RESUMEN
Meiotic crossovers shuffle parental genetic information, providing novel combinations of alleles on which natural or artificial selection can act. However, crossover events are relatively rare, typically one to three exchange points per chromosome pair. Recent work has identified three pathways limiting meiotic crossovers in Arabidopsis thaliana that rely on the activity of FANCM [Crismani W, et al. (2012) Science 336:1588-1590], RECQ4 [Séguéla-Arnaud M, et al. (2015) Proc Natl Acad Sci USA 112:4713-4718], and FIGL1 [Girard C, et al. (2015) PLoS Genet 11:e1005369]. Here we analyzed recombination in plants in which one, two, or all three of these pathways were disrupted in both pure line and hybrid contexts. The greatest effect was observed when combining recq4 and figl1 mutations, which increased the hybrid genetic map length from 389 to 3,037 cM. This corresponds to an unprecedented 7.8-fold increase in crossover frequency. Disrupting the three pathways did not further increase recombination, suggesting that some upper limit had been reached. The increase in crossovers is not uniform along chromosomes and rises from centromere to telomere. Finally, although in wild type recombination is much higher in male meiosis than in female meiosis (490 cM vs. 290 cM), female recombination is higher than male recombination in recq4 figl1 (3,200 cM vs. 2,720 cM), suggesting that the factors that make wild-type female meiosis less recombinogenic than male wild-type meiosis do not apply in the mutant context. The massive increase in recombination observed in recq4 figl1 hybrids opens the possibility of manipulating recombination to enhance plant breeding efficiency.
Asunto(s)
Arabidopsis/genética , Arabidopsis/fisiología , Cruzamiento , Intercambio Genético/genética , Recombinación Homóloga/genética , Genes de Plantas/genética , Mutación/genéticaRESUMEN
Meiosis, the basis of sex, evolved through iterative gene duplications. To understand whether subsequent duplications have further enriched the core meiotic "tool-kit," we investigated the fate of meiotic gene duplicates following whole genome duplication (WGD), a common occurrence in eukaryotes. We show that meiotic genes return to a single copy more rapidly than genome-wide average in angiosperms, one of the lineages in which WGD is most vividly exemplified. The rate at which duplicates are lost decreases through time, a tendency that is also observed genome-wide and may thus prove to be a general trend post-WGD. The sharpest decline is observed for the subset of genes mediating meiotic recombination; however, we found no evidence that the presence of these duplicates is counterselected in two recent polyploid crops selected for fertility. We therefore propose that their loss is passive, highlighting how quickly WGDs are resolved in the absence of selective duplicate retention.
Asunto(s)
Magnoliopsida/genética , Meiosis , Evolución Molecular , Duplicación de Gen , Genoma de Planta , Recombinación HomólogaRESUMEN
Mitochondria and chloroplasts (photosynthetic members of the plastid family of cytoplasmic organelles) in eukaryotic cells originated more than a billion years ago when an ancestor of the nucleated cell engulfed two different prokaryotes in separate sequential events. Extant cytoplasmic organellar genomes contain very few genes compared with their candidate free-living ancestors, as most have functionally relocated to the nucleus. The first step in functional relocation involves the integration of inactive DNA fragments into nuclear chromosomes, and this process continues at high frequency with attendant genetic, genomic, and evolutionary consequences. Using two different transplastomic tobacco lines, we show that DNA migration from chloroplasts to the nucleus is markedly increased by mild heat stress. In addition, we show that insertion of mitochondrial DNA fragments during the repair of induced double-strand breaks is increased by heat stress. The experiments demonstrate that the nuclear influx of organellar DNA is a potentially a source of mutation for nuclear genomes that is highly susceptible to temperature fluctuations that are well within the range experienced naturally.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Plantas/metabolismo , Estrés Fisiológico , Genes ReporterosRESUMEN
Endosymbiotic transfer of DNA and functional genes from the cytoplasmic organelles (mitochondria and chloroplasts) to the nucleus has been a major factor driving the origin of new nuclear genes, a process central to eukaryote evolution. Although organelle DNA transfers very frequently to the nucleus, most is quickly deleted, decays, or is alternatively scrapped. However, a very small proportion of it gives rise, immediately or eventually, to functional genes. To simulate the process of functional transfer, we screened for nuclear activation of a chloroplast reporter gene aadA, which had been transferred from the chloroplast to independent nuclear loci in 16 different plant lines. Cryptic nuclear activity of the chloroplast promoter was revealed, which became conspicuous when present in multiple nuclear copies. We screened â¼50 million cells of each line and retrieved three plants in which aadA showed strong nuclear activation. Activation occurred by acquisition of the CaMV 35S nuclear promoter or by nuclear activation of the native chloroplast promoter. Two fortuitous sites within the 3' UTR of aadA mRNA both promoted polyadenylation without any sequence change. Complete characterization of one nuclear sequence before and after gene transfer demonstrated integration by nonhomologous end joining involving simultaneous insertion of multiple chloroplast DNA fragments. The real-time observation of three different means by which a chloroplast gene can become expressed in the nucleus suggests that the process, though rare, may be more readily achieved than previously envisaged.
Asunto(s)
Núcleo Celular/genética , Cloroplastos/genética , Evolución Molecular , Genes de Plantas , Genoma de Plastidios , Secuencia de Bases , Reordenamiento Génico , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Proteínas de Plantas/genética , Nicotiana/genética , Activación Transcripcional , Transformación GenéticaRESUMEN
In higher plants, DNA transfer from the plastid (chloroplast) genome to the nucleus is a frequent, ongoing process. However, there has been uncertainty over whether this transfer occurs by a direct DNA mechanism or whether RNA intermediates are involved. Previous experiments utilising transplastomic Nicotiana tabacum (tp7 and tp17) enabled the detection of plastid-to-nucleus transfer in real time. To determine whether RNA intermediates are involved in this transfer, transplastomic lines (tpneoACG) were generated containing, in their plastid genomes, a nuclear promoter-driven kanamycin resistance gene (neo) with a start codon that required plastid RNA editing but otherwise identical to tp7 and tp17. Therefore it was expected that kanamycin resistance would only be acquired following RNA-mediated transfer of neo to the nucleus. Screening of tpneoACG progeny revealed several kanamycin-resistant plants, each of which contained the neo gene located in the nucleus. Surprisingly, neo was unedited in all these plants, indicating that neoACG was active in the absence of an edited start codon and suggesting that RNA intermediates were not involved in the transfers. However, analysis of tpneoACG revealed that only a low proportion of transcripts potentially able to mediate neo transfer were edited, thus precluding unequivocal conclusions regarding the role of RNA in plastid-to-nucleus transfer. The low proportion of edited transcripts was found to be due to predominant antisense neo transcripts, rather than to low editing efficiency of the sense transcripts. This study highlights a number of important considerations in the design of experiments utilising plastid RNA editing. The results also suggest that RNA editing sites reduce but do not eliminate functional plastid-to-nucleus gene transfer. This is relevant both in an evolutionary context and in placing RNA editing-dependent genes in the plastid genome as a means of transgene containment.
Asunto(s)
Núcleo Celular/metabolismo , Técnicas de Transferencia de Gen , Nicotiana/metabolismo , Plastidios , Edición de ARN , Transgenes , Secuencia de Bases , Datos de Secuencia Molecular , ARN Mensajero/genética , Nicotiana/genéticaRESUMEN
BACKGROUND: Chromosome pairing, recombination and DNA repair are essential processes during meiosis in sexually reproducing organisms. Investigating the bread wheat (Triticum aestivum L.) Ph2 (Pairing homoeologous) locus has identified numerous candidate genes that may have a role in controlling such processes, including TaMSH7, a plant specific member of the DNA mismatch repair family. RESULTS: Sequencing of the three MSH7 genes, located on the short arms of wheat chromosomes 3A, 3B and 3D, has revealed no significant sequence divergence at the amino acid level suggesting conservation of function across the homoeogroups. Functional analysis of MSH7 through the use of RNAi loss-of-function transgenics was undertaken in diploid barley (Hordeum vulgare L.). Quantitative real-time PCR revealed several T0 lines with reduced MSH7 expression. Positive segregants from two T1 lines studied in detail showed reduced MSH7 expression when compared to transformed controls and null segregants. Expression of MSH6, another member of the mismatch repair family which is most closely related to the MSH7 gene, was not significantly reduced in these lines. In both T1 lines, reduced seed set in positive segregants was observed. CONCLUSION: Results presented here indicate, for the first time, a distinct functional role for MSH7 in vivo and show that expression of this gene is necessary for wild-type levels of fertility. These observations suggest that MSH7 has an important function during meiosis and as such remains a candidate for Ph2.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Hordeum/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Secuencia de Aminoácidos , Secuencia de Bases , Diploidia , Fertilidad/genética , Genoma de Planta , Hordeum/crecimiento & desarrollo , Datos de Secuencia Molecular , Mutación , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Interferencia de ARN , Semillas/genética , Semillas/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Triticum/genéticaRESUMEN
DNA transfer to the nucleus from prokaryotic ancestors of the cytoplasmic organelles (mitochondria and plastids) has occurred during endosymbiotic evolution in eukaryotes. In most eukaryotes, organelle DNA transfer to nucleus is a continuing process. The frequency of DNA transposition from plastid (chloroplast) to nucleus has been measured in tobacco plants (Nicotiana tabacum) experimentally. We have monitored the effects of environmental stress on the rate of DNA transfer from plastid to nucleus by exploiting nucleus-specific reporter genes in two transplastomic tobacco lines. DNA migration from plastids to the nucleus is markedly increased by mild heat stress. In addition, insertions of mitochondrial DNA into induced double-strand breaks are observed after heat treatment. These results show that movement of organelle DNA to the nucleus is remarkably increased by heat stress.
Asunto(s)
Núcleo Celular/genética , Cloroplastos/genética , ADN de Cloroplastos/metabolismo , ADN Mitocondrial/metabolismo , Genoma de Planta , Respuesta al Choque Térmico/genética , Nicotiana/genética , Transporte Biológico , Genes Reporteros , Plantas Modificadas GenéticamenteRESUMEN
DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.
Asunto(s)
Arabidopsis/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Nicotiana/genética , Reacción en Cadena de la Polimerasa/métodos , ADN de Plantas/genéticaRESUMEN
The origin of new genes has long been considered a fundamental question in evolutionary biology. In eukaryotes, a major pathway for the 'birth' of new nuclear genes has been transfer of genes from the cytoplasmic organelles (mitochondria and plastids) to the nucleus. While the vast majority of gene transfer occurred shortly after endosymbiosis, the process continues today and is still driving the evolution of nuclear genomes. In tobacco (Nicotiana tabacum) a number of studies have indicated that DNA can transfer from the chloroplast to the nucleus at relatively high frequency. Less has been known, however, about how a newly transferred organelle gene can become activated in this new genetic environment. In a recent report we observed, in real-time, the activation of a plastid reporter gene newly transferred to the nucleus. A key observation from this study was that non-homologous repair is an important generator of novel sequence combinations which, in rare instances, can result in the nuclear activation of plastid genes. In addition, the activation of relocated genes can be aided by the fortuitous presence of plastid sequences able to promote nuclear expression.