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

Terol J, Ibañez V, Carbonell J, Alonso R, Estornell LH, Licciardello C, Gut IG, Dopazo J, Talon M - BMC Genomics (2015)

Bottom Line: A 7 kb Mutator-like element found in Clemenules was present in Arrufatina in inverted orientation flanking the 5' end of the deletion.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.

View Article: PubMed Central - PubMed

Affiliation: Centro de Genómica, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, 46113, Valencia, Spain. terol_javalc@gva.es.

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.

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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.
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Fig2: 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.

Mentions: Chromosomal rearrangements were investigated through 4 independent analyses based on read depth, copy number variation, pair-end reads and PCR. Coverage profile for a certain chromosome was remarkably similar in the three genomes except in a few stretches of ARR and NER that showed reduced depth, suggesting the occurrence of putative gross deletions. Reduced coverage was observed in a very similar fragment of chromosome 3 in both mutants, while NER showed three additional putative interstitial deletions (Figure 1A) two of them also in chromosome 3 and the third one in chromosome 8 (Figure 1B). Copy number variation analysis with CNV-seq [23] confirmed the occurrence of the four deletions insinuated with the coverage reduction. Thus, the deletions predicted by coverage analysis and CNV-seq were completely coincident. Further pair-end read analyses provided additional insights on rearrangements of chromosomes 3 and 8 in both mutated genotypes (Additional file 3: Table S3). While the orientation of pair reads indicated the nature of the rearrangement (deletion vs. inversion or translocation) the read percentage suggested the hemizygous condition of the event. The read pairing analyses ascertained the presence of the intriguing deletion of chromosome 3 common to the two mutated genotypes, and the NER deletion of chromosome 8. However, there was no pair read evidence supporting the two additional NER deletions of chromosome 3 spanned from positions 27.2 to 28.7 and 36.1 to 37.0 MB (Figure 1A), probably because these were located in low complexity areas and the corresponding reads were filtered during mapping. In ARR, the paired-end analyses identified a 6926 bp inversion of chromosome 3 (Figure 2A) apparently supported by a percentage of pair-end reads lower than the expected 50%, as shown in Additional file 3: Table S3. The structure indicated by this pairing, however, was clearly confirmed by PCR. There were other 3 sets of pair-end reads involving 3 different positions at chromosome 4. These pairings probably were mapping artifacts since no PCR evidence of their occurrence was obtained. This observation, however, indicated that the inverted sequence was repeated at least 4-fold in the clementine genome and prompted the suggestion that it might be a repetitive sequence. It is also worth to note that the presence of this inversion implies the occurrence of a deletion spanning from positions 6785295 to 8686355, a stretch very similar although not identical to the deletion identified in NER that spanned from positions 6782589 to 8724143. According to the data, the NER deletion of chromosome 8 was actually a double interstitial deletion composed of a gross and a small deletion separated by about 50 thousand bp (Figure 2B). In addition, the pair-end analyses provided evidence for a 721 bp translocation from the smaller deletion of chromosome 8 to chromosome 6, an event that also resulted in a 33 bp deletion of chromosome 6 (Additional file 3: Table S3).Figure 1


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

Terol J, Ibañez V, Carbonell J, Alonso R, Estornell LH, Licciardello C, Gut IG, Dopazo J, Talon M - BMC Genomics (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: 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.
Mentions: Chromosomal rearrangements were investigated through 4 independent analyses based on read depth, copy number variation, pair-end reads and PCR. Coverage profile for a certain chromosome was remarkably similar in the three genomes except in a few stretches of ARR and NER that showed reduced depth, suggesting the occurrence of putative gross deletions. Reduced coverage was observed in a very similar fragment of chromosome 3 in both mutants, while NER showed three additional putative interstitial deletions (Figure 1A) two of them also in chromosome 3 and the third one in chromosome 8 (Figure 1B). Copy number variation analysis with CNV-seq [23] confirmed the occurrence of the four deletions insinuated with the coverage reduction. Thus, the deletions predicted by coverage analysis and CNV-seq were completely coincident. Further pair-end read analyses provided additional insights on rearrangements of chromosomes 3 and 8 in both mutated genotypes (Additional file 3: Table S3). While the orientation of pair reads indicated the nature of the rearrangement (deletion vs. inversion or translocation) the read percentage suggested the hemizygous condition of the event. The read pairing analyses ascertained the presence of the intriguing deletion of chromosome 3 common to the two mutated genotypes, and the NER deletion of chromosome 8. However, there was no pair read evidence supporting the two additional NER deletions of chromosome 3 spanned from positions 27.2 to 28.7 and 36.1 to 37.0 MB (Figure 1A), probably because these were located in low complexity areas and the corresponding reads were filtered during mapping. In ARR, the paired-end analyses identified a 6926 bp inversion of chromosome 3 (Figure 2A) apparently supported by a percentage of pair-end reads lower than the expected 50%, as shown in Additional file 3: Table S3. The structure indicated by this pairing, however, was clearly confirmed by PCR. There were other 3 sets of pair-end reads involving 3 different positions at chromosome 4. These pairings probably were mapping artifacts since no PCR evidence of their occurrence was obtained. This observation, however, indicated that the inverted sequence was repeated at least 4-fold in the clementine genome and prompted the suggestion that it might be a repetitive sequence. It is also worth to note that the presence of this inversion implies the occurrence of a deletion spanning from positions 6785295 to 8686355, a stretch very similar although not identical to the deletion identified in NER that spanned from positions 6782589 to 8724143. According to the data, the NER deletion of chromosome 8 was actually a double interstitial deletion composed of a gross and a small deletion separated by about 50 thousand bp (Figure 2B). In addition, the pair-end analyses provided evidence for a 721 bp translocation from the smaller deletion of chromosome 8 to chromosome 6, an event that also resulted in a 33 bp deletion of chromosome 6 (Additional file 3: Table S3).Figure 1

Bottom Line: A 7 kb Mutator-like element found in Clemenules was present in Arrufatina in inverted orientation flanking the 5' end of the deletion.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.

View Article: PubMed Central - PubMed

Affiliation: Centro de Genómica, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, 46113, Valencia, Spain. terol_javalc@gva.es.

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.

Show MeSH
Related in: MedlinePlus