A segmental deletion series generated by sister-chromatid transposition of Ac transposable elements in maize

Abstract

Certain configurations of maize Ac/Ds transposon termini can undergo alternative transposition reactions leading to chromosome breakage and various types of stable chromosome rearrangements. Here, we show that a particular allele of the maize p1 gene containing an intact Ac element and a nearby terminally deleted Ac element (fAc) can undergo sister-chromatid transposition (SCT) reactions that generate large flanking deletions. Among 35 deletions characterized, all begin at the Ac termini in the p1 gene and extend to various flanking sites proximal to p1. The deletions range in size from the smallest of 12,567 bp to the largest of >4.6 cM; >80% of the deletions removed the p2 gene, a paralog of p1 located approximately 60 kb from p1 in the p1-vv allele and its derivatives. Sequencing of representative cases shows that the deletions have precise junctions between the transposon termini and the flanking genomic sequences. These results show that SCT events can efficiently generate interstitial deletions that are useful for in vivo dissection of local genome regions and for the rapid correlation of genetic and physical maps. Finally, we discuss evidence suggesting that deletions induced by alternative transposition reactions can occur at other genomic loci, indicating that this mechanism may have had a significant impact on genome evolution.

Figure 1.

Model for formation of deletions by sister-chromatid transposition. (For animated version, see supplemental material at http://www.genetics.org/supplemental/.) The diagram pertains to the structure of the p1-vv9D9A allele, which is the progenitor of the p1-ww deletion alleles described in the text. The two lines indicate sister chromatids joined at the centromere, which is indicated by a solid circle. The solid black boxes indicate the three exons of the p1 gene; the 5′-end of the p1 gene is closer to the centromere (Zhang and Peterson 1999). The red arrows indicate the Ac or fAc elements inserted into the second intron of the p1 gene, and the open and solid arrowheads indicate the 3′- and 5′-ends, respectively, of Ac/fAc. The short black line between Ac and fAc indicates a 112-bp rearranged p1 sequence (rP) present in the p1-vv9D9A allele (not to scale). (A) Following DNA replication, identical sister chromatids are joined at the centromere. Ac transposase (small circles) binds to the 5′ terminus of Ac in one chromatid and to the 3′ terminus of fAc in the sister chromatid. (B) Cuts are made at the Ac and fAc termini to excise the transposon ends. The two nontransposon ends join together at the site marked by the black X to form a chromatid bridge. (C) Reinsertion of the excised transposon ends into the chromatid bridge between b and c generates two reciprocal chromatids; one carries a deletion of c and the other carries an inverted duplication of c. (D) Same as for C, except that reinsertion between a and b generates one chromatid with a deletion of b c and one with an inverted duplication of b c. For simplicity, the model depicts fully replicated sister chromatids at the time of transposition. In reality, transposition may occur when the chromosomes are partially replicated.

Figure 2.

Detection of deletions by PCR analysis. (A) Structure of the p1-vv9D9A haplotype, including p1 (right) and its paralog p2 (left). Symbols have the same meaning as in Figure 1. Short horizontal arrows indicate the orientations and approximate positions of the primers used in PCR analysis. (B) Screening for deletions of sequences 5′ of the p1 gene using primer pair p1-1 + p1-2, which gives a 489-bp product in p1-vv9D9A. The primer pair Ac-6 + p1-8 detects a 313-bp band from the junction of the 3′-end of Ac with the 3′ sequence of p1 intron 2. PCR was performed using genomic DNA from plants of the genotypes indicated above each lane. The lane marked P1-wr contains DNA from the W22 inbred. The P1-wr allele has been previously shown to contain a tandem array of p1 genes (Chopra et al. 1998), whereas no p2 gene was detected in 16 diverse maize inbred lines containing P1-wr (Szalma et al. 2005). The negative result in the P1-wr lane would suggest that P1-wr alleles also lack (or are polymorphic for) the sequence upstream of p1 in p1-vv. The p1-ww1112 allele contains a deletion of p1 (Athma and Peterson 1991) and retains the p2 gene (Zhang et al. 2000). (C) Screening for deletions of the 5′-end of the p2 gene using primer pair p1-3 + p1-4, which gives a 420-bp product from the p2 gene and a 500-bp product from the p1 gene. As in B, primer pair Ac-6 + p1-8 detects a 313-bp band derived from the junction of the 3′-end of Ac with the 3′ sequence of p1 intron 2.

Figure 3.

Nucleotide sequences at endpoints of p1-ww495 and p1-ww2 deletion alleles. The top three lines show the structures of the indicated alleles and the locations of the sequences given below. Sequences a–d are from the progenitor allele p1-vv9D9A. Sequences e and f are from the derivative alleles p1-ww495 and p1-ww2, respectively. Sequences in italics and underlined represent Ac or fAc sequences. Note that the deletion endpoint in p1-ww495 is joined to the Ac 5′-end, while the deletion endpoint of p1-ww2 is joined to the 3′ fAc end. Small black boxes indicate the locations of sequences that hybridize with p1 genomic fragment 15. Other symbols have the same meaning as in Figure 1.

Figure 4.

Determination of the physical distance between p1 and p2 by CHEF gel analysis. Cells from plants of the indicated genotypes were protoplasted, embedded in agarose, and digested with NotI endonuclease. DNAs were separated by CHEF gel electrophoresis, transferred to membrane, and hybridized with genomic probe fragment 15 from the p1 gene (Figure 3). Left lane contains λDNA concatemers as size standards.

Figure 5.

Genomic DNA gel-blot analysis of the SCT-induced deletion alleles using probes linked to the p1 gene. (A) Genomic DNA of the genotypes indicated above each lane was digested with HindIII or SacI and hybridized with the indicated probes. See text for details. (B) Summary of endpoint mapping data of the SCT-generated p1-ww deletions. Schematic at top shows the positions of probe fragments (the solid boxes) and genetic distances in centimorgans, where known. Lines below show the extent of deletion found in the alleles indicated by the numbers to the right. Short vertical lines indicate deletion endpoints defined by cloned sequences. Dotted lines indicate the intervals into which those deletion(s) map. The relative order of ndp1 and npi286 cannot be determined on the basis of hybridization data reported here; the positions shown are based on prior recombination data showing a genetic distance from p1 of 3.5 and 3.6 cM for Ndp1 and npi286, respectively (http://www.maizegdb.org/cgi-bin/displaymaprecord.cgi?id=143431). See text for details.

Figure 5.

Genomic DNA gel-blot analysis of the SCT-induced deletion alleles using probes linked to the p1 gene. (A) Genomic DNA of the genotypes indicated above each lane was digested with HindIII or SacI and hybridized with the indicated probes. See text for details. (B) Summary of endpoint mapping data of the SCT-generated p1-ww deletions. Schematic at top shows the positions of probe fragments (the solid boxes) and genetic distances in centimorgans, where known. Lines below show the extent of deletion found in the alleles indicated by the numbers to the right. Short vertical lines indicate deletion endpoints defined by cloned sequences. Dotted lines indicate the intervals into which those deletion(s) map. The relative order of ndp1 and npi286 cannot be determined on the basis of hybridization data reported here; the positions shown are based on prior recombination data showing a genetic distance from p1 of 3.5 and 3.6 cM for Ndp1 and npi286, respectively (http://www.maizegdb.org/cgi-bin/displaymaprecord.cgi?id=143431). See text for details.

Figure 6.

Hypothetical transposition involving the fAc 3′-end and the Ac 5′-end on the same chromatid in p1-vv9D9A (symbols have the same meaning as in Figure 1). This type of transposition reaction would result in reorientation of the sequences hybridizing to oligonucleotide primers 6 (Ac-6) and 7 (Ac-7) (compare A and C) and thus could be detected by PCR. No such products were detected among 10 p1-ww alleles tested. See text for details. (A) Ac transposase binds to a fAc 3′-end and an Ac 5′-end in the same chromatid. (B) Cuts are made at the Ac and fAc termini; sequences at which the Ac and fAc termini were formerly inserted are joined together at the site marked by the X. (C) The excised transposon ends reinsert at a site between a and b. The DNA between fAc and the insertion site is inverted.