Contrasted patterns of crossover and non-crossover at Arabidopsis thaliana meiotic recombination hotspots.

The vast majority of meiotic recombination events (crossovers (COs) and non-crossovers (NCOs)) cluster in narrow hotspots surrounded by large regions devoid of recombinational activity. Here, using a new molecular approach in plants, called "pollen-typing", we detected and characterized hundreds of CO and NCO molecules in two different hotspot regions in Arabidopsis thaliana. This analysis revealed that COs are concentrated in regions of a few kilobases where their rates reach up to 50 times the genome average. The hotspots themselves tend to cluster in regions less than 8 kilobases in size with overlapping CO distribution. Non-crossover (NCO) events also occurred in the two hotspots but at very different levels (local CO/NCO ratios of 1/1 and 30/1) and their track lengths were quite small (a few hundred base pairs). We also showed that the ZMM protein MSH4 plays a role in CO formation and somewhat unexpectedly we also found that it is involved in the generation of NCOs but with a different level of effect. Finally, factors acting in cis and in trans appear to shape the rate and distribution of COs at meiotic recombination hotspots.

Integrative epigenomic mapping defines four main chromatin states in Arabidopsis.

Post-translational modification of histones and DNA methylation are important components of chromatin-level control of genome activity in eukaryotes. However, principles governing the combinatorial association of chromatin marks along the genome remain poorly understood. Here, we have generated epigenomic maps for eight histone modifications (H3K4me2 and 3, H3K27me1 and 2, H3K36me3, H3K56ac, H4K20me1 and H2Bub) in the model plant Arabidopsis and we have combined these maps with others, produced under identical conditions, for H3K9me2, H3K9me3, H3K27me3 and DNA methylation. Integrative analysis indicates that these 12 chromatin marks, which collectively cover ∼90% of the genome, are present at any given position in a very limited number of combinations. Moreover, we show that the distribution of the 12 marks along the genomic sequence defines four main chromatin states, which preferentially index active genes, repressed genes, silent repeat elements and intergenic regions. Given the compact nature of the Arabidopsis genome, these four indexing states typically translate into short chromatin domains interspersed with each other. This first combinatorial view of the Arabidopsis epigenome points to simple principles of organization as in metazoans and provides a framework for further studies of chromatin-based regulatory mechanisms in plants.

Comparison of library preparation methods reveals their impact on interpretation of metatranscriptomic data.

BACKGROUND: Metatranscriptomics is rapidly expanding our knowledge of gene expression patterns and pathway dynamics in natural microbial communities. However, to cope with the challenges of environmental sampling, various rRNA removal and cDNA synthesis methods have been applied in published microbial metatranscriptomic studies, making comparisons arduous. Whereas efficiency and biases introduced by rRNA removal methods have been relatively well explored, the impact of cDNA synthesis and library preparation on transcript abundance remains poorly characterized. The evaluation of potential biases introduced at this step is challenging for metatranscriptomic samples, where data analyses are complex, for example because of the lack of reference genomes. RESULTS: Herein, we tested four cDNA synthesis and Illumina library preparation protocols on a simplified mixture of total RNA extracted from four bacterial species. In parallel, RNA from each microbe was tested individually. cDNA synthesis was performed on rRNA depleted samples using the TruSeq Stranded Total RNA Library Preparation, the SMARTer Stranded RNA-Seq, or the Ovation RNA-Seq V2 System. A fourth experiment was made directly from total RNA using the Encore Complete Prokaryotic RNA-Seq. The obtained sequencing data were analyzed for: library complexity and reproducibility; rRNA removal efficiency and bias; the number of genes detected; coverage uniformity; and the impact of protocols on expression biases. Significant variations, especially in organism representation and gene expression patterns, were observed among the four methods. TruSeq generally performed best, but is limited by its requirement of hundreds of nanograms of total RNA. The SMARTer method appears the best solution for smaller amounts of input RNA. For very low amounts of RNA, the Ovation System provides the only option; however, the observed biases emphasized its limitations for quantitative analyses. CONCLUSIONS: cDNA and library preparation methods may affect the outcome and interpretation of metatranscriptomic data. The most appropriate method should be chosen based on the available quantity of input RNA and the quantitative or non-quantitative objectives of the study. When low amounts of RNA are available, as in most metatranscriptomic studies, the SMARTer method seems to be the best compromise to obtain reliable results. This study emphasized the difficulty in comparing metatranscriptomic studies performed using different methods.

Genome-wide crossover distribution in Arabidopsis thaliana meiosis reveals sex-specific patterns along chromosomes.

In most species, crossovers (COs) are essential for the accurate segregation of homologous chromosomes at the first meiotic division. Their number and location are tightly regulated. Here, we report a detailed, genome-wide characterization of the rate and localization of COs in Arabidopsis thaliana, in male and female meiosis. We observed dramatic differences between male and female meiosis which included: (i) genetic map length; 575 cM versus 332 cM respectively; (ii) CO distribution patterns: male CO rates were very high at both ends of each chromosome, whereas female CO rates were very low; (iii) correlations between CO rates and various chromosome features: female CO rates correlated strongly and negatively with GC content and gene density but positively with transposable elements (TEs) density, whereas male CO rates correlated positively with the CpG ratio. However, except for CpG, the correlations could be explained by the unequal repartition of these sequences along the Arabidopsis chromosome. For both male and female meiosis, the number of COs per chromosome correlates with chromosome size expressed either in base pairs or as synaptonemal complex length. Finally, we show that interference modulates the CO distribution both in male and female meiosis.

Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA.

Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5'UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules.

Characterization of meiotic non-crossover molecules from Arabidopsis thaliana pollen.

Meiotic recombination is essential for proper segregation of homologous chromosomes and thus for formation of viable gametes. Recombination generates either crossovers (COs), which are reciprocal exchanges between chromosome segments, or gene conversion not associated with crossovers (NCOs). Both kinds of events occur in narrow regions (less than 10 kb) called hotspots, which are distributed along chromosomes. While NCOs may represent a large fraction of meiotic recombination events in plants, as in many other higher eukaryotes, they have been poorly characterized due to the technical difficulty of detecting them. Here, we present a powerful approach, based on allele-specific PCR amplification of single molecules from pollen genomic DNA, allowing detection, quantification and characterization of NCO events arising at low frequencies at recombination hotspots.

Hot regions of noninterfering crossovers coexist with a nonuniformly interfering pathway in Arabidopsis thaliana.

In most organisms that have been studied, crossovers formed during meiosis exhibit interference: nearby crossovers are rare. Here we provide an in-depth study of crossover interference in Arabidopsis thaliana, examining crossovers genome-wide in >1500 backcrosses for both male and female meiosis. This unique data set allows us to take a two-pathway modeling approach based on superposing a fraction p of noninterfering crossovers and a fraction (1 - p) of interfering crossovers generated using the gamma model characterized by its interference strength nu. Within this framework, we fit the two-pathway model to the data and compare crossover interference strength between chromosomes and then along chromosomes. We find that the interfering pathway has markedly higher interference strength nu in female than in male meiosis and also that male meiosis has a higher proportion p of noninterfering crossovers. Furthermore, we test for possible intrachromosomal variations of nu and p. Our conclusion is that there are clear differences between left and right arms as well as between central and peripheral regions. Finally, statistical tests unveil a genome-wide picture of small-scale heterogeneities, pointing to the existence of hot regions in the genome where crossovers form preferentially without interference.