Alternative Transposition Generates New Chimeric Genes and Segmental Duplications at the Maize p1 Locus

Abstract

The maize Ac/Ds transposon family was the first transposable element system identified and characterized by Barbara McClintock. Ac/Ds transposons belong to the hAT family of class II DNA transposons. We and others have shown that Ac/Ds elements can undergo a process of alternative transposition in which the Ac/Ds transposase acts on the termini of two separate, nearby transposons. Because these termini are present in different elements, alternative transposition can generate a variety of genome alterations such as inversions, duplications, deletions, and translocations. Moreover, Ac/Ds elements transpose preferentially into genic regions, suggesting that structural changes arising from alternative transposition may potentially generate chimeric genes at the rearrangement breakpoints. Here we identified and characterized 11 independent cases of gene fusion induced by Ac alternative transposition. In each case, a functional chimeric gene was created by fusion of two linked, paralogous genes; moreover, each event was associated with duplication of the ∼70-kb segment located between the two paralogs. An extant gene in the maize B73 genome that contains an internal duplication apparently generated by an alternative transposition event was also identified. Our study demonstrates that alternative transposition-induced duplications may be a source for spontaneous creation of diverse genome structures and novel genes in maize.

Keywords: Ac/Ds alternative transposition; chimeric gene; exon shuffling; segmental duplication; translocation.

Figure 1

RET of Ac/fAc produces reciprocal chromosome translocation and segmental duplications. Black and blue lines indicate maize chromosome 1 and 10 sequences, respectively. Red lines with arrowheads indicate Ac and fAc elements; open and solid red arrowheads indicate 3′ and 5′ Ac termini, respectively. Black and gray boxes indicate exons of maize p1 and p2 genes, respectively. This legend applies to all figures. Photographs of kernels of plants heterozygous for each allele are presented alongside their schematic structures. (A) Upper (black line): chromosome 1 containing p1 and p2 loci with Ac/fAc elements in progenitor allele p1-ovov454. Lower (blue line): chromosome 10 with transposition target site indicated by vertical orange line. (B) Following chromosome replication, Ac and fAc termini are excised from p1 locus and inserted into target site on chromosome 10. (C) Translocation structure of the p1-vvD103 allele. Asterisk marks the 5.9-kb duplication at the chromosome 1-10 translocation junction in p1-vvD103. (C′) Following DNA replication, the reciprocal translocation chromosomes contain two identical sister chromatids. The ∼70-kb region of the ensuing duplication is indicated by gray-shaded arrows. (D) The reverse-oriented Ac and fAc termini excise from the p1 locus of the upper chromatid and insert into the target site (green vertical line) of p2 gene on the sister chromatid. (E) Schematic structure of the P1P2 allele with associated chromosomes. Upper line indicates translocation chromosome 1-10 containing ∼70-kb direct duplications indicated as gray-shaded arrows. Lower line indicates reciprocal translocation chromosome 10-1.

Figure 2

The p1-vvD103 allele is associated with a chromosome 1-10 reciprocal translocation. (A) Schematic structures of p1-vvD103 reciprocal translocation chromosomes 1-10 (upper) and 10-1 (lower). All symbols as in Figure 1. Positions of primers D5, Ac5, P3, and D3 are indicated by the short horizontal arrows. (B) Gel analysis of PCR amplification by primers Ac5 + D5. Lanes 1–4: P1P2-2, p1-vvD103, p1-ovov454; p1-ww[4Co63]. Both p1p2 and p1-vvD103 alleles show a band at ∼5 kb. (C) Gel analysis of PCR amplification by primers D3 + P3. Lanes 2–4: p1-vvD103, p1-ovov454; p1-ww[4Co63]. The p1-vvD103 allele shows a band at 2.5 kb. (D) PCR analysis of genomic DNA from oat–maize addition lines using primers D3 + D5 to identify reciprocal translocation chromosome. Lane 1, oat; lane 2, maize p1-ww[4Co63]; lanes 3–12, oat–maize addition lines with maize chromosomes 1–10, respectively; lane 13, maize p1-ovov454. Lane 12, oat–maize addition line containing maize chromosome 10 shows a band of the same size (800 bp) as produced by maize p1-ww[4Co63] and allele p1-ovov454. (E) Maize sporocyte nucleus (pachytene stage) of p1-vvD103/p1-ww[4Co63]. The short and long arms of chromosome 1 and chromosome 10 are designated 1S, 10S, 1L, and 10L. “10C” indicates the centromere of chromosome 10. The breakpoint of desynapsis in chromosome 10 is in the short arm near the end, and the breakpoint in chromosome 1 is in the short arm near the middle.

Figure 3

The P1P2 alleles contain tandem direct duplications with breakpoints in the p2 gene. (A) Schematic structures of P1P2 fusion allele on chromosome 1-10 and reciprocal translocation region on chromosome 10-1. Bracketed segments indicate HindIII restriction fragments, and the open boxes represent the positions of probe fragment pp1. Black arrowheads indicate positions of PCR primers. (B) Gel profiles of PCR products from duplication junctions. Primers Ac5 + P1 amplified template DNA from J, 454, D, and P1P2-1–P1P2-5. Alleles P1P2-6 to P1P2-10 were amplified using primers Ac5 + P2, and alleles P1P2-12 and P1P2-14 were amplified using primers Ac5 + P3 (from a separate gel). (C) DNA gel blot analysis of P1P2 alleles hybridized with probe fragment pp1. Arrows indicate bands common to multiple templates. Bands of varying size (ranging from 7.9 to 3.9 kb) are derived from duplication junctions specific for each allele. Lanes in B and C: J—p1-ww[4Co63]; 454—P1-ovov454; D—p-vvD103. Lane 7f contains DNA from a derivative of P1P2-7 in which Ac has transposed and a newly formed fAc is present; hence its PCR patterns are somewhat different from those of lane 7.

Figure 4

Structures of chimeric P1P2 fusion genes. (A) Exon/intron structure of p2 gene with Ac insertion sites specific to each P1P2 alelle indicated by numbered red triangles. Arrowhead marked “ATG” indicates translation start codon; stop sign indicates stop codon. (B) Fine structures of P1P2 fusion genes. Each chimeric allele contains p1 exons 1 and 2 and partial intron 2, fused via Ac with the p2 gene. Sequences shown are from the Ac/p2 fusion junctions; sequences from p2 exons 1, 2, and 3 are shown in gray boxes.

Figure 5

The chimeric P1P2 alleles are expressed in maize pericarp. (A) Diagram of predicted structure of P1P2 transcripts, indicating positions of primers P4 and P5. (B) Gel analysis of RT-PCR products produced by primer P4 from p1 exon 1 and P5 from p2 exon 3 (620 bp), with the internal control of a tubulin gene fragment (322 bp). Total RNA extracted from developing kernel pericarp was used as template for RT-PCR. Depending on whether plants were homozygous or heterozygous at the fusion locus, different intensities of 620-bp bands were obtained for different samples of P1P2 alleles.

Figure 6

RET model for generation of duplications in maize gene AC234515.1. (A) Structure of maize gene AC234515.1_FGT002 downloaded from the Maize Genome Database (http://www.maizegdb.org). (B) AC234515.1 contains two directly duplicated segments of 1054 and1056 bp (gray-shaded arrows), including exons 8, 9, 10, 11, intron sequences, and duplicated PIF-Harbinger MITE elements. The MITEs (blue lines) have degraded 5′-end sequences (truncated solid arrowheads) and intact 3′ ends (complete open arrowheads). Both MITEs are flanked by TSDs (“TAA”) shown as black vertical lines. (C) Prior to RET, the progenitor gene may consist of 10 exons (labeled here as 1–9, plus 12). An intact 5′ end (blue triangle) from a PIF-Harbinger MITE element is predicted to be located downstream of exon 12. (D) The reverse-oriented 3′ and 5′ MITE termini (circled in cyan color) flanking exon 12 undergo RET beginning with excision of the termini. The intertransposon segment containing exon 12 is circularized and presumably lost. The excised termini reinsert into intron 7 on the sister chromatid, generating TSDs indicated by vertical orange lines. (E) The expected gene structure generated by RET. The gene contains identical duplicated segments (gray-shaded arrows) containing exons 8 and 9 and MITE elements located precisely at the duplication endpoints. The 5′ MITE end located downstream of exon 12 may be subsequently excised or segregated from the AC234515.1 gene.

Figure 7

Template switching followed by NHEJ explains the 5.9-kb duplication at the p1-vvD103 translocation junction. (A) Replicated chromosomes 1 (black) and 10 (blue) in progenitor p1-ovov454 allele are shown; chromosome 1 contains p1 and p2 loci with Ac/fAc insertions indicated in red. The segments to be duplicated are shaded in pink (1601 nt from p1 locus) and blue (4331 nt from chromosome 10). (B) Excision of fAc and Ac termini by Ac transposase generates DSBs and circularizes the small intertransposon segment. Ac transposase also cuts at the insertion target site (orange vertical line) on chromosome 10, which will join with the fAc end to generate the chromosome 10-1 translocation. (C) Repair of DSBs. The DSB at the Ac 5′ end primes repair replication using the sister chromatid as template, copying p1 and partial fAc sequences. Similarly, the DSB at the distal side of chromosome 10 uses sister chromatid as replication template. (D) The two extended chromatids are joined by NHEJ using the 3-bp microhomology site “GTA,” producing a chromosome 1-10 translocation junction containing a 5.9-kb duplication composed of 1601 nt from chromosome 1 and 4331 nt from chromosome 10. (E) Final structure of translocation chromosomes in p1-vvD103 allele.