Polymorphic inverted repeats near coding genes impact chromatin topology and phenotypic traits in Arabidopsis thaliana.

Transposons are mobile elements that are commonly silenced to protect eukaryotic genome integrity. In plants, transposable element (TE)-derived inverted repeats (IRs) are commonly found near genes, where they affect host gene expression. However, the molecular mechanisms of such regulation are unclear in most cases. Expression of these IRs is associated with production of 24-nt small RNAs, methylation of the IRs, and drastic changes in local 3D chromatin organization. Notably, many of these IRs differ between Arabidopsis thaliana accessions, causing variation in short-range chromatin interactions and gene expression. CRISPR-Cas9-mediated disruption of two IRs leads to a switch in genome topology and gene expression with phenotypic consequences. Our data show that insertion of an IR near a gene provides an anchor point for chromatin interactions that profoundly impact the activity of neighboring loci. This turns IRs into powerful evolutionary agents that can contribute to rapid adaptation.

Genetic diversity loss in the Anthropocene.

Anthropogenic habitat loss and climate change are reducing species' geographic ranges, increasing extinction risk and losses of species' genetic diversity. Although preserving genetic diversity is key to maintaining species' adaptability, we lack predictive tools and global estimates of genetic diversity loss across ecosystems. We introduce a mathematical framework that bridges biodiversity theory and population genetics to understand the loss of naturally occurring DNA mutations with decreasing habitat. By analyzing genomic variation of 10,095 georeferenced individuals from 20 plant and animal species, we show that genome-wide diversity follows a mutations-area relationship power law with geographic area, which can predict genetic diversity loss from local population extinctions. We estimate that more than 10% of genetic diversity may already be lost for many threatened and nonthreatened species, surpassing the United Nations' post-2020 targets for genetic preservation.

Isolation, Library Preparation, and Bioinformatic Analysis of Historical and Ancient Plant DNA.

The ability to sequence DNA retrieved from ancient and historical material plays a crucial role in reinforcing evolutionary and anthropological inference. While the focus of the field is largely on analyzing DNA from ancient hominids and other animals, we have also learned from plant ancient DNA (aDNA), in particular, about human farming practices, crop domestication, environment management, species invasion, and adaptation to various environmental conditions. In the following protocols, we outline best practices for plant aDNA isolation, preparation for sequencing, bioinformatic processing, and authentication. We describe the process all the way from processing of archaeological or historical plant material to characterizing and authenticating sequencing reads. In alternative protocols, we include modifications to this process that are tailored to strongly degraded DNA. Throughout, we stress the importance of precautionary measures to successfully analyze aDNA. Finally, we discuss the evolution of the archaeogenomics field and the development of new methods, which both shaped this protocol. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of aDNA Alternate Protocol 1: Isolation of ultra-short DNA (Dabney modification) Support Protocol 1: Preparation of PTB-based mix Support Protocol 2: Preparation of binding buffer Basic Protocol 2: Preparation of genomic libraries Alternate Protocol 2: Preparation of genomic libraries with uracil removal Basic Protocol 3: Bioinformatic processing and authentication of aDNA.

Hybridization ddRAD-sequencing for population genomics of nonmodel plants using highly degraded historical specimen DNA.

Species' responses at the genetic level are key to understanding the long-term consequences of anthropogenic global change. Herbaria document such responses, and, with contemporary sampling, provide high-resolution time-series of plant evolutionary change. Characterizing genetic diversity is straightforward for model species with small genomes and a reference sequence. For nonmodel species-with small or large genomes-diversity is traditionally assessed using restriction-enzyme-based sequencing. However, age-related DNA damage and fragmentation preclude the use of this approach for ancient herbarium DNA. Here, we combine reduced-representation sequencing and hybridization-capture to overcome this challenge and efficiently compare contemporary and historical specimens. Specifically, we describe how homemade DNA baits can be produced from reduced-representation libraries of fresh samples, and used to efficiently enrich historical libraries for the same fraction of the genome to produce compatible sets of sequence data from both types of material. Applying this approach to both Arabidopsis thaliana and the nonmodel plant Cardamine bulbifera, we discovered polymorphisms de novo in an unbiased, reference-free manner. We show that the recovered genetic variation recapitulates known genetic diversity in A. thaliana, and recovers geographical origin in both species and over time, independent of bait diversity. Hence, our method enables fast, cost-efficient, large-scale integration of contemporary and historical specimens for assessment of genome-wide genetic trends over time, independent of genome size and presence of a reference genome.

RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis.

The RNA exosome is a key 3'-5' exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.

Alternative use of miRNA-biogenesis co-factors in plants at low temperatures.

Plants use molecular mechanisms to sense temperatures, trigger quick adaptive responses and thereby cope with environmental changes. MicroRNAs (miRNAs) are key regulators of plant development under such conditions. The catalytic action of DICER LIKE 1 (DCL1), in conjunction with HYPONASTIC LEAVES 1 (HYL1) and SERRATE (SE), produces miRNAs from double-stranded RNAs. As plants lack a stable internal temperature to which enzymatic reactions could be optimized during evolution, reactions such as miRNA processing have to be adjusted to fluctuating environmental temperatures. Here, we report that with decreasing ambient temperature, the plant miRNA biogenesis machinery becomes more robust, producing miRNAs even in the absence of the key DCL1 co-factors HYL1 and SE. This reduces the morphological and reproductive defects of se and hyl1 mutants, restoring seed production. Using small RNA-sequencing and bioinformatics analyses, we have identified specific miRNAs that become HYL1/SE independent for their production in response to temperature decrease. We found that the secondary structure of primary miRNAs is key for this temperature recovery. This finding may have evolutionary implications as a potential adaptation-driving mechanism to a changing climate.

Using herbaria to study global environmental change.

During the last centuries, humans have transformed global ecosystems. With their temporal dimension, herbaria provide the otherwise scarce long-term data crucial for tracking ecological and evolutionary changes over this period of intense global change. The sheer size of herbaria, together with their increasing digitization and the possibility of sequencing DNA from the preserved plant material, makes them invaluable resources for understanding ecological and evolutionary species' responses to global environmental change. Following the chronology of global change, we highlight how herbaria can inform about long-term effects on plants of at least four of the main drivers of global change: pollution, habitat change, climate change and invasive species. We summarize how herbarium specimens so far have been used in global change research, discuss future opportunities and challenges posed by the nature of these data, and advocate for an intensified use of these 'windows into the past' for global change research and beyond.

A Role for the F-Box Protein HAWAIIAN SKIRT in Plant microRNA Function.

As regulators of gene expression in multicellular organisms, microRNAs (miRNAs) are crucial for growth and development. Although a plethora of factors involved in their biogenesis and action in Arabidopsis (Arabidopsis thaliana) has been described, these processes and their fine-tuning are not fully understood. Here, we used plants expressing an artificial miRNA target mimic (MIM) to screen for negative regulators of miR156. We identified a new mutant allele of the F-box gene HAWAIIAN SKIRT (HWS; At3G61590), hws-5, as a suppressor of the MIM156-induced developmental and molecular phenotypes. In hws plants, levels of some endogenous miRNAs are increased and their mRNA targets decreased. Plants constitutively expressing full-length HWS-but not a truncated version lacking the F-box domain-display morphological and molecular phenotypes resembling those of mutants defective in miRNA biogenesis and activity. In combination with such mutants, hws loses its delayed floral organ abscission ("skirt") phenotype, suggesting epistasis. Also, the hws transcriptome profile partially resembles those of well-known miRNA mutants hyl1-2, se-3, and ago1-27, pointing to a role in a common pathway. We thus propose HWS as a novel, F-box dependent factor involved in miRNA function.