The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR, and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection.

Although dysploidy and polyploidisation events are known to be important drivers in the evolutionary history of the genus Crocus, only a few examples of natural hybrid origins have so far been documented. Here, we describe the phylogenetic affinities of five taxa in the Crocus vernus species complex from the Carpathian Basin in Central Europe. Genetic variability was evaluated using chloroplast DNA sequences of the accD-psaI intergenic spacer and amplified fragment length polymorphism (AFLP) in combination with karyological observations. Genetic diversity and differentiation of the seven investigated Crocus species were also evaluated using AFLP data. We created a phylogenetic hypothesis using both sequences and AFLP fingerprinting data under maximum parsimony (MP). AFLPs were also analysed by means of multivariate statistics using principal coordinate (PCoA) analysis and Bayesian clustering (BC) to test for hybridity. Both the AFLP and plastid phylogenetic trees separated the taxa into two groups: (1) a ‘Balkan’ clade, and; (2) an ‘Adriatic’ clade. The Balkan clade contained Crocus heuffelianus samples and one Crocus vittatus from Croatia as well as Crocus tommasinianus samples from Hungary; the Adriatic clade included Crocus vittatus and Crocus vernus samples from Hungary and Croatia as well as Crocus neapolitanus from Italy. A hard incongruence was found in the placement of the Slovakian Crocus scepusiensis which clustered inside the Balkan clade on our plastid tree, while it fell in the Adriatic clade on the AFLP tree. The same populations occupied intermediate position on the PCoA plot. The BC analysis assigned all Crocus scepusiensis specimens as F1 hybrids, while Crocus vernus and Crocus heuffelianus were assigned as parental species. Together with our cytological investigation that determined a 2n=18 chromosome number of Crocus scepusiensis, we conclude an allopolyploid hybrid origin for this Northern Carpathian taxon as the result of a cross between members of the 2n=10 Balkan clade (e.g., Crocus heuffelianus from Transylvania) and the 2n=8 Adriatic clade (Crocus vernus). A similar origin is postulated for the North Balkan Crocus vittatus (2n=18), which was clustered as an F1 hybrid in the BC analysis; thus, parallel evolution may have taken place in the northern and southern part of the Carpathian Basin beginning from the same parental species but leading to different allopolyploid derivatives.

Arabidopsis NODULIN HOMEOBOX (NDX) is a nuclear protein described as a regulator of specific euchromatic genes within transcriptionally active chromosome arms. Here we show that NDX is primarily a heterochromatin regulator that functions in pericentromeric regions to control siRNA production and non-CG methylation. Most NDX binding sites coincide with pericentromeric het-siRNA loci that mediate transposon silencing, and are antagonistic with R-loop structures that are prevalent in euchromatic chromosomal arms. Inactivation of NDX leads to differential siRNA accumulation and DNA methylation, of which CHH/CHG hypomethylation colocalizes with NDX binding sites. Hi-C analysis shows significant chromatin structural changes in the ndx mutant, with decreased intrachromosomal interactions at pericentromeres where NDX is enriched in wild-type plants, and increased interchromosomal contacts between KNOT-forming regions, similar to those observed in DNA methylation mutants. We conclude that NDX is a key regulator of heterochromatin that is functionally coupled to het-siRNA loci and non-CG DNA methylation pathways.

The authors would like to correct erroneous text relating to the Step-by-step protocol of the best-performing DRIP experiment (exp. 5) section in the Supplemental Material. The corrected text is as follows and has been updated in the Revised Supplemental Material online: "Cells were lysed in the lysis buffer provided by the NucleoSpin Tissue kit (Macherey-Nagel) at 65°C for 7 h (according to the kit protocol), or at 37°C overnight (where indicated in the main text)."

Arabidopsis NODULIN HOMEOBOX (NDX) is a plant-specific transcriptional regulator whose role in small RNA biogenesis and heterochromatin homeostasis has recently been described. Here we extend our previous transcriptomic analysis to the flowering stage of development. We performed mRNA-seq and small RNA-seq measurements on inflorescence samples of wild-type and ndx1-4 mutant (WiscDsLox344A04) Arabidopsis plants. We identified specific groups of differentially expressed genes and noncoding heterochromatic siRNA (hetsiRNA) loci/regions whose transcriptional activity was significantly changed in the absence of NDX. In addition, data obtained from inflorescence were compared with seedling transcriptomics data, which revealed development-specific changes in gene expression profiles. Overall, we provide a comprehensive data source on the coding and noncoding transcriptomes of NDX-deficient Arabidopsis flowers to serve as a basis for further research on NDX function.