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1.  Arabidopsis meiotic crossover hotspots overlap with H2A.Z nucleosomes at gene promoters 
Nature genetics  2013;45(11):10.1038/ng.2766.
PRDM9 directs human meiotic crossover hotspots to intergenic sequence motifs, whereas budding yeast hotspots overlap low nucleosome density regions in gene promoters. To investigate hotspots in plants, which lack PRDM9, we used coalescent analysis of Arabidopsis genetic variation. Crossovers increase towards gene promoters and terminators, and hotspots are associated with active chromatin modifications, including H2A.Z, histone H3K4me3, low nucleosome density and low DNA methylation. Hotspot-enriched A-rich and CTT-repeat DNA motifs occur upstream and downstream of transcriptional start respectively. Crossovers are asymmetric around promoters and highest over CTT-motifs and H2A.Z-nucleosomes. Pollen-typing, segregation and cytogenetic analysis show decreased crossovers in the arp6 H2A.Z deposition mutant, at multiple scales. During meiosis H2A.Z and DMC1/RAD51 recombinases form overlapping chromosomal foci. As arp6 reduces DMC1/RAD51 foci, H2A.Z may promote formation or processing of meiotic DNA double-strand breaks. We propose that gene chromatin ancestrally designates hotspots within eukaryotes and PRDM9 is a derived state within vertebrates.
doi:10.1038/ng.2766
PMCID: PMC3812125  PMID: 24056716
2.  Guidelines for Genome-Wide Association Studies 
PLoS Genetics  2012;8(7):e1002812.
doi:10.1371/journal.pgen.1002812
PMCID: PMC3390399  PMID: 22792080
3.  A Meiotic Tapas Menu 
PLoS Genetics  2006;2(2):e19.
doi:10.1371/journal.pgen.0020019
PMCID: PMC1378127  PMID: 16738707
4.  Consent and Internet-Enabled Human Genomics 
PLoS Genetics  2010;6(6):e1000965.
doi:10.1371/journal.pgen.1000965
PMCID: PMC2891701  PMID: 20585615
5.  The DNA Replication Factor RFC1 Is Required for Interference-Sensitive Meiotic Crossovers in Arabidopsis thaliana 
PLoS Genetics  2012;8(11):e1003039.
During meiotic recombination, induced double-strand breaks (DSBs) are processed into crossovers (COs) and non-COs (NCO); the former are required for proper chromosome segregation and fertility. DNA synthesis is essential in current models of meiotic recombination pathways and includes only leading strand DNA synthesis, but few genes crucial for DNA synthesis have been tested genetically for their functions in meiosis. Furthermore, lagging strand synthesis has been assumed to be unnecessary. Here we show that the Arabidopsis thaliana DNA REPLICATION FACTOR C1 (RFC1) important for lagging strand synthesis is necessary for fertility, meiotic bivalent formation, and homolog segregation. Loss of meiotic RFC1 function caused abnormal meiotic chromosome association and other cytological defects; genetic analyses with other meiotic mutations indicate that RFC1 acts in the MSH4-dependent interference-sensitive pathway for CO formation. In a rfc1 mutant, residual pollen viability is MUS81-dependent and COs exhibit essentially no interference, indicating that these COs form via the MUS81-dependent interference-insensitive pathway. We hypothesize that lagging strand DNA synthesis is important for the formation of double Holliday junctions, but not alternative recombination intermediates. That RFC1 is found in divergent eukaryotes suggests a previously unrecognized and highly conserved role for DNA synthesis in discriminating between recombination pathways.
Author Summary
Meiotic recombination is important for pairing and sustained association of homologous chromosomes (homologs), thereby ensuring proper homolog segregation and normal fertility. DNA synthesis is thought to be required for meiotic recombination, but few genes coding for DNA synthesis factors have been studied for possible meiotic functions because their essential roles in the mitotic cell cycle make it difficult to study their meiotic functions due to the lethality of corresponding null mutations. Current models for meiotic recombination only include leading strand DNA synthesis. We found that the Arabidopsis gene encoding the DNA REPLICATION FACTOR C1 (RFC1) important for lagging strand synthesis promotes meiotic recombination via a specific pathway for crossovers (COs) that involves the formation of double Holliday Junction (dHJ) intermediates. Therefore, lagging strand DNA synthesis is likely important for meiotic recombination. Because DNA synthesis is a highly conserved process and meiotic recombination is highly similar among budding yeast, mammals, and flowering plants, the proposed function of lagging strand synthesis for meiotic recombination might be a general feature of meiosis.
doi:10.1371/journal.pgen.1003039
PMCID: PMC3493451  PMID: 23144629
6.  Deep Genome-Wide Measurement of Meiotic Gene Conversion Using Tetrad Analysis in Arabidopsis thaliana 
PLoS Genetics  2012;8(10):e1002968.
Gene conversion, the non-reciprocal exchange of genetic information, is one of the potential products of meiotic recombination. It can shape genome structure by acting on repetitive DNA elements, influence allele frequencies at the population level, and is known to be implicated in human disease. But gene conversion is hard to detect directly except in organisms, like fungi, that group their gametes following meiosis. We have developed a novel visual assay that enables us to detect gene conversion events directly in the gametes of the flowering plant Arabidopsis thaliana. Using this assay we measured gene conversion events across the genome of more than one million meioses and determined that the genome-wide average frequency is 3.5×10−4 conversions per locus per meiosis. We also detected significant locus-to-locus variation in conversion frequency but no intra-locus variation. Significantly, we found one locus on the short arm of chromosome 4 that experienced 3-fold to 6-fold more gene conversions than the other loci tested. Finally, we demonstrated that we could modulate conversion frequency by varying experimental conditions.
Author Summary
During the production of gametes, most sexually reproducing organisms undergo meiotic recombination. The most familiar form of meiotic recombination is crossing-over, which results in the reciprocal exchange of DNA between parental chromosomes and is important for chromosome segregation as well as generating new allelic combinations in progeny. The same molecular mechanisms that facilitate crossing-over can also enable the non-reciprocal exchange of genetic information between chromosomes in the process called gene conversion. Understanding gene conversion is important because it influences allele frequencies and has been implicated in human diseases. Unfortunately, it has been difficult until now to measure directly except in organisms, like fungi, that group their gametes after meiosis. In this study we have developed a novel assay system that enables us to measure gene conversion directly in the model multi-cellular eukaryote A. thaliana (a flowering plant). Using this assay system we measured gene conversion frequencies across the Arabidopsis genome in more than 1 million meioses and also demonstrated that we can manipulate those frequencies by varying experimental conditions.
doi:10.1371/journal.pgen.1002968
PMCID: PMC3464199  PMID: 23055940
7.  Epigenetic Remodeling of Meiotic Crossover Frequency in Arabidopsis thaliana DNA Methyltransferase Mutants 
PLoS Genetics  2012;8(8):e1002844.
Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes.
Author Summary
The majority of eukaryotes reproduce via a specialized cell division called meiosis, which generates gametes with half the number of chromosomes. During meiosis, homologous chromosomes pair and undergo a process of reciprocal exchange, called crossing-over (CO), which generates new combinations of genetic variation. The relative chance of a CO occurring is variable along the chromosome, for example COs are suppressed in the centromeric regions that attach to the spindle during chromosome segregation. These patterns correlate with domains of epigenetic organization along chromosomes, including methylation of the DNA and histones. DNA methylation occurs most densely in the centromeric regions of Arabidopsis thaliana chromosomes, where it is required for transcriptional suppression of repeated sequences. We demonstrate that mutants that lose DNA methylation (met1) show epigenetic remodeling of crossover frequencies, with increases in the centromeric regions and compensatory changes in the chromosome arms, though the total number of crossovers remains the same. As crossover numbers and distributions are subject to homeostatic mechanisms, we propose that these drive crossover remodeling in met1 in response to epigenetic change in the centromeric regions. Together these data demonstrate how domains of epigenetic organization are important for shaping patterns of crossover frequency along eukaryotic chromosomes.
doi:10.1371/journal.pgen.1002844
PMCID: PMC3410864  PMID: 22876192
8.  Fluorescence-Tagged Transgenic Lines Reveal Genetic Defects in Pollen Growth—Application to the Eif3 Complex 
PLoS ONE  2011;6(3):e17640.
Background
Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis.
Methodology/Principal Findings
Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation.
Conclusion/Significance
eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis.
doi:10.1371/journal.pone.0017640
PMCID: PMC3049774  PMID: 21408229
9.  Genetic Interference: Don’t Stand So Close to Me 
Current Genomics  2010;11(2):91-102.
Meiosis is a dynamic process during which chromosomes undergo condensation, pairing, crossing-over and disjunction. Stringent regulation of the distribution and quantity of meiotic crossovers is critical for proper chromosome segregation in many organisms. In humans, aberrant crossover placement and the failure to faithfully segregate meiotic chromosomes often results in severe genetic disorders such as Down syndrome and Edwards syndrome. In most sexually reproducing organisms, crossovers are more evenly spaced than would be expected from a random distribution. This phenomenon, termed interference, was first reported in the early 20th century by Drosophila geneticists and has been subsequently observed in a vast range of organisms from yeasts to humans. Yet, many questions regarding the behavior and mechanism of interference remain poorly understood. In this review, we examine results new and old, from a wide range of organisms, to begin to understand the progress and remaining challenges to understanding the fundamental unanswered questions regarding genetic interference.
doi:10.2174/138920210790886835
PMCID: PMC2874225  PMID: 20885817
Meiosis; recombination; crossover; Double-strand break; synaptonemal complex; chromosome Spo11; Rec8; Pch2.
10.  The CYCLIN-A CYCA1;2/TAM Is Required for the Meiosis I to Meiosis II Transition and Cooperates with OSD1 for the Prophase to First Meiotic Division Transition 
PLoS Genetics  2010;6(6):e1000989.
Meiosis halves the chromosome number because its two divisions follow a single round of DNA replication. This process involves two cell transitions, the transition from prophase to the first meiotic division (meiosis I) and the unique meiosis I to meiosis II transition. We show here that the A-type cyclin CYCA1;2/TAM plays a major role in both transitions in Arabidopsis. A series of tam mutants failed to enter meiosis II and thus produced diploid spores and functional diploid gametes. These diploid gametes had a recombined genotype produced through the single meiosis I division. In addition, by combining the tam-2 mutation with AtSpo11-1 and Atrec8, we obtained plants producing diploid gametes through a mitotic-like division that were genetically identical to their parents. Thus tam alleles displayed phenotypes very similar to that of the previously described osd1 mutant. Combining tam and osd1 mutations leads to a failure in the prophase to meiosis I transition during male meiosis and to the production of tetraploid spores and gametes. This suggests that TAM and OSD1 are involved in the control of both meiotic transitions.
Author Summary
In the life cycle of sexual organisms, a specialized cell division—meiosis—reduces the number of chromosomes from two sets (2n, diploid) to one set (n, haploid), while fertilization restores the original chromosome number. Meiosis reduces ploidy because it consists of two divisions following a single DNA replication. In this study, we identified genes that control the entry into the first and the second meiotic division in the model plant Arabidopsis thaliana. Plants lacking the CYCA1;2 gene execute a single division during meiosis producing functional diploid gametes and polyploid plants in the next generation. By combining this mutation with two others that affect key meiotic processes, we generated plants that produce diploid gametes through a mitotic-like division that are genetically identical to their parents. Furthermore, plants lacking CYCA1;2 and another previously described gene (OSD1) undergo no divisions during male meiosis, producing tetraploid pollen grains.
doi:10.1371/journal.pgen.1000989
PMCID: PMC2887465  PMID: 20585549
11.  Scientists←Editors←Scientists: The Past, Present, and Future of PLoS Genetics 
PLoS Genetics  2009;5(7):e1000580.
doi:10.1371/journal.pgen.1000580
PMCID: PMC2712074  PMID: 19649314
12.  Meiotic Transmission of an In Vitro–Assembled Autonomous Maize Minichromosome 
PLoS Genetics  2007;3(10):e179.
Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.
Author Summary
The production of transgenic maize has traditionally used techniques that integrate DNA fragments into a host chromosome. This can disrupt important native genes or can lead to poor expression of the added gene; consequently, large numbers of transgenic plants must be screened to find one suitable for commercial use. Further, there is a limit to the amount of DNA that can be integrated, making it difficult to add multiple genes at one time. Here, we describe a new system for delivering genes to maize. We constructed a minichromosome vector that remains separate, or autonomous, from the plant's chromosomes when introduced into maize cells. These minichromosomes were constructed from DNA sequences that naturally occur in maize centromeres, the chromosomal regions needed for inheritance. We characterized the behavior of Maize Minichromosome 1 (MMC1) through four generations, showing that it is efficiently inherited and that the genes it carries are expressed. This work makes it possible to design minichromosomes that carry several genes, enhancing the ability to engineer plant processes, including improving disease resistance, drought tolerance, or the production of complex biochemicals.
doi:10.1371/journal.pgen.0030179
PMCID: PMC2041994  PMID: 17953486
13.  The Role of AtMUS81 in Interference-Insensitive Crossovers in A. thaliana 
PLoS Genetics  2007;3(8):e132.
MUS81 is conserved among plants, animals, and fungi and is known to be involved in mitotic DNA damage repair and meiotic recombination. Here we present a functional characterization of the Arabidopsis thaliana homolog AtMUS81, which has a role in both mitotic and meiotic cells. The AtMUS81 transcript is produced in all tissues, but is elevated greater than 9-fold in the anthers and its levels are increased in response to gamma radiation and methyl methanesulfonate treatment. An Atmus81 transfer-DNA insertion mutant shows increased sensitivity to a wide range of DNA-damaging agents, confirming its role in mitotically proliferating cells. To examine its role in meiosis, we employed a pollen tetrad–based visual assay. Data from genetic intervals on Chromosomes 1 and 3 show that Atmus81 mutants have a moderate decrease in meiotic recombination. Importantly, measurements of recombination in a pair of adjacent intervals on Chromosome 5 demonstrate that the remaining crossovers in Atmus81 are interference sensitive, and that interference levels in the Atmus81 mutant are significantly greater than those in wild type. These data are consistent with the hypothesis that AtMUS81 is involved in a secondary subset of meiotic crossovers that are interference insensitive.
Author Summary
Meiosis is a specialized type of cell division in which one diploid progenitor cell divides into four haploid cells that are subsequently used for fertilization during sexual reproduction. During meiosis, chromosomes pair, synapse, and exchange genetic information, all of which are required for proper chromosome segregation during subsequent stages. Failure to properly segregate meiotic chromosomes often leads to genetic defects such as aneuploidy. Using the model plant A. thaliana, we have developed a powerful system for the visual analysis of meiotic recombination directly in the pollen, in which the four products of individual meioses are fused together in a tetrad. We have used this system to characterize the gene AtMUS81 and show that Atmus81 mutants have a moderate reduction in meiotic crossovers and are sensitive to a wide range of DNA-damaging agents. Importantly, the remaining crossovers in Atmus81still exhibit crossover interference, a phenomenon whereby one crossover inhibits the occurrence of other nearby crossovers. Our results suggest that AtMUS81 mediates a subset of meiotic recombination events in Arabidopsis that are insensitive to crossover interference.
doi:10.1371/journal.pgen.0030132
PMCID: PMC1941751  PMID: 17696612
14.  Who's driving the centromere? 
Journal of Biology  2004;3(4):17.
Centromere function is remarkably conserved between species, yet the satellite sequences that make up centromeric DNA are highly divergent. Proteins that bind these sequences appear to be evolving under positive selection, supporting a model wherein the interplay between centromeric repeats and the proteins that bind them creates an opportunity for an intriguing phenomenon known as centromere-based meiotic drive.
doi:10.1186/jbiol15
PMCID: PMC549717  PMID: 15485584

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