An increasing number of studies have revealed that long non-coding RNAs (lncRNAs) play important roles in gene regulation and nuclear organization. Although the mechanisms are still largely unknown, many lncRNAs have been shown to interact with chromatin. Thus, one approach to understanding the function of these lncRNAs is to identify their sites of genomic interaction. Hybridization capture methods using oligonucleotide probes have been used for years to study chromatin-associated RNA. Recently, several groups have developed novel methods based on proximity ligation to investigate RNA–chromatin interactions at a genome-wide scale. This review discusses these technologies and highlights their advantages and disadvantages for the consideration of potential users. XIST lncRNA, responsible for X chromosome inactivation in mammals [XIST lncRNA is expressed from only one of the two X chromosomes. It coats the X chromosome and recruits multiple complexes including epigenetic modifiers that establish gene silencing and formation of facultative heterochromatin.Chromatin-associated RNAs (chrRNAs) have important roles in gene regulation, genomic organization, and chromatin structure maintenance. Although the majority of chrRNAs are nascent transcripts attached to chromatin by RNA polymerase II (Pol II) , chrRNAs mammals ,3. XIST cis-acting. However, it is often hard to distinguish the mature RNA molecules that regulate gene expression from transcribed lncRNAs involved in gene regulation independently of their RNA sequence. For example, the Airn lncRNA gene, which overlaps with Igf2r in antisense orientation in the mammalian genome, is required for silencing the paternal genes in the Igf2r cluster. It has been reported that transcription through the Igf2r promoter, rather than the sequence of the mature Airn RNA, leads to Igf2r silencing [Airn RNA molecules recruit repressive chromatin modifiers to distant, non-overlapped genes like Slc22a2, Slc22a3, and Pde10a to cause silencing [Airn lncRNA gene has two ways to repress gene expression in the Igf2r cluster. Although XIST is considered to be a cis-acting lncRNA, it can act over long genomic distances through travel of XIST RNA to distal regions of the X chromosome. Thus, the mode of action of cis-acting lncRNAs can be further subclassified.ChrRNAs (or lncRNAs) are generally categorized into two groups based on their mode of action. LncRNAs that regulate the expression of nearby genes are defined as ilencing . Subsequilencing . Thus, ttrans-acting lncRNAs. HOTAIR is one of the trans-acting lncRNAs reported. HOTAIR, is expressed from the HOXC locus and has been reported to silence HOXD genes by recruiting the repressive chromatin modifiers PRC2, LSD1, and CoREST/REST [HOTAIR in the MDA-MD-231 breast cancer cell line represses several genes independently of PRC2 [HOTAIR trans-regulates HOXD cluster genes owing to inconsistencies in the results of studies using Hotair mutant mice [The other group of chrRNAs, which regulate gene expression on a genome-wide scale, consists of EST/REST ,7,8. How of PRC2 . In addiant mice ,11,12,13XIST RNA was found to spread on the X chromosome at an initial stage of X-chromosome inactivation [MALAT1 and NEAT1, were also identified by using one-to-all methods [The functions of chrRNAs (or lncRNAs) have been reviewed comprehensively elsewhere ,15. Thistivation ,17. Many methods . However methods  and that methods . In addi methods , preventTo investigate lncRNA binding sites on chromatin, in 2011 two groups developed methods using biotinylated DNA probes. These methods are termed ChIRP-seq (chromatin isolation by RNA purification)  and CHARroX2, a gene involved in dosage compensation in Drosophila and associated with many regions on the X chromosome in Drosophila cells [HOTAIR-associated DNA, demonstrating that HOTAIR lncRNA preferentially associated with regions containing a GA-rich DNA motif [ChIRP-seq uses 20-mer biotin-conjugated DNA oligonucleotide probes to recognize the target lncRNA. Cells are crosslinked by glutaraldehyde or formaldehyde to stabilize RNA-chromatin interaction, sonicated for DNA fragmentation and then hybridized with the probes. To ensure specificity, multiple probes are designed and split into two separate pools (referred to as the “odd” and “even” pools) that identify common targets. After the reversal of crosslinking, genomic DNA is analyzed by high-throughput sequencing to identify genomic regions associated with the lncRNAs. To test the reliability of ChIRP-seq, the authors performed ChIRP-seq targeting la cells . They alNA motif . ChIRP-sroX2 in Drosophila [Xist genome-wide binding regions [CHART-seq, developed by Simon et al., is conceptually similar to ChIRP-seq . This prosophila , and was regions .Xist transcript (~17 kb)). The genomic regions are isolated followed by high-throughput DNA sequencing. Using RAP-DNA, the authors showed that Xist RNA is initially transferred to chromosome regions of the X chromosome that have high contact frequencies with the Xist transcription site as determined by genome-wide Chromatin Conformation Capture (Hi-C), and then gradually spreads to other accessible locations [RAP-DNA is another method that can be used to identify chromatin regions that interact with RNAs . RAP-DNAocations .In 2017, two groups reported the first “all-to-all” methods to detect RNA–chromatin interactions genome-wide. These methods were termed MARGI (mapping RNA–genome interactions) and GRID-seq  ,25. In 28 cells fixed in 1% formaldehyde. After nuclear isolation, all proteins are biotinylated using iodoacetyl PEG2 biotin (IPB) in preparation for subsequent purification using streptavidin beads. This step can ease the numerous buffer changes required in MARGI. In iMARGI, the ligation steps are performed in situ instead of on streptavidin beads. This modification may contribute to the lower number of input cells required for iMARGI (~5 × 106) owing to less diffusion of RNA molecules or to less non-specific ligation in nuclei. Both MARGI and iMARGI use a biotinylated bivalent DNA linker to ligate RNA and DNA in formaldehyde-fixed nuclei. In MARGI, streptavidin beads require to be blocked with D-biotin before RNA ligation. Because both proteins and the linker are biotinylated this is done to prevent free linkers from binding to streptavidin beads. The 5′ end of this linker is pre-adenylated with Mth RNA ligase. T4 RNA ligase 2, truncated KQ is used for RNA ligation, which ligates the pre-adenylated 5′ end of the linker to the 3′ OH end of the RNA. The double-stranded end of the linker ligates with T4 DNA ligase to the digested and end prepared genomic DNA (digested by HaeIII for MARGI and by AluI for iMARGI). It is critical to keep track of the directionality of the linker to identify the RNA or DNA end for subsequent data analysis. As discussed later  that is pre-adenylated at the 5′ end. T4 RNA ligase 2, truncated KQ is used for RNA ligation, which can ligate pre-adenylated 5′end of DNA or RNA to the 3′ OH end of the RNA. However, the authors have not reported which linker with pre-adenylated DNA or RNA could be ligated to RNA more efficiently and why they used the linker with pre-adenylated RNA [MmeI is then used to cut 18–20 bases away from its recognition sites in the linker to generate paired-end tags (cDNA (18–20 bp)—linker-DNA—genomic DNA (18–20 bp)). After separation by PAGE, digested products of the correct size are purified from the gel and then ligated to the Y-shaped sequencing adapters, which preserve the directionality of the library for PCR amplification.GRID-seq uses a biotinylated bivalent linker to ligate RNA and DNA in situ in nuclei double-fixed in formaldehyde and disuccinimidyl glutarate. Approximately 2 × 10ated RNA ,29. AfteDpnII and DNA ligation reactions are performed. Unlike in GRID-seq, the ligated molecules in ChAR-seq are sonicated to generate smaller fragments for library construction. ChAR-seq has been tested initially with Drosophila cells. Later the authors reported the use of human embryonic stem cells (hESCs), human retinal pigmented epithelial cells (hTERT RPE-1) and nuclei isolated from Xenopus laevis embryos in their protocol paper although they did not provide any data [Drosophila cells because the genome size of mammals is much larger than that of Drosophila. If so, mammalian cells will require more high-throughput sequencing reads to generate usable sequence reads.ChAR-seq is another method to capture RNA–chromatin interactions based on proximity ligation. First, RNA is ligated to a biotinylated bivalent linker. After second-strand synthesis using one of the DNA strands in the linker as a primer, genomic DNA is digested by any data . Random 6 mammalian cells (mouse ES cells (mESCs) and mouse oligodendrocyte progenitor cells (mOPCs)) fixed in formaldehyde (1% or 2%). One end of the linker is single-stranded DNA that is pre-adenylated at the 5′ end for RNA ligation and the other end is double-stranded DNA that is ligated to genomic DNA ends digested by DNase I. Similar to the GRID-seq linker, the RADICL-seq linker contains two restriction enzyme recognition sites  that direct cutting within the RNA and DNA ends of the ligation product to generate tags of 25–27 bp each. After separation by PAGE, digested products of the correct size are purified from the gel. The Y-shaped sequencing adapters are then ligated to the biotin-purified fragments for library construction. One unique feature of RADICL-seq is the use of RNase H treatment to reduce abundant nascent transcripts. As an additional control, actinomycin D, which inhibits Pol II, is used before cell fixation to stop Pol II transcription and thus eliminate nascent transcripts.RADICL-seq is the latest all-to-all sequencing method and was published in 2020 . DespiteAll four all-to-all methods described above are conceptually similar because they are based on proximity ligations; however, they have some differences in experimental conditions and data processing. In brief, they differ in four main technical aspects: (1) number of cells required, (2) crosslinking conditions, (3) method used to digest genomic DNA, and (4) use of long reads versus reads trimmed by restriction enzyme digestion . We discDrosophila cells. GRID-seq has been tested with both Drosophila and mammalian cells. Because mESCs were analyzed using both GRID-seq and RADICL-seq, these methods can be compared directly. The original MARGI protocol uses ~4 × 108 cells (human embryonic kidney (HEK) and H9 (human ESC)), which is a fairly large number of cells compared with other methods. Development of iMARGI reduced the required cell quantity to a more treatable number, i.e., ~5 × 106 human foreskin fibroblasts (HFFs). GRID-seq used ~2 × 106 mammalian cells (human MDA-MB-231 (breast cancer cells), MM.1S (multiple myeloma cells) and mouse ESC) or 1 × 107Drosophila cells (S2). ChAR-seq was successfully used with 1-1.5 × 108Drosophila cells (CME-W1-cl8+ and Kc167). RADICL-seq was successfully performed with 2 × 106 mammalian cells (mouse ESCs and OPCs). Thus, GRID-seq and RADICL-seq could be achieved with only 2 × 106 cells, whereas MARGI and ChAR-seq require higher numbers of cells  in the fixation step and additional disuccinimidyl glutarate (DSG), a strong protein–protein crosslinker. Although the authors did not show the data, they mentioned that they found more small RNAs when using 3% formaldehyde + DSG than with weaker crosslinking conditions  . The RADHaeIII (iMARGI: AluI); GRID-seq, AluI; ChAR-seq, DpnII).All four protocols begin with the nucleus isolation step by using a lysis buffer containing non-ionic detergent (0.2% NP-40), after which the nuclei are permeabilized by SDS. Except for RADICL-seq, which uses DNase I to digest the genomic DNA, the protocols use restriction enzymes  [Bonetti et al.  found thregions) . In contregions) . Thus, dMmeI and EcoP15I, respectively) to digest the ligated products into fixed-size fragments. In ChAR-seq, the ligation products are sonicated to obtain smaller fragments for library construction. One of advantages of iMARGI is that longer sequencing reads can be obtained. On the other hand, in GRID-seq and RADICL-seq the entire linker must be sequenced to identify which end of the sequence corresponds to RNA and which corresponds to DNA. To obtain fixed-size fragments can be sequenced, GRID-seq uses MmeI, which cuts both the RNA and DNA ends to generate tags of 18–20 bp each, and only these 18–20 bp will be used for mapping. RADICL-seq instead uses EcoP15I, which digests 25–27 bp away from its recognition sites. RADICL-seq therefore results in longer reads for mapping and about three times as many uniquely mapped reads than GRID-seq (45% vs. 14% of mappable reads to the mouse genome). The restriction-enzyme-based methods such as GRID-seq and RADICL-seq ensure that all reads contain both RNA and DNA sequences; in contrast, MARGI and ChAR-seq do not produce fixed-size fragments and require more sequencing reads to obtain comparable numbers of RNA and DNA interactions. Future technology development should consider methods to achieve even longer reads to further complete the RNA/DNA interactome with more broadly repeated sequences of the genome, including retrotransposons.After RNA and DNA ligation to the bivalent linker, the methods differ in how the ligation products are treated. In MARGI, the ligated molecules are circularized for library construction. GRID-seq and RADICL-seq use a linker that contains two recognition sites for type II and III restriction enzymes, respectively , moderate with ChIRP-seq (25–30%) and lowest with CHART-seq (10%) [The major limitation of all-to-all methods is the requirement for a large number of sequencing reads to detect RNA species with low expression and to generate interpretable interaction data. To validate the specificity of the detected biological interactions, each all-to-all method should be compared with one or more one-to-all methods using several RNAs as positive controls. hIRP-seq –30% and Here, we have reviewed several one-to-all and all-to-all technologies used to study RNA–chromatin interactions. In particular, we have argued that the proximity-based all-to-all methods are more powerful for understanding the role of chromatin-associated RNAs and genome organization. Zhou et al. also pointed out that Hi-C was a main technology to study genome organization before developing of the proximity-based all-to-all methods to capture RNA–chromatin interactions. The all-to-all methods have more advantages to study cell-type-specific genomic interactions because RNAs play roles for cell-type-specific gene regulation even though DNA-DNA contacts detected by Hi-C are not varied . As discIn conclusion, these technologies to study RNA–chromatin interactions have great potential to enable exploration of chrRNAs and identify their functions in gene regulation and genome organization. By building on these current protocols, researchers may develop more advanced protocols, perhaps even capable of analyzing single cells. Recently RIC-seq (RNA in situ conformation sequencing), which is also a technology based on proximity ligation was reported . This te


N = 248) took part in a common resource game in groups of four under each of the three choice systems. We examined the average percentage taken of the resource as well as satisfaction and fairness ratings depending on the choice system in interaction with (a) whether the distribution of opportunities among group members was symmetric versus asymmetric, and (b) the status of an actor (advantaged versus disadvantaged) within asymmetric groups. Both implemented collective choice systems (median choice and majority voting) increased sustainable resource management, especially in asymmetric groups, by restricting overconsumption of advantaged individuals, as well as satisfaction and fairness ratings. Collective choice increased collective welfare by increasing profits of disadvantaged individuals and members of symmetric groups. The results indicate that in the presence of asymmetric distribution of opportunities, collective choice is a means to reconcile the conflict between individual and collective interests in social dilemmas and to foster sustainable resource management.Asymmetric distribution of opportunities among actors can reinforce the conflict between individual and collective interests in social dilemma situations. The present study investigates the interplay of asymmetric distribution of opportunities to consume resources and three choice systems: individual choice, median choice, and majority voting. Participants ( Prior work revealed that asymmetric distribution of opportunities within groups is associated with lower levels of cooperation10 and increases the risk of failing to reach a collective goal, such as jointly raising a target sum of money to avoid losing the money altogether11.The consumption of limited, shared resources represents a social dilemma, a situation where individual and collective interests conflict. Often the groups of actors, be that of individuals or collectives like states, are characterised by asymmetric distribution of opportunities , such that some actors have privileged access to common resources while others are disadvantagedAs asymmetry between actors can be a challenge for cooperation, a serious question emerges about how to establish cooperation in the presence of asymmetric distribution of opportunities. We propose implementing a decision process at the collective level to foster sustainable resource management in the presence of asymmetric opportunities. Specifically, the current investigation examines the effectiveness of two collective choice systems in fostering sustainable management of common resources in a common resource game, when involved actors’ opportunities to harvest from the resource are distributed symmetrically versus asymmetrically. Additionally, we examine how satisfied the actors are with each choice system in interaction with (a) whether actors within groups have symmetric versus asymmetric opportunities to harvest from the resource, and (b) the status of an actor (advantaged or disadvantaged) within asymmetric groups.13. Although groups of cooperative individuals, who restrict their consumption behaviour to benefit the collective, profit more than groups of defectors, individuals can earn most if they do not cooperate but instead exploit the common resource14. Usually, the consumption of resources is determined by individual choice, that is, individuals decide simultaneously and in isolation. However, individual choice fosters overexploitation of common resources15 and failure to reach a collective goal11. Evidently, the question is whether collective  choice may facilitate pursuing interests of the collective by forcing individuals to reach a decision at the group level. Indeed, voting on an extraction level has been documented as one feasible way to increase sustainable resource use17.The consumption of shared resources constitutes a social dilemma, because sustaining the resource (so that many can profit from it) requires individual restraint20 with considerable consequences for cooperation. An asymmetric distribution of opportunities in social dilemma situations can induce ambiguity on how to act—should all group members behave in the same way or should the asymmetry be reflected in their behaviour? Although individuals seem to strive for equal costs and payoffs23, asymmetry induces uncertainty about the fair solution27. In fact, asymmetric opportunities promote egocentric assessments of fairness28. Overall, cooperation levels are typically lower in groups characterised by asymmetric distribution of opportunities among actors29. Voting on a binding common extraction level could overcome the negative effects of asymmetry as outlined in what follows.So far, most studies on social dilemma situations involved groups of individuals with symmetric opportunities. However, actors in the real world in many cases do not have the same preconditions31. The specific voting system influences the likelihood of reaching a decision and its outcomes. For example, agreement is more often reached when determined by a majority rule compared to an unanimity rule31. In general, cooperation depends on whether the actors reach an agreement31 and whether the voting is binding or transgressions can be punished32.Voting is a commonly used mechanism to reach a collective decision as a function of individual interests. Empirical evidence suggests that voting on a common contribution or extraction level can increase cooperation and the sustainable use of common resources30 and the implementation of a majority voting system33. In the implemented median choice system, each group member suggests an extraction level, and the median of these suggestions is implemented for all group members. The system leads to sustainable resource use when there is a majority of cooperators who can overrule a minority of non-cooperators14. In the implemented majority voting system, each group member proposes an individual extraction level for each group member, including themselves. All group members then vote on these proposals. If a majority votes for the same proposal, it is implemented. If no proposal reaches a majority, each group member can decide individually how much to extract in that period.In the present study, we examine two specific forms of collective choice, the implementation of a median choice system21, suggesting equal extraction levels is probably more likely to reach a majority in contrast to suggestions that favour individual group members. Independent of the extraction level, the implemented median voting system guarantees equal payoffs for all group members, rendering irrelevant the factor of distribution among group members. When there is a majority of cooperators, both systems should lead to more sustainable resource management compared to individual choice. However, asymmetric opportunities could make it more difficult to reach a common decision in the majority voting system, so the median choice system could be most efficient in promoting sustainable resource use. Regarding the evaluation, on the other hand, the majority voting system with the two-step process of proposing extraction levels for every group member and then voting on these proposals offers more participation in the decision process and might therefore be perceived more positively than the median choice system.Extraction decisions should take into account two factors: (a) development of the resource, and (b) distribution of extraction levels across group members. Long-term interest should always be to sustain the resource preserving enduring extraction possibilities. Therefore, the group should not exceed the sustainable rate of consumption that allows the resource to regenerate. The question remains about how extractions from the resource should be distributed among group members. The fairness principle of equality suggests equal rates for every group member; however, it is the interest of every group member to maximise his or her own share. Therefore, individuals could win the most if they voted on a solution that granted them the maximum while excluding other group members from consumption . The majority voting system implemented in the present study in principle allows for differences in group members’ extraction levels, but as individuals usually strive for equal payoffs20. We examine how asymmetric opportunities interact with collective choice systems regarding sustainable behaviour in a common resource game. Asymmetry likely influences the effectiveness of structural solutions to dilemmas : Symmetric actors profit equally from a collective decision; in asymmetric groups, however, different group members might be advantaged by different choice systems, which could make it more difficult to reach a common decision in asymmetric groups. On the other hand, we propose that in the presence of asymmetric opportunities in particular, collective choice can be a means of improving resource management by preventing over-consumption by advantaged group members. We examine sustainable resource management within three specific choice systems—individual choice, median choice, and majority voting—across groups with symmetric versus asymmetric opportunities. We argue that the median choice system is effective if there are enough cooperative individuals within the group. However, the median choice system is restrictive simply by excluding the extremes, and it does not allow for any variations among individuals’ profit. The majority voting system, on the other hand, seems to be a more realistic alternative, allowing more participation in the decision process and variation in individuals’ profit. However, whereas a common decision in the median choice system is guaranteed, the majority voting system leaves room for failure to reach a common decision. Therefore, we seek to answer the question regarding which of the implemented collective choice system is best suited to foster sustainable resource management in the presence of asymmetric opportunities.The present investigation advances the understanding of behaviour in social dilemmas by highlighting the importance of considering asymmetric opportunities of actors in interaction with situational constraints. In the real world, there are huge differences in  opportunities between individuals or other parties like states or communities that influence resource consumptionFrom an applied perspective, we want to emphasise that it is not sufficient to demonstrate that a system is effective in promoting sustainable resource management for it to be implemented; it is also necessary that the actors involved support the system. We therefore include in our analyses an evaluation of each system regarding satisfaction with the outcome and fairness perceptions as a second dependent variable. It remains an open question how collective choice systems that promote a common decision on how to behave in a dilemma are being evaluated and how these evaluations are influenced by symmetric versus asymmetric opportunities within the group, on one hand, and by the status of the actor (advantaged versus disadvantaged) on the other.Asymmetry within groups fosters unsustainable resource use.Both collective choice systems, median choice and majority voting, foster more sustainable resource use compared to individual choice.To examine the interplay of asymmetric opportunities within groups and different choice systems on sustainable resource management, participants took part in a common resource game in groups of four see “” section(c)The collective choice systems have a greater effect in fostering sustainable resource use in asymmetric compared to symmetric groups.(d)Collective choice should in particular restrict the resource consumption of advantaged group members compared to disadvantaged group members.We further propose that the collective choice systems can overcome negative effects of asymmetry and therefore can prove especially effective in asymmetric groups. Both collective choice systems diminish the influence of the extreme—in the median choice system the two extreme proposals are discarded, and in the majority voting system proposals that favour individual group members are not likely to receive a majority. Therefore, both collective choice systems should be especially effective in altering behaviour of advantaged group members within asymmetric groups, for which more extreme extraction behaviour should be observed under individual choice. Regarding the interaction of asymmetry and choice system, we therefore expected the following:28, advantaged group members might evaluate the individual choice system more favourably, while disadvantaged group members might prefer the collective choice systems. On the other hand, individuals seem to strive for equal costs and payoffs23, so all group members could evaluate the collective choice systems more favourably than individual choice if these systems promote more equal profits. Overall, we expected higher satisfaction and fairness ratings in the collective choice systems compared to individual choice, as these systems foster sustainable resource management and therefore higher profits in the long run.Additionally, we explored how each choice system is being evaluated by assessing ratings on outcome satisfaction and fairness perception after each choice system. One could expect differential effects of asymmetric opportunities on satisfaction and fairness ratings depending on choice system. As asymmetry fosters egocentric assessment of fairnessAs the resource doubled in size between periods, all group members could extract a rate of 12.5% in each period while sustaining the resource (see Methods section for more details). In all three choice systems, the average percentage taken significantly exceeded this threshold, which led to an overall decrease of the resource over the six periods ; this was increased significantly to 14.24% in the asymmetric condition . Advantaged group members across choice systems took a significantly higher percentage of the resource available  than did disadvantaged group members  in the individual choice system, and they took significantly less in the median choice system  and in the majority voting system , as well as individual choice and majority voting  was different from zero in the expected direction. Whereas the average percentage taken decreased under median choice and majority voting for the majority of participants, we also observed cases where the average percentage taken did not change or even increased, which is reflected in the random effects for person  =  − 7.08, p < 0.001), and a small increase for disadvantaged group members  = 1.66, p = 0.097; the conditional effects describe the difference in means between two conditions under a certain condition: here, difference between average percentage taken in the median choice system and the individual choice system by disadvantaged group members). In the majority voting system, advantaged group members’ extraction level decreased  =  − 4.02, p < 0.001), whereas the average percentage taken did not change for disadvantaged group members  = 0.22, p = 0.828). Therefore, collective choice mainly reduced over-consumption by advantaged group members, which fostered the overall lower percentage taken. The median choice by its nature eliminated the difference in extraction levels between advantaged and disadvantaged group members, yet advantaged group members still extracted significantly more than disadvantaged group members in the majority voting system  = 5.25, p < 0.001). In sum, median choice, as well as majority voting, fostered sustainable resource management in comparison to individual choice in both symmetric and asymmetric groups.The analysis examining the interplay of asymmetric opportunities and collective choice on extraction behaviour revealed no evidence that the collective choice systems had differential effects on symmetric versus asymmetric groups  and stipulated mostly equal extraction rates for all group members . Elected proposals in asymmetric groups were equally sustainable , but varied significantly more between group members . In symmetric and asymmetric groups, proposals that were not elected were on average also close to the sustainable rate , but proposed extraction rates differed substantially between group members  = -2.98, p = 0.003). In asymmetric groups, the variation in proposed extraction levels between group members was reflected in higher proposed extraction levels for advantaged and lower proposed extraction levels for disadvantaged group members  = 0.90), the more the average extraction rate deviated from the sustainable rate of 12.50 in either direction , and the higher the variation among group members .Analysing the voting proposals over periods one to five revealed that in symmetric groups elected proposals were on average below the fair and sustainable rate of 12.5% (SD = 1.68). In periods with a successful voting decision, the average percentage taken was 11.86% (SD = 2.84) compared to 15.42%  after unsuccessful voting  decisions. After a successful voting decision, the average percentage taken was thus decreased compared to extraction under individual choice  = − 6.67, p < 0.001), whereas the average percentage taken was even slightly increased compared to extractions under individual choice when no proposal was elected  = 2.51, p = 0.012; for more details see Supplementary Information). After successful voting decisions, there were no differences in the average percentage taken between symmetric and asymmetric groups  =  − 0.47, p = 0.638) or between advantaged and disadvantaged group members  = 0.91, p = 0.364). If no proposal was elected, the average percentage taken differed between symmetric and asymmetric groups  = 2.16, p = 0.032) and between advantaged and disadvantaged group members  = 5.58, p < 0.001).To further explore the effect of the majority voting system on sustainable resource use, we examined the impact of successful voting decisions which ranged over periods one to five from 0  to 5  with a mean of 2.69  than individuals in groups with a symmetric distribution of opportunities  compared to disadvantaged group members . Satisfaction and fairness ratings were significantly increased in both collective choice systems  compared to individual choice , as well as individual choice and majority voting  was different from zero in the expected direction.Individuals in groups with an asymmetric distribution of opportunities across all choice systems rated satisfaction and fairness significantly lower  = 7.46, p < 0.001) but only to a lesser extent in asymmetric groups  = 3.88, p < 0.001). In an attempt to further explore this pattern, we tested whether asymmetric opportunities lead to fewer successful voting decisions, and whether this in turn predicts satisfaction and fairness ratings. We indeed found that for the majority voting system the number of successful voting decisions  partially mediated the effect of asymmetry on satisfaction and fairness ratings . Asymmetry led to fewer successful voting decisions , whereas the number of successful voting decisions predicted satisfaction and fairness ratings . The order in which participants completed the choice systems had an impact on satisfaction and fairness ratings. Specifically, symmetric groups rated individual choice more favourably when it was the first system they experienced compared to when they first experienced one of the collective choice systems. Therefore, the increase in satisfaction and fairness ratings was larger in symmetric groups when they started with collective choice. For asymmetric groups, the increase in satisfaction and fairness ratings in the majority voting system was larger when they started with individual choice (see Supplementary Information for details).The analysis including the interaction of the choice systems and the asymmetry contrasts revealed no interaction of asymmetry with median choice  = 3.77, p < 0.001) than for disadvantaged  = 7.02, p < 0.001) group members  = 1.58, p = 0.114) compared to disadvantaged group members  = 3.90, p < 0.001), although the interaction term did not reach significance (p = 0.051).The increase in satisfaction and fairness ratings in the median choice system depended on group members’ status as advantaged or disadvantaged, indicating a smaller increase for advantaged  = 3.87, p < 0.001) and not significant for asymmetric groups  = 1.54, p = 0.124), whereas the effect of median choice did not differ between symmetric and asymmetric groups  =  − 0.68, p = 0.494; majority voting: b = -0.96, t(490) =  − 0.30, p = 0.761) but a large increase in profit for the latter  = 6.19, p < 0.001; majority voting: b = 7.82, t(490) = 2.48, p = 0.013; see Fig. Although in both collective choice systems participants took on average a lower percentage of the resource, both median choice and majority voting led to overall higher profits compared to individual choice  the resource would decrease to contain only 60 units after period six, whereas with the average percentage taken under median choice (13.32%) the resource would still contain 109 units. If every group member extracted as advantaged group members did under individual choice (17.44%), the resource would decrease to only 9 units within six periods, whereas if every group member extracted as disadvantaged group members did under individual choice (12.92%), it would still contain 130 units, which is fourteen times the amount.Our findings highlight the importance of taking into consideration asymmetric distributions of opportunities among actors in social dilemma situations. In line with other findings34. Accordingly, there is good reason to assume that implementation of collective choice systems will find support among the involved actors.Relevant to our second outcome, satisfaction and fairness ratings, both collective choice systems were evaluated more favourably compared to individual choice. However, satisfaction and fairness ratings in the majority voting system compared to individual choice increased only in symmetric groups, which might be explained by a lower rate of successful voting decisions in asymmetric groups. The increase in satisfaction and fairness ratings in the collective choice systems did not depend on whether group members were advantaged or disadvantaged. Although advantaged individuals extracted more under individual choice, they did not rate this system more favourably than the collective choice systems. These findings suggest that many individuals involved in resource dilemmas support a shift from individual to collective choice, even if that might restrict their own extraction behaviour as advantaged group members. It would have been an interesting additional dependent variable to ask participants directly which system they prefer for another round of play. With regard to different punishment systems, it has been shown that outcome satisfaction and fairness perception predicted the willingness to continue under the present systemOverall, profits were increased in both collective choice systems compared to individual choice. This was characterised by disadvantaged individuals and symmetric groups gaining significantly under the collective choice systems, while advantaged individuals profited equally from all three systems. Thus, collective choice fosters sustainable resource management and improves the welfare of the collective. Notably, however, in both collective choice systems profit as well as satisfaction and fairness ratings were still significantly lower in asymmetric compared to symmetric groups. This implies that research should not focus just on (collective) choice systems to foster sustainable resource management, but also search for solutions to reduce asymmetry between actors. In the following section, we outline additional ideas for future research along with limitations of our study.21. In this study, we aimed to examine whether installing a majority voting system increases sustainable resource use without the requirement of reaching a successful voting decision. Previous research suggests that the process of deciding as a group can increase cooperation even after unsuccessful voting decisions17. However, extraction behaviour depended on whether or not groups reached an agreement35, that is, extraction levels after successful voting decisions were substantially decreased compared to individual choice while extraction levels after unsuccessful voting decisions were even slightly increased compared to extractions under individual choice. Although the majority voting system overall improved sustainable resource management, it still resulted in differences in extraction level and profit between advantaged and disadvantaged group members. In fact, the variation in proposed extraction levels between group members in asymmetric groups reflected overall higher proposed extraction levels for advantaged and lower proposed extraction levels for disadvantaged group members, whereas there were on average no differences in proposed extraction levels for group members of symmetric groups. Voting proposals that were supported by a majority in symmetric and asymmetric groups mainly suggested equal extraction rates for all group members and were on average sustainable grow). In asymmetric groups the variation in elected voting proposals was increased compared to symmetric groups, but still small compared to proposals that were not elected. Therefore, the differences in extraction levels among group members originated mainly from unsuccessful voting decisions followed by individual extraction.In the majority voting system, a voting decision was only reached in half of the trials, which is comparable to rates observed in other studiesWhether or not a common decision was reached in the majority system also affected satisfaction and fairness ratings. Asymmetric groups often failed to find agreement in the majority voting system, which in turn predicted lower satisfaction and fairness ratings. It is important to interpret these results with caution, as the differences between extraction after successful and unsuccessful voting decisions could be due to the decision process via majority voting but could also  be explained by the fact that more cooperative groups more often reached a common decision. Of note, groups contributed unequal numbers of observations regarding the extraction after successful and unsuccessful voting decisions . Therefore, it is an interesting topic for future research to explore how to increase the rate of successful voting decisions, for instance by allowing more than one preference or voting round to answer the question about whether reaching a common decision via majority voting per se can increase sustainable behaviour.36. It remains an open question to what extent the findings with this manipulation of asymmetry can be generalised to contexts where inequality reflects meritocratic or other social rules. It is possible that advantaged group members who feel they earned their status would evaluate collective choice systems that restrict their extraction behaviour more negatively than advantaged group members who were randomly assigned to this role. Another characteristic of our manipulation of asymmetry are the equal rates of advantaged and disadvantaged group members within asymmetric groups. This precludes that members of one group determine which proposal is elected in the majority voting system. It remains possible that proposals with more variation in extraction levels among group members would be elected if one group (advantaged or disadvantaged) constituted a majority. Varying the group composition regarding advantaged and disadvantaged group members is therefore yet another important entry on the agenda for future research in this field.Regarding the generalisability of the results, some specificities of the implemented experimental conditions should be considered. The first concerns the manipulation of asymmetry: In the present study, we tested the effect of artificially and randomly created differences in opportunity to access a common resource. Even in this setting, we find that advantaged group members overuse the common resource. This effect could be even stronger when differences in opportunities are established based on prior efforts37. It is possible that these participants considered the level of incentives to be rather low, however there is evidence from other economic games showing that behaviour under high and low stakes is comparable39.A similar rationale applies to the collective choice systems, as we implemented two specific forms of median choice and majority voting. It may prove useful for future research to vary single aspects of the choice systems to provide information on what specific aspects of the choice systems foster sustainable resource management . Also, the study design included a fixed number of six periods under each choice system. Our data suggests that knowing the common resource game would end after six periods influenced behaviour, particularly in the sixth and therefore last period, in which many participants exploited the resource completely. It poses an interesting question whether the definite ending after six periods also influenced behaviour in the periods before and participants would have extracted more sustainably if the number of periods was undisclosed. Also, there may be individual differences in how the resource game was construed (as finitely or indefinitely repeated). However, as the resource size directly determined the maximum extraction possibilities, we suppose that differences in how the resource game was construed  would play out primarily in the sixth period. Another interesting topic for future research would be to examine the variability in extraction levels and the effects of the collective choice systems between individuals (random effects) including potentially underlying characteristics . It should also be noted that we used a student sample (mostly other than psychology students), recruited from a western university, and thus should be cautious with generalisations to populations with more diverse backgroundsTo conclude, the present investigation demonstrates that asymmetric opportunities within groups foster unsustainable resource management. Both implemented collective choice systems, median choice and majority voting, increase sustainable resource management in the presence of asymmetry, as they restrict the over-consumption behaviour of advantaged group members. This leads to overall higher profits as reflected in an increase for disadvantaged group members, whereas advantaged group members profit equally from all three systems. In addition, both collective choice systems are evaluated more favourably than individual choice independent of asymmetry. Thus, we identified collective choice as an effective means to overcome a social dilemma and to foster sustainable resource management in the presence of asymmetric opportunities.It is our understanding that only approbated psychologists conducting clinical studies are legally obligated to obtain ethical oversight (according to the Declaration of Helsinki). For psychological studies without any clinical measures, the American Psychological Association (APA) and the German Association of Psychologists (DGPs) provide guidelines for the responsible treatment of participants. We conducted our study in full accordance with these guidelines. The implementation of economic games to study behaviour in social dilemma situations was approved by the University of Ulm’s Central University Research Ethics Committee (application number 297/16). The implementation of follow-up studies applying methods that have been approved before does not, at our institute, require completion of an entire approval process. There was no deception of participants. Participants gave informed consent before starting the study protocol, and the study was conducted in accordance with relevant guidelines and regulations.40. Decisions in the resource game were incentivised, as participants’ extracted units were transformed into real money at the end of the experiment (for every unit extracted they received 0.01€). Earnings from the common resource game ranged between 1.24€ and 5.30€. When informed about their final income at the end of the experiment, participants were told that they would receive a minimum of 3€ even if their earnings fell below that.Participants played a total of 18 periods of a common resource game in groups of four. In the first period of each choice system the resource contained 160 units. After the group members made their extraction decision, the remaining units were doubled for the next period. Thus, if groups took exactly half of the units available, the resource would remain the same. If they took more than half of the units, the resource would decrease. If they took less than half of the units, the resource would increase. The resource could, however, never exceed the original number of 160 units. Participants were always informed about how many units the resource contained and the extraction limit before making their decision. All interactions were computer-mediated using z-Treemedian choice system, each group member indicated how many units he or she wanted to extract. The median of those proposals was extracted for each group member14. For instance, if the four group members wanted to extract 10, 12, 12, and 16 units, the median of 12 units would be automatically extracted for each group member. If they wanted to extract 10, 12, 14 and 16 units, the mean of the two middle proposals would be computed. The median was automatically extracted for all group members, independent of their status as advantaged or disadvantaged. Thus, the extraction in the median system could exceed the extraction limit for disadvantaged group members . After making their decision, participants were informed about the proposals of each group member, the median, and how many units were left for the next period. In the majority voting system, participants were asked to propose how many units each group member (including themselves) should extract. They were then presented the four proposals and asked to vote on which one should be implemented. Each participant was granted a single vote and no one could not abstain from voting. Votes for identical proposals were combined. If one proposal received a majority (at least three of four votes), it was implemented. If no proposal received a majority, each group member would decide individually how much to extract in that period. After voting, participants were informed if a proposal received a majority, and if so, how many units each group member received and how many units the resource contained for the next period. If no proposal received a majority, participants were informed that they could extract individually in that period, about the extraction decision of all group members, and how many units the resource contained for the next period.In the individual choice system, participants decided how many units to extract from the resource individually and simultaneously. After they made their decision, they were informed how many units the group extracted in total and how many units were left for the next period. In the n = 72) or disadvantaged (n = 72) within asymmetric groups was constant over the three choice systems.Participants were randomly assigned to one of two asymmetry conditions. In symmetric groups, all group members could extract up to one fourth (25%) of the resource in each period. In asymmetric groups, two advantaged group members could extract up to one third (33%) of the resource, while two disadvantaged group members could only extract up to one sixth (17%) of the resource. The asymmetry condition and the role assignment as advantaged . After giving informed consent, participants read the instructions for the common resource game including information on the basic structure, the choice systems, and the asymmetry conditions. The order of the systems was randomised across groups. Participants were then informed if their group was assigned to the symmetric (n = 104) or asymmetric condition (n = 144) and what percentage of the resource they could extract in each period.A priori power analyses in multi-level settings require detailed information  that was not available. We therefore planned on including at least 220 participants in the study. Within time and budget restrictions, we were able to recruit a total of 248 participants on campus of a German university  ranged between − 16.26 and 61.39 with a mean of 27.15 (SD = 15.49). Therefore, the vast majority of participants (N = 164) was characterised as prosocial, whereas 79 could be described as individualistic, 3 as altruistic, and 2 as competitive. Due to power issues, however, this measure was not included in any of the analyses for the present contribution.For exploratory purposes, we first measured participants’ social value orientation (SVO) using the slider measureParticipants sat in cubicles to ensure privacy and anonymity. The number assigned to each group member was randomly shuffled after each period: The participant who appeared as “group member 1” in period 1 could be “group member 3” in period 2. Therefore, participants could not deduce behaviour of a specific person across periods to prevent that previous behaviour of specific group members influenced later decisions . Note that despite this procedure, advantaged group members could infer something of the behaviour of other advantaged group members in cases where the other extracted more than one sixth of the resource. Additionally, to prevent order effects, the numbers were shifted for each group member, so that participant 1 would see the decision of participant 2 as “group member 1,” the decisions of participant 3 as “group member 2,” and the decisions of participant 4 as “group member 3.” For participant 2, the decisions of participant 3 were displayed as “group member 1,” the decisions of participant 4 as “group member 2,” and the decisions of participant 1 as “group member 3.”34  influenced my income.”) Participants responded on a scale ranging from 1 = I strongly disagree to 7 = I strongly agree. After all 18 periods were finished, we assessed demographic information  before informing participants about their final income. Participants received a minimum of 3€ (≈ $3.40) plus the amount they earned beyond that in the common resource game and the SVO measure .After each system, we assessed participants’ outcome satisfaction and their perception of fairness by three itemsThe first dependent variable was extraction behaviour. To account for the changing number of units available, we calculated the percentage taken by each individual relative to the size of the resource available. For the regression analyses, we averaged the percentage taken over all periods within one system. As participants behaved differently in the sixth period , we calculated the average percentage taken across periods one to five as the central dependent variable. The second outcome was the evaluation reflecting outcome satisfaction and fairness perception. Principal axis factor analyses revealed that both scales were represented by a common factor; we therefore combined all six items into one measure of satisfaction and fairness . To compare the choice systems, we used dummy variables for median choice and majority voting with individual choice as reference group. To examine the moderating role of asymmetry, we contrast coded the conditions to compare symmetric versus asymmetric groups on the one hand and (within asymmetric groups) advantaged versus disadvantaged group members on the other.43. For the average percentage taken, the intra class correlation (ICC) indicated that a substantial proportion of variance was explained by the participants as level-2 units (ICC = 0.25), and only a small percentage was additionally explained by the groups as level-3 units (ICC = 0.05). For satisfaction and fairness ratings, there was substantial variance due to participants (ICC = 0.14) and groups (ICC = 0.14). We therefore included random effects for participants for the average percentage taken and random effects for participants and groups for satisfaction and fairness ratings in the regression analyses. The fixed effects describe the average effect of choice systems across participants, whereas the random slopes indicate the variation in intercept and slope between participants.To account for the nested structure of the data, we applied multilevel mixed regressions using the R package nlmeSupplementary information.


Zea mays) centromeres, and back-spliced RNAs were generated from CRM1. We identified 3 types of CRM1-derived circular RNAs with the same back-splicing site based on the back-spliced sequences. These circular RNAs bound to the centromere through R-loops. Two R-loop sites inside a single circular RNA promoted the formation of chromatin loops in CRM1 regions. When RNA interference (RNAi) was used to target the back-splicing site of the circular CRM1 RNAs, the levels of R-loops and chromatin loops formed by these circular RNAs decreased, while the levels of R-loops produced by linear RNAs with similar binding sites increased. Linear RNAs with only one R-loop site could not promote chromatin loop formation. Higher levels of R-loops and lower levels of chromatin loops in the CRM1 regions of RNAi plants led to a reduced localization of the centromeric H3 variant (CENH3). Our work reveals centromeric chromatin organization by circular CRM1 RNAs via R-loops and chromatin loops, which suggested that CRM1 elements might help build a suitable chromatin environment during centromere evolution. These results highlight that R-loops are integral components of centromeric chromatin and proper centromere structure is essential for CENH3 localization.In most plants, centromeric DNA contains highly repetitive sequences, including tandem repeats and retrotransposons; however, the roles of these sequences in the structure and function of the centromere are unclear. Here, we found that multiple RNA sequences from centromeric retrotransposons (CRMs) were enriched in maize ( Circular retrotransposon RNAs generated by back-splicing bind to chromatin and mediate dynamic regulation of R-loops and centromeric chromatin loops in maize, driving centromeric chromatin organization and CENH3 localization. Z. mays), several centromeres  have been well sequenced [Centromeres are located in the primary constriction of chromosomes and enable the correct separation of chromosomes during mitosis and meiosis. Active centromeres are marked by a centromeric specific histone H3 variant, named CENH3 in plants ,2 and ceequenced ,9, and mThere are 4 kinds of CRMs in maize, CRM1–CRM4 ,12, withSaccharomyces cerevisiae), the topological structure of the centromere can be induced by the sequence of the single centromeric nucleosome [The complex composition and arrangement of centromeric repeats in most species make it difficult to fill centromeric gaps during genome assembly and recognize their roles in centromere organization . Many stcleosome . During cleosome . Not allDrosophila melanogaster) [in trans to regulate centromere activity, with too much or too little centromeric RNA leading to a centromere malfunction [Xenopus laevis is required for the localization and activation of the chromosomal passenger complex in the inner centromeric regions [Centromere transcription processes have been reported to play roles in centromere assembly ,25. The ogaster) . RNAPII-ogaster) . Centromogaster) ,30, and ogaster) . Specifiogaster) ,33. Furtogaster) , and traogaster) . In buddfunction . Long no regions . In maiz regions . Previou regions . CentromArabidopsis thaliana [Flowering Locus C in Arabidopsis takes part in the formation of chromatin loops to regulate gene expression [R-loops containing RNA:DNA hybrids and single-strand DNA (ssDNA) can also influence chromatin activity . In yeasthaliana . R-loopsthaliana . Chromatthaliana ,46; for pression . HoweverArthrobacter luteus (Alu) repeats in the flanking introns can lead to exon circularization [Oryza sativa) and polyploid cotton (Gossypium sp.) [Arabidopsis, circular RNA from exons was shown to bind to the target gene through the formation of R-loops, resulting in depressed gene transcription [Circular RNA is formed by fusing the downstream 3′-splice site and the upstream 5′-splice site of a pre-messenger RNA, through the process of back splicing . For cirrization . In planium sp.) ,51. Manyium sp.) –54; for cription . Unlike This study addresses the interaction between CRMs and centromere structure and function. Three types of circular RNAs are produced from CRM1 in maize, each with different sizes but the same back-splicing site. These circular RNAs can bind to centromeric chromatin through R-loops. The two R-loop sites inside a single circular RNA promote the formation of chromatin loops. When RNAi was performed to target the back-splicing site of the circular RNAs, the levels of R-loops and chromatin loops formed by the circular RNAs decreased; however, the R-loops of the two kinds of linear RNAs with similar R-loop formation sites increased. The high levels of R-loops and low levels of chromatin loops in the CRM1 regions led to a reduced CENH3 localization in the RNAi plants. The process of back splicing in retrotransposons was also found to be conserved in numerous crop species.For detailed analysis of centromeric RNA, we used anti-CENH3 RIP in the maize inbred line B73 to capture RNA bound to the centromeric regions, using both high-throughput sequencing and cDNA library screening methods . CentromThe commonly used mapping programs for high-throughput sequence analyses, including tophat2, hisat2, bwa, bowtie2 and STAR, could not handle the repeat sequences well; therefore, extensively used BLAST software was performed to detect all the reads mapped to the centromeric repeats . AbundanThe reads containing long terminal repeat (LTR) sequences were classified as LTR reads. Aside from these, the reads aligned to only 1 CRM element site were identified as nonspliced reads, while those aligned to 2 separate sites were referred to as spliced reads. LTR and nonspliced reads accounted for almost all of the reads from the CRM elements and were distributed across the CRM regions . A few sThe RNAs from nonspliced and LTR reads were directly transcribed from the CRM elements, which could be confirmed easily according to their DNA sequences. Many previous studies have explored the functions of the direct transcripts from the centromeres –36. CRM2Among all the back-spliced reads mapped to CRM1 in the RIP and input data, 4 and 6 reads were found to have the same back-splicing sites, suggesting that these reads may be derived from the same RNA . This baDespite sharing a back-splicing site, the lengths of these 10 reads from CRM1 were different; we only obtained an RNA sequence less than 250 nt in size after merging all the reads, which may not cover the whole length of the original back-spliced RNA . We thenTwo discontinuous portions of the 354-nt sequence, which we refer to as the 269-nt and 85-nt sequences, were exactly matched to the CRM1 element . The dow×) and Illumina (100×) whole-genome sequencing data to the assumed 354-bp DNA, and no reads in the public datasets were matched to the back-splicing junction site (× to 900×), together with 1 input sequencing (input-seq) dataset (genome coverage 3×), were BLAST-searched with the unassigned 354-bp DNA sequence as the template. No reads from the 3 public datasets and the input-seq dataset were found to match the region containing the back-splicing site. Only one read from our anti-CENH3 ChIP-seq dataset was mapped to the 354-bp sequence containing the back-splicing site -RNA/rRNA-RNA , which dTwo divergent primers (F4+R2 and F3+R3) for the 354-nt RNA were used to show the direct fusion of the 3′-splice site in the 85-nt sequence to the 5′-splice site in the 269-nt sequence Figs , and S2CAdditionally, the primers F2+R2 could be used to amplify a type of shorter sequence with an 8- to 26-nt fragment of the 85-nt sequence fused to the 5′-splice site in the 269-nt sequence Figs  and S2G.To confirm the full lengths of these circular RNAs, we performed northern blotting using RNAs purified with 354-bp biotinylated sense and antisense oligos. Digoxin-labeled 25-bp ssDNA probes from the 269-bp sequence were used for northern blotting. The 354-nt and 277- to 296-nt circular RNAs could be detected using both the sense and antisense probes; however, the 607-nt circular RNAs could only be detected using the sense probe . The RNaArabidopsis, the ribosomal DNA (rDNA) regions are highly enriched with R-loops, and the gene AT1G24510—which encodes a chaperonin-60/T-complex protein (TCP-1/cpn60) chaperonin family protein—showed no R-loop formation [How do the circular RNAs bind to the centromeric chromatin? First, we wondered whether the circular RNAs bind to the centromeres through R-loops. RNase H was used to treat B73 chromatin-binding RNAs to detect the formation of RNA:DNA hybrids by these circular CRM1 RNAs . The 354ormation . The conThe R-loop formation sites of these circular RNAs could adopt different forms, such as one circular RNA binding to only one single site (case 1), multiple circular RNAs binding to several sites inside one CRM1 element (case 2), or one circular RNA binding to two nearby sites simultaneously (case 3) . BecauseDpnII restriction endonuclease digestion to check whether chromatin loops were formed in CRM1 regions  containing the CRM1 elements and the internal reference gene SAM . High cAccording to the anti-CENH3 RIP data, the amount of nonspliced RNA from the CRM elements is much larger than that of the back-spliced RNA at the centromeric regions . To distAnti-S9.6 RIP of circular and linear CRM1 RNAs was conducted using chromatin-binding RNA samples Figs  and S3G,Agrobacterium tumefaciens–mediated transformation. The RNAi target sequence was designed to cover the back-splicing site of the circular RNAs, including a 204-bp sequence of the 269-bp region and the 85-bp sequence . Two positive transgenic events were obtained (RNAi 5 and 18). The levels of the 354-, 607-, and 277- to 296-nt circular RNAs were dramatically decreased in the T1 generation of the RNAi plants compared to the wild-type HiII plants (used for the transgenic transformation)  . The levi plants . The undf growth .1 generation of RNAi plants 5 and 18 compared to those of the HiII plants  was selected for the protoplast preparation due to its ability to stably transmit maize chromosomes [O. sativa), common wheat (Triticum aestivum), and sorghum (Sorghum bicolor) were also used for protoplast transformations. Alternative back splicing of the CRM1 RNA occurred in the samples transcribing the sense strand of all 3 species (Glycine max)  Figs  and S5H.T. urartu (We next checked the endogenous back-spliced RNAs from retrotransposons according to the RNA sequencing (RNA-seq) data. A 323-nt endogenous RNA showing a back-splicing pattern was identified from the wheat retrotransposon RLG_Sumaya_116F2-1, based on the lncRNA sequencing (lncRNA-seq) data of . urartu . The 323. urartu . An alte. urartu . The dis. urartu . These rDuring maize evolution, specific retrotransposons were inserted and rearranged in the centromeric regions . Many stCse4 (Chromosome segregation protein 4) deposition [Arabidopsis, CENH3 deposition occurs at the G2 phase [Many centromeric RNAs have been detected in different species ,29–37. Iposition ,63. In AG2 phase ; howeverG2 phase . In our The level of back-spliced CRM RNA is low in the anti-CENH3 RIP sample. In most cases, circular RNAs are transcribed at low levels compared to linear transcripts . One recIn maize, we observed the formation of the R-loops by CRM1 RNA and their regulation in the centromere. Studies of yeast demonstrated that R-loops are linked to H3-Ser10 phosphorylation and chromatin condensation, especially at centromeric and pericentromeric regions . The chrChromatin loops can be induced in the centromeric regions by circular CRM1 RNAs . The chrCRM1 is extensively distributed along the centromere, as are the R-loops and chromatin loops associated with the CRM1 elements. Each centromere with a specific CRM1 composition should have its own distinguishing centromere architecture and CENH3 localization patterns, which may be important for centromere pairing during the early meiotic stage  and dynaDuring de novo centromere formation, CENH3 may initially be deposited in many positions –21. AccoIn the centromeric region, abundant RNAs from the centromeric CRM elements were identified. Because the repeat sequences are present in many copies, especially centromeric repeats, the dedicated tools for the detection of circular RNA from the next-generation sequencing data are not suitable for analyzing circular RNA originating from repeats . InsteadThe inbred line B73 was used for all the analyses performed in maize. The first generation of the hybrid HiII A × B was used for the RNAi transgene transformation. The rice restorer line Minghui63, sorghum inbred line BTx623, common wheat cultivar Chinese Spring, oat cultivar Baiyin11, and soybean cultivar Williams82 were used for the protoplast transformations.https://github.com/jstjohn/SeqPrep) with the parameter “-q 30 -L 25,” and the merged reads with remaining unmerged paired-end reads underwent BLAST to the assumed 354-bp sequence using the parameter “-e value 1 × 10−5.” The positions of matched reads were compared to the location of the junction site to detect whether there are reads covering the back-splicing site. The total centromere sizes in maize were estimated with the CENH3-binding regions using the ChIP-seq data; centromere coverage was determined according to the total number of sequencing reads divided by the centromere sizes.This experiment was conducted as previously described . About 1The paired-end reads generated from the anti-CENH3 ChIP-seq dataset are available under the Gene Expression Omnibus (GEO) database (GSE124242). The public anti-CENH3 ChIP-seq datasets were obtained from GEO database with accession numbers SRR3018834, SRR2000635, and SRR2000640, and the input data were obtained with accession number SRR2000648. The public raw reads of Pacbio and Illumina whole-genome sequencing datasets were from SRX1472849 and SRX1452310.Young leaves were cross-linked in 0.4 M sucrose, 10 mM Tris-HCl (pH 8), 1 mM EDTA, 1 mM PMSF, and 1% formaldehyde in a vacuum for 30 min, after which the reaction was terminated by adding 2 M glycine. The leaves were washed 3 times with RNase-free water, and Kimwipes  were used to remove water drops from the leaves. The leaves were then transferred to liquid nitrogen and ground into a fine powder. The following experiment was carried out using the native ChIP protocol as previously described with slight modification . The chrFor the clone selection, the RNA was reverse transcribed with Superscript III , and first-strand cDNA was generated using random primers. The PCR products were ligated to the T-vector using a pEASY-T1 Simple Cloning Kit . The primers are listed in The methods to join the paired-end reads from anti-CENH3 RIP and input data and BLAST to different sequences were the same for the ChIP-seq reads as mentioned earlier. The number of mapped reads for each unit of the different repeat sequences was counted, and the relative enrichments of each sequence in RIP samples were calculated using the ratios normalized by input reads. The paired-end reads from the anti-CENH3 RIP-seq dataset have been made available, and the input dataset was available in the GEO dataset (GSE137701).T. urartu (GSE137701).The same strategy was applied to discover the back-spliced RNAs from RNA-seq datasets of wheat. Wheat retrotransposons were identified using LTR_STRUC software. The method used to identify the novel back-spliced retrotransposon RNAs from wheat was performed as described previously. The paired-end reads used for wheat were obtained from the lncRNA-seq dataset of The genomic DNA of B73 was extracted using hexadecyltrimethyl ammonium bromide (CTAB). Nuclear DNA was obtained from unbroken nuclei extracted using the 3C method . The nucTotal RNA was extracted using TRIzol reagent, and the first-strand cDNA was produced using MMLV reverse transcriptase . The RIP-RNA and protoplast-transformed RNA was extracted using TRIzol reagent and reverse transcribed using Superscript III reverse transcriptase. Chromatin RNA was extracted using the same method applied for ChIP. The chromatin was treated with Proteinase K in RNA elution buffer  overnight at 37°C and then extracted using TRIzol reagent.For the detection of circular RNA, the total RNA was treated to remove mRNA and rRNA, as previously described . A totalhttps://www.guttmanlab.caltech.edu/protocols-RAP.php) and other previously published methods [The experimental procedures for RNA antisense purification followed the protocols developed by Jesse Engreitz  was used for signal detection in northern blotting, following the protocol supplied with the kit. Digoxin-labeled sense probes were used in northern blotting to detect the RNA purified by the biotinylated sense oligos. The digoxin-labeled antisense probes were used in northern blotting to detect the RNA purified using the biotinylated antisense oligos. The RNA was run on a 3% denaturing formaldehyde agarose gel. The probes are listed in Actin was used as an internal reference gene for normalization. The primers used are listed in To confirm that circular RNAs can form RNA:DNA hybrids, 4 μg of chromatin-binding RNAs was treated with 120 U of RNase H  at 37°C for 3 h. The RNA was then purified using a phenol-chloroform extraction, and Superscript III reverse transcriptase was used for reverse transcription. For the RT-qPCR, RNA not treated with RNase H was used as a control, and 2O was added to the samples that did not receive RNase H or the antibody treatment. The RNA was then precleaned using Protein A/G Plus Agarose  in IP buffer  for 4 h, prior to the addition of 4 μg S9.6 antibody  and an overnight incubation. A 100-μl aliquot of Protein A/G Plus Agarose was used to purify the antibody–RNA complex during 2-h incubation. After washing the Protein A/G Plus Agarose 3 times with IP buffer, the bound RNA was eluted twice using RNA elution buffer  at 65°C for 15 min. The RNA was extracted in a phenol-chloroform extraction and reverse transcribed using Superscript III. The primers used for qPCR are listed in Chromatin RNA was extracted according to the method described earlier. The dsRNA was removed using RNase III . A total of 5 μg RNA with or without RNase H treatment was used for each immunoprecipitation (IP) experiment, and another 5 μg RNA without antibody binding was used as the control. A 3-μl aliquot of RNase H was added to the RNase H–treated sample, and 3 μl DEPC-treated HNuclear DNA was extracted as described earlier. A total of 5 μg of nuclear DNA was treated with 0, 1,000, and 2,000 U of S1 nuclease  for 3 h at 37°C. The samples were extracted using a phenol-chloroform extraction. The qPCR primers used to detect the ssDNA region are listed in Nuclear DNA was extracted as described earlier. A total of 5 μg of nuclear DNA was treated with 100 U of T7 endonuclease I overnight at 37°C. The DNA polymerase I large (Klenow) fragment  was added to fill in the ends and incubated at 25°C for 15 min. After that, the enzyme was inactivated at 75°C for 20 min. T4 DNA ligase  was supplemented to ligate the DNA fragments at 16°C overnight, which could then be used as a PCR template to detect the shorter sequence generated by T7 endonuclease I  cleavage. The primers are listed in 2 was incubated on the surface of freshly cleaved mica for 5 min, rinsed with 200 μl of Milli-Q filtered ultrapure water, and dried with a gentle stream of nitrogen gas. All images were obtained under ambient air conditions using a Bruker MultiMode 8 AFM with a nanoscope IIIa controller in ScanAsyst mode. The heights, widths, and circumferences of these circular RNAs were calculated with this custom software. Estimating lengths of circular RNAs from the AFM images was performed according to a previously described method [Circular CRM1 RNAs were prepared by purification from the total RNAs using biotinylated sense oligos targeting the back-splicing site and were treated with 5 U of RNase R at 37°C for 2 h. For AFM preparation, all samples were performed in a solution of 10 mM Tris-HCl (pH 7.5) with the RNA concentration of 3 ng/μl. A 20 μl sample containing 5 mM MgCld method .DpnII . The quantity and quality of the DNA samples were normalized to the internal reference gene SAM [DpnII-digested and ligated centromeric BAC (ZMMBBb0497C16) DNA containing CRM1 and the PCR products of the SAM locus amplified from the 3C DNA sample [The 3C sample was produced according to a previously described method , and thegene SAM . The 3C A sample . The no-A sample . PrimersXhoI  and BglII  as well as BamHI  and SalI  digestion. The constructed sequence was then transferred into a pCambia3301 vector via a PstI  digestion for the Agrobacterium-mediated transformation of maize. The primers are listed in The RNAi vector was generated by adding a 289-bp sequence containing the back-splicing site to the pUC-RNAi vector , using XXbaI  and HindIII  digestion. A BamHI digestion site was added to the 269-bp region, and an EcoRI digestion site was added to the 85-bp region. The plasmid was then linearized using XhoI for in vitro transcription. In vitro transcription was performed according to the protocol given in the manual for T7 RNA polymerase . The primers are listed in The 1,671-bp CRM1 DNA sequence amplified from maize genomic DNA was cloned into the pET-30a vector using Maize protoplast transformation was performed as previously described . A totalFISH and immunostaining assays were performed as previously described . The 354t test.Thirty cells from interphase and 15 cells from mitosis were examined for each transgenic line to identify the centromere fluorescence intensity of the CENH3 signals with ImageJ software . SignifiS1 Figx axis in the first panel represents the positions along Chromosome 2 (panel E) and Chromosome 5 (panel F). The x axis in the second panel represents the enlarged view of cen2 (panel E) and cen5 (panel F) as illustrated by CENH3 enrichment. The first track of each panel represents the centromeric region. The other 4 tracks represent the distributions of the 354-bp, CRM1, CRM2, and CentC, respectively. The red box indicates the centromeric region. The peak heights in each track represent the RPM value (0–1). (G) Anti-CENH3 ChIP-qPCR shows the enrichment of the 269-bp and 253-bp DNA in CENH3 binding regions. “right-300 bp” represents the 300-bp DNA on the right of the 607-bp sequence. Actin was used as an internal reference gene. The columns and error bars represent the relative value and standard error of the means (n = 3), respectively. P values were determined by Student t test: *P < 0.05, **P < 0.01. The data underlying this figure can be found in https://github.com/sxx-ying/maize-centromere-circRNA).(A) The strategy of joining the paired-end reads for BLAST searching the centromere retrotransposons. (B) Relative enrichment of reads from the anti-CENH3 RIP-seq data associated with CRM elements, CentC repeats, 2 expressed centromeric genes (Zm00001d030471 and Zm00001d004256), and the 2 unexpressed genes (Zm00001d004248 and Zm00001d030471) located in centromere regions near CRM1 elements. The dotted line represents the value of no enrichment. (C) DNA-FISH of the 354-bp clone sequence. Blue indicates DAPI. Green indicates the sequence. Bar = 10 μm. (D) RT-PCR analysis of the 354-nt RNA without and with reverse transcription. (E and F) The distribution of the 354-bp sequence on Chromosome 2 (panel E) and Chromosome 5 (panel F). The (TIF)Click here for additional data file.S2 Fig(A) The sequences amplified using primers F4+R2. The first 2 lines represent 65-nt sequence from the 269-nt region and the 85-nt sequence, respectively. The third line represents the amplified 97-nt sequence. (B) Divergent primers F3+R3 were used to detect the existence of the 354-nt circular RNA. (C) The sequences amplified using primers F3+R3. In (A) and (C), the upper 2 lines are 65-nt sequence in the 269-nt and 85-nt sequence, respectively. The third line is the amplified sequence. (D) The sequences amplified using primers F2+R2. The first line represents the amplified 590-nt sequence, and the second line represents the 607-nt sequence. (E) Divergent primers F1+R1 were used to detect the existence of the 607-nt circular RNA. (F) The sequences amplified using primers F1+R1. The first line is the amplified sequence and the second line is the 607-nt sequence. (G) The shorter sequences amplified with primers F2+R2. Five sequences are shown. (H) Divergent primers F2+R3 confirmed the existence of the 277- to 296-nt circular RNA. The 17- to 27-nt sequence from the 85 nt was connected to the 5′ end of the 269-nt sequence in the amplified sequences. In (B), (E), and (H), the upper panel represents the positions of primers on the 354-nt sequence, and the lower panel represents the amplified sequences. (I) The sequences amplified with primers F2+R3. Five sequences are shown. The green bars and green lines represent the 269-nt sequence in the 354-nt and the amplified sequence, respectively. The red bars and red lines represent the 85-nt sequence in the 354-nt and the amplified sequence, respectively. The purple lines represent the intermediate 253-nt sequence. (J) Distribution of the heights, widths, and circumferences of the circular RNAs. Each point indicates a circular RNA. About 30 molecules are calculated. Mean and standard error of mean indicated. The data underlying this figure can be found in (TIF)Click here for additional data file.S3 FigDpnII digestion sites on the 607-bp region and the surrounding regions. (F) RNA-85, RNA-269, and RNA-85+269 were sensitive to RNase R treatment. The right panel shows the positions of the primers. (G) Anti-S9.6 RIP-qPCR was used to confirm the R-loop formation by linear CRM1 RNAs. Chromatin-binding RNA was used for RIP. (H) The percentages of chromatin-binding RNA in the total RNA for the CRM1 RNAs. In (A) and (G), Actin was used as an internal reference gene, the columns and the error bars represent the relative value and standard error of the means (n = 3). P values were determined by Student t test: *P < 0.05, **P < 0.01. In (B), (D), (E), and (F), the red, yellow, and green bars represent the 85-bp, 253-bp, and 269-bp region, respectively. The black lines represent the left and right sides of the 607-bp region. The data underlying this figure can be found in (A) RNA:DNA hybrids formed by circular CRM1 RNAs were checked by RNase H treatment. Chromatin-binding RNA was used for confirmation. (B) The potential cases for R-loop formation by circular CRM1 RNAs. The blue circles represent circular CRM1 RNAs. (C) Detection of the R-loop structure by T7 endonuclease I digestion and subsequent ligation. The red arrows show the shortened sequences. (D) Shortened sequence (showed by the rectangle with dotted line) obtained after T7 endonuclease I treatment and DNA ligation. The arrows on 2 sides show the primer positions. The detailed sequences of the shorter PCR bands were shown in the lower panel. The upper 3 tracks show the sequences of CRM1, the 85 bp, and the 269 bp, respectively. The fourth track shows the shorter sequence amplified by Primer 1. The last 2 tracks show the shorter sequences amplified by Primer 3. (E) The (TIF)Click here for additional data file.S4 Fig1 generation of RNAi plants and HiII. Thirty cells from interphase (D) and 15 cells in mitosis (E) were measured to quantify the CENH3 signals. (F) Anti-CENH3 ChIP-qPCR analysis showed reduced CENH3 localization in CRM1 regions in the T1 generation of RNAi plants 5. (G) Seedling phenotypes of the T2 generation of the RNAi plants. The left 3 seedlings are from HiII and the right 3 seedlings are from the T2 generation plants in each panel. (H) FISH detection of the RNAi vector in the T1 and T2 generation of the RNAi plants. Blue indicates DAPI. Green indicates the vector signals. Bar = 10 μm. (I) The RNA levels of CRM1 circular RNAs in the T2 generation of RNAi plants. In (A), (C), (D), (E) and (I), HiII is the control. In (A), (F), and (I), Actin was used as an internal reference gene. In (A), (C), (D), (E), (F), and (I), the columns and error bars represent the relative values and standard error of the means, respectively. P values were determined by Student t test: *P < 0.05, **P < 0.01. The data underlying this figure can be found in (A) RT-qPCR shows that chromatin-binding levels of the 354-nt, 607-nt, and 277- to 296-nt circular RNAs are reduced in the RNAi plants. (B) The regions chosen for detecting the ssDNA sites are marked as 85–1, 253–1, 253–2, 253–3, 269–1, and 269–2. The red bars show the ssDNA regions confirmed in (C). (C) The ssDNA sites confirmed by 1,000 U S1 nuclease treatment. The DNA with no S1 nuclease treatment was used as the control template; 607-left was used as an internal reference gene. (D and E) Quantification of the CENH3 fluorescence intensity in the T(TIF)Click here for additional data file.S5 Figx axes in each panel represent positions along Chromosome 1A, 1B, and 1D. The first 3 tracks of each panel represent distributions of the 323-bp, 188-bp, and 135-bp sequence along the whole chromosome. The other 3 tracks represent several detailed positions. The data underlying this figure can be found in https://github.com/sxx-ying/maize-centromere-circRNA). The sense-strand–transcribed CRM1 RNA can be spliced into the 354-nt–like back-spliced RNA after being transformed into oat (A), rice (B), wheat and sorghum (E), and soybean (H) protoplasts. (C and F) The 354-nt–like back-spliced RNAs from rice (C) and wheat and sorghum (F) protoplast transformation. The red frames mark the labeled digestion sites. The first track shows the 354-nt RNA and the other 3 tracks show the back-spliced sequences in protoplasts. (D and G) Detailed process of the back splicing in rice (D) as well as wheat and sorghum (G) protoplast. The red arrows show the upstream sequence, and the green arrows show the downstream sequence. The left panel shows the sequence positions on the 1,671-nt sequence, and the right panel shows the final spliced sequences. (I) The distributions of the 323-bp sequence (consisted of 188-bp and 135-bp sequence) in the AA, BB, and DD subgenome of common wheat. The (TIF)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.


We present stated preference data for improved forest management from seven Swiss municipalities in the Cantons of Grisons and Valais. The data was collected between October 2019 and February 2020 using an online questionnaire. We invited 10289 households to participate and received 939 responses. The online questionnaire consisted of two main parts – (1) an online choice experiment and (2) questions on the sociodemographic characteristics of the responding households. The choice experiment confronted households with twelve consecutive choice tasks. Each choice task consisted of three options with a varying degree of avalanche and rock fall risk reduction due to improved forest management. The options further differed with respect to the way the charges for the improved forest management are determined and assigned to the households. We additionally included a cost attribute to estimate the respondents' willingness to pay. At the end of the choice experiment we asked five de-briefing questions and eight attitudinal questions. Additionally, we asked the responding households to state their willingness to take risks. The sociodemographic characteristics collected in the second part of the questionnaire allowed us to analyse their impact on the choices we observed in the first part of the questionnaire. An analysis of the choice data and further interpretive insights are presented in the article “Determining the insurance value of ecosystems: A discrete choice study on natural hazard protection by forests”. Specifications TableValue of the Data-The data allow for eliciting preferences of households for funding improved forest management that is geared towards reducing avalanche and rock fall risk. Additionally, it helps to see how variations in sociodemographic characteristics affect these preferences. Eventually, the data provide a demand side perspective on the insurance value of ecosystems.-The data helps to inform policy makers as well as the private sector (insurance companies) when it comes to incorporating ecosystem services into climate change adaptation and disaster risk management agendas.-The data can be used to operationalize the insurance value of ecosystems. In particular, this dataset enables a demand side perspective on the insurance value of ecosystems and helps to analyze preferences and the willingness to pay based on sociodemographic characteristic.-Preferences and willingness to pay estimates on the demand side can be linked with insights from the supply side (the provisioning of ecosystem services) in order to identify feasible strategies and policies.1“completely unwilling to take risks” and 10 to “very willing to take risks”. The here presented data is thus a combination of the choices respondents made within the choice experiment and their stated sociodemographic characteristics. For reasons of confidentiality we anonymized the data by removing all fields that would enable personal identification . The complete questionnaire, the dataset and data description are available on the Environmental Data Platform EnviDat of the Swiss Federal Institute for Forest, Snow and Landscape Research WSL (doi:10.16904/envidat.175). “DIVES_Questionnaire.pdf” shows the complete questionnaire. “DIVES_choice_data.csv” is the dataset and “data_description.pdf” provides its detailed description.We conducted a discrete choice study to elicit the preferences of Swiss households for forest management that is geared towards the reduction of natural hazards. We focused on seven municipalities in the Cantons of Grisons and Valais. In each of these municipalities, households face avalanche and rock fall risks. The first part of our online questionnaire consisted of a discrete choice study in which respondents face 12 consecutive choice tasks. In each choice task, respondents are asked to choose one among three alternatives. Each of these alternatives had five attributes with two to six attribute levels. The attributes and their respective levels are presented in 2In October 2019 we sent invitation letters to all households in our seven case study municipalities in the Cantons of Grisons, GR, and Valais, VS . For the households in GR and the municipality of Täsch, we received the address data of all registered households from the municipal authorities. Due to the large number of households in Davos we only invited households where the main person registered was born in an even year. To contact the households in Zermatt, Grächen and St. Niklaus we used bulk mailing. Overall, we invited 10,289 households. The online questionnaire was open between October 2019 and February 2020. In these five months we received 939 responses. We administered the online questionnaire with the hosting service provided by Sawtooth. For the layout of the questionnaire we used Sawtooth's survey software Lighthouse Studio.The questionnaire consisted of two main parts: (1) A choice experiment with twelve consecutive choice tasks and the associated de-briefing and attitudinal questions. (2) Questions on the sociodemographic characteristics of the household.2.1The choice experiment consisted of twelve consecutive choice tasks. In each choice task respondents had to choose between three alternatives. Options 1 and 2 described situations in which better forest management leads to reduced avalanche and rock fall risk. The status quo in each choice task described a situation without improved forest management, hence there is no change in the avalanche and rock fall risk faced. To identify relevant attributes and credible attribute levels we collaborated with insurance and re-insurance professionals and academics from different disciplines. In the course of workshops and project meetings we identified five relevant attributes and the respective levels. These are shown in We used Ngene to generate a D-efficient design that varies the attribute levels in Options 1 and 2 and has a constant status quo across twelve choice tasks Subsequent to the choice experiment we asked the responding household five de-briefing questions to understand whether the households understood the experiment and considered it realistic. For each question the possible response ranged from “strongly disagree” to “strongly agree” with the opportunity not to state any answer. Next, we asked eight attitudinal questions to get an idea of the subjective risk exposure of the responding households as well as subjective valuations and opinions on natural hazards, forest management and their link to climate change. They are shown in “absolutely no willingness to take risks” and 10 to “very high willingness to take risks”.Finally, we asked the responding household to indicate their willingness to take risks. Following ,2, we us2.2In the second part of the questionnaire we asked the responding households to share some sociodemographic characteristics. The questions and the respective answer options are presented in The participation in the survey was voluntary and respondents were informed that the data will be analyzed anonymously. In participating in the survey and submitting the questionnaire each respondent gave her/his informed consent.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.