Natural insertions in rice commonly form tandem duplications indicative of patch-mediated double-strand break induction and repair

The insertion of DNA into a genome can result in the duplication and dispersal of functional sequences through the genome. In addition, a deeper understanding of insertion mechanisms will inform methods of genetic engineering and plant transformation. Exploiting structural variations in numerous rice accessions, we have inferred and analyzed intermediate length (10–1,000 bp) insertions in plants. Insertions in this size class were found to be approximately equal in frequency to deletions, and compound insertion–deletions comprised only 0.1% of all events. Our findings indicate that, as observed in humans, tandem or partially tandem duplications are the dominant form of insertion (48%), although short duplications from ectopic donors account for a sizable fraction of insertions in rice (38%). Many nontandem duplications contain insertions from nearby DNA (within 200 bp) and can contain multiple donor sources—some distant—in single events. Although replication slippage is a plausible explanation for tandem duplications, the end homology required in such a model is most often absent and rarely is >5 bp. However, end homology is commonly longer than expected by chance. Such findings lead us to favor a model of patch-mediated double-strand-break creation followed by nonhomologous end-joining. Additionally, a striking bias toward 31-bp partially tandem duplications suggests that errors in nucleotide excision repair may be resolved via a similar, but distinct, pathway. In summary, the analysis of recent insertions in rice suggests multiple underappreciated causes of structural variation in eukaryotes.Genomic DNA insertion causes genome expansion and, potentially, the rearrangement and diffusion of protein domains and regulatory elements throughout the genome (1, 2). Additionally, genetic engineers generally aim to integrate specific DNA into the nuclear genome, so the natural mechanisms by which this integration occurs may serve as a starting point to elaborate and improve genome modification (3, 4). Common causes of gene-sized insertions are unequal recombination (5), transposable element replication (1), and ectopic recombination stimulated by double-strand breaks (DSBs) in the genome (2, 6). Shorter events are less well characterized, but it appears that these can be created by similar processes (7). Still, high-throughput sequencing of DSB repair events in humans (8) and plants (9) suggests that insertions related to induced breaks are very rare and very short.Although the processes described above can produce duplications at distant genetic loci, the most common form of non-microsatellite-associated insertions in humans is tandem duplications (10). Once created, tandem duplications can be dramatically expanded by unequal recombination or replication slippage. Such duplications may be deleterious, or they may be promoted by selection for a novel or expanded function (11, 12).Although tandem repeats are ubiquitous in eukaryotic genomes, the mechanisms for their origin are still in question. Early analysis of human indel mutations indicated that replication slippage was the most effective model to explain the origin of assorted repeats (13). In other studies, longer, de novo tandem duplications were also hypothesized to be caused by slippage because, out of 85 insertions producing such duplications, 50 were associated with flanking repeats >2 bp (14). Replication slippage would presumably require a preexisting short repeat because priming must occur between the end of the loop that will become the duplication and the position to where replication slips. Authors of more recent work investigating insertions across the human genome suggest alternatives to replication slippage on the grounds that homology is often either nonexistent or very short, whereas the length of homology and the length of insertion are not correlated (10). These researchers favor a model based on DSBs being repaired by nonhomologous end-joining (NHEJ). However, conventional models of DSB repair are strained to predict tandem duplications >10 bp, much less >100 bp. Such models require extensive single-stranded, complementary ends to be preserved during the break. Moreover, DSBs produced by Tal-effector nucleases in humans do not yield insertions that form tandem repeats, despite the fact that the breaks generate a 5′ overhang (15). Thus, this common class of mutations currently lacks a firm molecular explanation.Similar to tandem duplications, short duplications are commonly found within 100 bp of one another, but with unique intervening DNA (16). By comparing human polymorphisms with chimp sequence, Thomas et al. (16) inferred that the repeats were recent insertions. As discussed by the authors and herein, a mechanism for such duplications is even less forthcoming than for tandem duplications.In this study, we used extensive population-scale rice resequencing data to confirm that tandem duplications are also abundant natural polymorphisms in the plant kingdom. Additionally, we found that many insertions in rice, although not perfectly tandem, are from a ∼50-bp window around the insertion site. We rarely found the end homology in tandem repeats that is expected for replication slippage, although we did note a bias toward short microhomology between insertion ends and insertion site. These data led us to elaborate on the DSB model of tandem duplication, proposing that long patch base excision repair (BER) on complementary strands commonly leads to such patterns (17). Additionally, we characterized common forms of nontandem, but local, duplication.