PCR analysis of the twinned alleles with primers 1+2+Ac5.

<p>Lane 1: DNA ladder; lanes 2 and 5: water (negative control); lanes 3 and 4: <i>P1-ovov454</i>, <i>P1-rr-T1</i>; lanes 6 and 7: <i>P1-ovov454</i>, <i>P1-rr-T481</i>. Note that primers 1 and 2 are specific for each allele.</p

Tandem direct duplications in maize generated by Reversed-Ends Transposition.

<p>Red lines with arrowhead(s) indicate the <i>dhAT</i> family elements associated with each duplication; solid and open arrowheads indicate the transposon 5′ and 3′ ends, respectively. The truncated solid arrowhead in <i>dhAT-Zm13</i> indicates a deletion of 12 bp from the 5′ TIR. The blue lines represent duplicated segments. The blue triangles indicate the transposon target site duplications. Numbers above each line indicate the length of that segment. Sequences and genomic positions are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003691#pgen.1003691.s009" target="_blank">Text S2</a>.</p

Phenotypes and Gene Structures of <i>P-oo</i> Alleles

<div><p>(A) The kernel pericarp pigmentation phenotypes specified by the indicated alleles.</p><p>(B) Genomic Southern blot. Genomic DNA from plants homozygous for the indicated alleles was cut with KpnI and HindIII, and hybridized with probes 15 or 8B from the <i>p1</i> gene. Lanes marked <i>P-oo32</i> contain approximately twice as much DNA as lanes marked <i>P1-rr11;</i> this DNA overloading enables the detection of the 7.6-kb band in the KpnI 8B blot, but also results in the intense 6.5-kb band in the HindIII 15 blot.</p><p>(C) Restriction map. The solid and gray boxes are exons 1, 2, and 3 (left to right) of <i>p1</i> and <i>p2,</i> respectively. Red triangles indicate <i>Ac</i> or <i>fAc</i> insertions, and the open and solid arrowheads indicate the 3′ and 5′ ends, respectively, of <i>Ac/fAc</i>. Sequences hybridizing with Southern blot probes are indicated by the solid bars above (probe 8B) and below (probe 15) the map. The short horizontal arrows indicate the orientations and approximate position of PCR primers. Primers are identified by numbers below the arrows. The sequence of the junction of each fusion allele is shown here; the black letters indicate <i>p2</i> sequence, while the red letters indicate <i>fAc</i> sequence. K, KpnI; H, HindIII. Lines below the map indicate the restriction fragments produced by digestion with KpnI or HindIII and hybridizing with the indicated probe; asterisks indicate HindIII restriction sites located within <i>Ac</i> or <i>fAc</i> sequences.</p></div

Reversed <i>Ac</i> ends transposition generates direct duplication.

<p>The two lines indicate sister chromatids of maize chromosome 1, joined at the centromere (black). The blue boxes are exons 3, 2, and 1 (left to right) of the <i>p1</i> gene. Red lines with arrowhead(s) indicate <i>Ac/fAc</i> insertions, and the open and solid arrowheads indicate the 3′ and 5′ ends, respectively, of <i>Ac</i>/<i>fAc</i>. The short horizontal arrows show the orientations and approximate positions of PCR primers, and the numbers below are the primer names. The green/black triangles indicate the transposon target site sequences and target site duplications. (<i>A</i>) <i>Ac</i> transposase cleaves the lower chromatid at the 3′ end of <i>fAc</i> and the 5′ end of <i>Ac</i> (arrows). (<i>B</i>) Following transposase cleavage, the internal <i>p1</i> genomic sequences are joined to form a circle. Dotted lines indicate the insertion of the <i>fAc</i> and <i>Ac</i> termini into the a/b site on the sister chromatid. (<i>C</i>) Transposon ends insert into the upper sister chromatid at a proximal site. (<i>D</i>) The <i>Ac</i> 5′ end joins to the distal side (green) of the target site and the <i>fAc</i> 3′ end joins to the proximal side (black) of the target site to generate a proximal deletion (upper chromatid) and a direct duplication (lower chromatid). The shaded arrows encompass the duplicated segments. For animation, please see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003691#pgen.1003691.s006" target="_blank">Movie S1</a>.</p

Deletions by Reversed <i>Ac</i> Ends Transposition Generate Chimerical Genes

<div><p>The solid circle indicates the centromere, the short vertical line indicates the target site, and the other symbols have the same meaning as those in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020164#pgen-0020164-g001" target="_blank">Figure 1</a>. (For animated version, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020164#pgen-0020164-sv001" target="_blank">Video S1</a>).</p><p>(A) Ac transposase (blue oval) binds to the 5′ end of <i>Ac</i> and 3′ end of <i>fAc</i>.</p><p>(B) As in ordinary transposition, the <i>Ac</i> 5′ end and the <i>fAc</i> 3′ end are excised by transposase cleavage, and the sequences flanking the <i>Ac/fAc</i> ends join together to form a ~13-kb circle. The X mark at the junction indicates the transposon footprint.</p><p>(C) The excised transposon ends insert into a site in intron 2 of <i>p2</i>. The <i>Ac</i> 5′ end joins to the distal side of the insertion site to form a circle, and the <i>fAc</i> 3′ end joins to the proximal side of the insertion site to generate a chimeric gene containing exon 1 and exon 2 of <i>p2</i> and exon 3 of <i>p1</i>.</p><p>This study reports the isolation of the progenitor (A) and deletion products (C). Note that the hypothetical structures shown in (B) are transient in nature and would not be amenable to physical isolation.</p></div

RT-PCR Analysis of <i>P-oo</i> Transcripts

<p>RNA was extracted from kernel pericarp (20 DAP), reverse transcribed, and PCR-amplified using primers complementary to both <i>p1</i> and <i>p2</i> transcripts. The progenitor allele <i>(P1-rr11)</i> shows amplification of a 605-bp band from <i>p1.</i> The <i>p-ww2</i> and <i>P-oo</i> alleles show amplification of a 522-bp band characteristic of the 5′ region of the <i>p2</i> gene. The <i>p1-ww1112</i> allele has a deletion of <i>p1;</i> the native <i>p2</i> gene is intact in this allele, but is not expressed in kernel pericarp.</p

PCR primers.

<p>PCR primers.</p

Breakpoint sequences of reciprocal duplication/deletion alleles <i>P1-rr-T1</i> and <i>p1-ww-T1</i> generated by Reversed Ends Transposition.

<p>(<i>A</i>) Diagram shows the structure of the progenitor <i>P1-ovov454</i> allele prior to RET. Two sister chromatids are shown, with symbols as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003691#pgen-1003691-g001" target="_blank">Figure 1</a>. The dotted box shows the a/b target site region, whose sequence is indicated above. The color of the letters in the sequences matches the chromatid line color. (<i>B</i>) PCR amplification of the deletion/duplication breakpoints in <i>p1-ww-T1</i> and <i>P1-rr-T1</i> with primers 2+Ac3 (upper panel) or 1+Ac5 (lower panel). Lane 1: DNA ladder, lane 2: <i>p1-ww[4Co63]</i>, lane 3: <i>P1-ovov454/p1-ww[4Co63]</i>, lane 4: <i>p1-ww-T1/p1-ww[4Co63]</i>, lane 5: <i>P1-rr-T1/p1-ww[4Co63]</i>. (<i>C</i>) Sister chromatid structures of <i>p1-ww-T1</i> (upper) and <i>P1-rr-T1</i> (lower). Sequences of the deletion and duplication breakpoints (dotted boxes) are shown in color matching the chromatid line color. Note that each breakpoint has a copy of the 8 bp TSD GCGCTTTA which is present in a single copy at the a/b target site in the progenitor allele.</p

Ears with twinned sectors T1 (left) and T481 (right).

<p>The white and red phenotypic twinned sectors are outlined. The remainder of the ear has the orange-variegated phenotype specified by the progenitor <i>P1-ovov454</i> allele.</p

DNA gel blot analysis of the twinned alleles.

<p>(<i>A</i>) Structure of progenitor <i>P1-ovov454</i> allele (upper), and predicted structures of the <i>P1-rr-Twin</i> (duplication) and <i>p1-ww-Twin</i> (deletion) alleles (lower) generated via reversed <i>Ac</i> ends transposition. Blue lines and boxes indicate <i>p1</i> sequences, green lines indicate sequences proximal to <i>p1</i>, and gray boxes indicate sequences homologous to probe 15. The short vertical black lines indicate <i>Sal</i>I sites, and red asterisks (*) mark methylated <i>Sal</i>I sites. The other symbols have the same meaning as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003691#pgen-1003691-g001" target="_blank">Figure 1</a>. (<i>B</i>) DNA gel blot. Genomic DNA was digested with <i>Sal</i>I and hybridized with <i>p1</i> genomic probe 15 (gray boxes in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003691#pgen-1003691-g005" target="_blank">Figure 5<i>A</i></a>). Lane 1: <i>p1-ww[4Co63]</i>, Lane 2: <i>P1-ovov454</i>/<i>p1-ww[4Co63]</i>, Lane 3: <i>P1-rr-T1</i>, Lane 4: <i>p1-ww-T1/p1-ww[4Co63]</i>, Lane 5: <i>P1-rr-T481</i>.</p