engineering in maize as well as other plant species.

Fig. 1.

Chromosomal truncation constructs pWY76 and pWY86 and the control construct pWY96. LB, T-DNA left border; RB, T-DNA right border; Tvsp, terminator from soybean vegetative storage protein gene; Bar, bialophos resistance gene as a selection marker gene; TEV, tobacco etch virus 5′ untranslated region; P35S, 2× 35S promoter from cauliflower mosaic virus; Tnos, Nos terminator from Agrobacterium; Tmas, Mas terminator from Agrobacterium; Pnos, Nos promoter from Agrobacterium; Pmas1′, Mas promoter from Agrobacterium; lox and FRT, site-specific recombination sites; HPT, hygromycin B-resistance gene; GFP, green fluorescent protein gene; DsRed, red fluorescent protein gene; FLP, recombinase gene; Telomeres, telomere units of pAtT4 isolated from Arabidopsis thaliana (2). Arrows designate the direction of transcription.

Fig. 2.

Cytological detection of chromosomal truncations. Metaphase chromosomes were hybridized with pWY96 probe (red) and mixtures of repetitive sequence probes, CentC (green), knob (green), subtelomere 4–12-1 clone (green), and Cent4 (white). Arrows denote the transgene truncation sites (white arrows) and the corresponding sites on the homologues (gray arrows). (A) pWY86 transgenic event B77 with a chromosome 3 short-arm truncation. (Inset) The chromosome 3 pair with (Left) and without (Right) the transgene (red). (B) pWY76 transgenic event T87 with a chromosome 1 short-arm terminal-knob truncation. (Inset) Chromosome 1 homologues with (Upper) and without (Lower) the transgene (red). (C and D) pWY86 transgenic events B37 and B44 with truncations of chromosome 4 long-arm terminal subtelomeric 4–12-1 sequence (green). (Insets) Chromosome 4 homologues with (Upper) and without (Lower) the transgene (red). Chromosome 4 can be identified by the Cent4 hybridization (white) at their centromeres. (Scale bar, 10 μm.)

Fig. 3.

Chromosomal truncations revealed by a Southern blot with multiple enzymatic digestions. (A) Restriction map of the T-DNA region of the pWY86 construct with restriction sites (top line) and their positions relative to the right border (RB) (bottom line). Bars show the position of bar and FLP probes. (B) Restriction mapping of the positions of four transgenes by a Southern blot. Genomic DNAs from pWY86 events B37 (lanes 1–3), B2 (–6), B77 (lanes 7–9), and B21 (lanes 10–12) were digested with HindIII (lanes 1, 4, 7, and 10), EcoRV(lanes 2, 5, 8, and 11), and SmaI (lanes 3, 6, 9, and 12). The Southern blot was hybridized with the ³²P-labeled FLP probe. DNA fragment sizes were indicated at the left-hand side. Telomere smears are indicated by arrowheads. Discrete bands found for B37 and B77 events likely result from other copies of the transgene inserted elsewhere in the genome.

Fig. 4.

Telomere sequences detected in internal chromosome locations. Metaphase chromosomes of a pWY86 transgenic line were probed with a pWY96 probe (red) and a telomere probe (green). Chromosomes were stained with DAPI. Arrowheads indicate the internal telomere (green) and transgene (red) signals. Arrowheads in the enlarged images from the top to the bottom panels denote merged transgene (red) and telomere (green) signals, and telomere only (green) and transgene only (red). (Scale bar, 10 μm.)