Involvement of a citrus meiotic recombination TTC-repeat motif in the formation of gross deletions generated by ionizing radiation and MULE activation

Abstract

Background: Transposable-element mediated chromosomal rearrangements require the involvement of two transposons and two double-strand breaks (DSB) located in close proximity. In radiobiology, DSB proximity is also a major factor contributing to rearrangements. However, the whole issue of DSB proximity remains virtually unexplored.

Results: Based on DNA sequencing analysis we show that the genomes of 2 derived mutations, Arrufatina (sport) and Nero (irradiation), share a similar 2 Mb deletion of chromosome 3. A 7 kb Mutator-like element found in Clemenules was present in Arrufatina in inverted orientation flanking the 5' end of the deletion. The Arrufatina Mule displayed "dissimilar" 9-bp target site duplications separated by 2 Mb. Fine-scale single nucleotide variant analyses of the deleted fragments identified a TTC-repeat sequence motif located in the center of the deletion responsible of a meiotic crossover detected in the citrus reference genome.

Conclusions: Taken together, this information is compatible with the proposal that in both mutants, the TTC-repeat motif formed a triplex DNA structure generating a loop that brought in close proximity the originally distinct reactive ends. In Arrufatina, the loop brought the Mule ends nearby the 2 distinct insertion target sites and the inverted insertion of the transposable element between these target sites provoked the release of the in-between fragment. This proposal requires the involvement of a unique transposon and sheds light on the unresolved question of how two distinct sites become located in close proximity. These observations confer a crucial role to the TTC-repeats in fundamental plant processes as meiotic recombination and chromosomal rearrangements.

Figure 1

Sequencing coverage and copy number variation (CNV). The sequence coverage and the CNVs along chromosome 3 (A) and chromosome 8 (B) in three clementines, CLE, ARR and NER are shown. Read depths of each chromosome are depicted as black profiles in unitless scales. CNVs are shown as red points at a genome level log2 ratio between CLE, the original variety and either ARR or NER, the two mutations. The red color gradient sections represents log10 p calculated on each of ratios.

Figure 2

Chromosomal rearrangements in ARR and NER. A) Representation of the position and orientation of the pair-end reads (red, green and purple arrows) supporting the deletion of a similar fragment in chromosome 3 (green bars) of ARR and NER. Positions of breakpoints (bp) and the mapping of the pair reads are shown in the reference CLE genome. White bars flanking green bars represent fragments in CLE that are deleted in ARR and/or NER. The inversion found in ARR is shown as a pink big arrow. The different elements are not drawn to scale. B) Representation of the position and orientation of the pair-end reads (represented by colored arrows) supporting several rearrangements in chromosomes 8 (gray bars) and 6 (brown bars) of NER. Positions of breakpoints (bp) and the mapping of the pair reads are shown in the reference CLE genome. Red and blue pair-ends indicate the occurrence of two consecutive but separate deletions. Green and purple pair-ends support the occurrence of both a translocation from a small fragment of one of the initially deleted stretches of chromosome 8 to chromosome 6, and a small deletion in chromosome 6. White bars flanking either gray or brown bars represent fragments in CLE that are deleted in NER. The gray bar flanking brown bars represents the fragment in CLE that was translocated from chromosome 8 to chromosome 6. The different elements are not drawn to scale.

Figure 3

Structure of 4 CitMul elements. Transposable CitMul elements contain a 5 exon transcript, with a largest exon 1 including predicted protein sequences coding for a FAR1 DNA binding domain, a SWIM-type zinc finger motif and a MULE transposase domain. Introns are depicted as lines connecting exons.

Figure 4

Conserved motifs in the Cit_Mule_1 terminal region. A) Schematic representation of the terminal 5′ sequence of Cit-Mule_1 showing the TSD (light blue), a fragment with the terminal inverted repeats (yellow), two translin recognition sites (brown), TC stretches (green), GGG triplets (dark blue), a putative topoisomerase II-like motif (red) and two specific DNA motifs of recombination hot spots identified in Schizosaccharomyces pombe (pink) and Escherichia coli (magenta). Arrows show the first nucleotide of the promoter, the first one of the 5′UTR and the first ATG of the protein according to the ORF prediction. B) Sequences of the 100 terminal-most nucleotides of CitMule_1. The “1” sequence represents the upstream terminal end of CitMule_1 and the “2” sequence the downstream end. The double-underlined sequences indicate tracks with 88% similarity, while the underlined sequences denote tracks with lower similarity (78%).

Figure 5

Consensus sequence of insertion sites of CitMule elements. Consensus sequence of insertion sites of CitMule elements. Consensus sequences were obtained with the Weblogo analysis (Crooks et al.2004) using the 7 available target site sequences including the three different 9 bp TSDs of CitMule_1, CitMule_2 and CitMule_4, and the 4 different 9 bp “dissimilar” TDs of CitMule 3 and CitMule_ARR (i.e. the 2 different sequences flanking CitMule 3 and the 2 different sequences flanking CitMule_ARR). For the consensus analysis, the immediate 13 positions upstream and downstream of CitMule termini were examined. The preferred insertion site of CitMule are the bendable AT-repeat triplets represented in the positions 2 to 4 and 10 to 12 and the precise insertion takes place between the positions 2 and 3 (or 10 and 11) in one strand and 10 and 11 (or 2 and 3) in the other. The y-axis represents the strength of the information.

Figure 6

Frequency of the alternative allele on chromosome 3. Frequency of alternative allele in 100 bp windows in the 3 stretches of chromosome 3 corresponding to the deletions identified in ARR and NER. Frequency was calculated as the number of reads of the alternative allele with respect the total number of reads. The partner of shift detected in the allelic frequency of the 6, 7–8, 6 Mb deletion in both ARR and NER indicates the occurrence of a meiotic recombination event in the genome of reference (www.phytozome.net).

Figure 7

A proposed model for the ARR TE-mediate deletion. A. Structure of the chromosome 3 (gray bar) of the original variety, CLE, showing the occurrence of CitMule (position 6785327; red track) with the transposon inverted repeats (TIRs, green and yellow arrows) and a putative recombination hotspot (position 7797540; blue star) formed by a GAA/TTC track. B. A triplex DNA structure is formed in ARR at the recombination hotspot generating a loop that eventually brings the upstream end of CitMule near to the vicinity of position 8686356. Two transposase monomers bind to the transposon inverted repeats. C. Looping of the transposon brings the two ends of the transposable element close together. Transposase monomers form a dimer generating a paired-end complex. The complex recruits other involved proteins such as topoisomerases (blue object) and translins (purple object) resulting in the formation of a synaptic complex. Other putative trans-factors are not represented. D. Transposase, first, cuts the CitMule away from the flanking donor DNA and after cleavage the transposase/complex is released. E. In normal insertions, it is expected that transposase encounters a unique target side for insertion, generating the same TSD sequence in each side of the insertion. In ARR, the transposase/complex recognized two different CitMule target sites separated by approximately 2 Mb. One of these was the target site at position 8686356 and the other one was at position 6785296. Note that this position and the original position of CitMule are separated by 35 bp. F. Transposase catalyzes the inverted insertion of the transposon between these 2 target sites provoking in this way the release of the fragment spanning from one target site to the other one. Therefore, these events resulted in both an inverted insertion of CitMule and a 2 MB deletion in chromosome 3 as observed in ARR.