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Local adaptation is key for ecotypic differentiation and species evolution1. Understanding the underlying genomic patterns allows the prediction of future maladaptation and ecosystem stability2. Here we report the whole-genome resequencing of 865 individuals from 100 range-wide populations of European beech (Fagus sylvatica), which is one of the most important forest tree species in Europe. We show that genetic variation closely mirrors geography. Adaptive variation identified by genotype-environment associations exhibits highly polygenic architectures, involving thousands of associated sequence variants across the genome. By modelling the ‘genomic offset’ of these sequence variants under projected future climate conditions, we identify broad- and fine-scale variation highlighting geographic regions as well as populations at potential elevated risk of mortality or local extinction. Our results emphasize the importance of considering natural genetic variation for forest conservation under climate change conditions.

Hundreds of land plant lineages have independently evolved separate sexes in either gametophytes (dioicy) or sporophytes (dioecy), but 43% of all dioecious angiosperms are found in just 34 entirely dioecious clades, suggesting that their mode of sex determination evolved a long time ago. Here, we review recent insights on the molecular mechanisms that underlie the evolutionary change from individuals that each produce male and female gametes to individuals specializing in the production of just one type of gamete. The canonical model of sex chromosome evolution in plants predicts that two sex-determining genes will become linked in a sex-determining region (SDR), followed by expanding recombination suppression, chromosome differentiation and, ultimately, degeneration. Experimental work, however, is showing that single genes function as master regulators in model systems, such as the liverwort Marchantia and the angiosperms Diospyros and Populus. In Populus, this type of regulatory function has been demonstrated by genome editing. In other systems, including Actinidia, Asparagus and Vitis, two coinherited factors appear to independently regulate female and male function, yet sex chromosome differentiation has remained low. We discuss the best-understood systems and evolutionary pathways to dioecy, and present a meta-analysis of the sizes and ages of SDRs. We propose that limited sexual conflict explains why most SDRs are small and sex chromosomes remain homomorphic. It appears that models of increasing recombination suppression with age do not apply because selection favours mechanisms in which sex determination depends on minimal differences, keeping it surgically precise.

Many dioecious angiosperms are trees, which only flower after years of vegetative development and do not usually exhibit marked secondary sexual dimorphism. Nevertheless, if the genetic basis of sex determination is known, the sex of an individual can be determined using molecular markers. Here, we report that in the genus Populus sect. Populus an XY system of sex determination, which is found in P. tremula and P. tremuloides, likely re-evolved from a ZW system present in P. alba, P. adenopoda and P. qiongdaoensis. Strikingly, this new XY system is mechanistically identical to the older system found in several species of the Populus sections Tacamahaca, Aigeiros and Turanga demonstrating a remarkable example of convergent evolution. In both XY systems, male-specific inversely repeated sequences appear to silence the ARR17 gene, which functions as a sex switch, via small interfering RNAs and DNA methylation. In the ZW system, female-specific copies of ARR17 appear to regulate dioecy. With this detailed information on the genetic basis of sex determination it was possible to develop molecular markers that can be utilized to determine the sex in seedlings and non-flowering trees of different poplar species. We used the female-specific ARR17 gene to develop a sex marker for P. alba and P. adenopoda. For P. grandidentata, we employed the male-specific ARR17 inverted repeat. Finally, we summarize previously described markers for P. tremula, P. tremuloides, P. trichocarpa, P. deltoides and P. nigra. These markers can be useful for poplar ecologists, geneticists and breeders.

Although hundreds of plant lineages have independently evolved dioecy (that is, separation of the sexes), the underlying genetic basis remains largely elusive1. Here we show that diverse poplar species carry partial duplicates of the ARABIDOPSIS RESPONSE REGULATOR 17 (ARR17) orthologue in the male-specific region of the Y chromosome. These duplicates give rise to small RNAs apparently causing male-specific DNA methylation and silencing of the ARR17 gene. CRISPR-Cas9-induced mutations demonstrate that ARR17 functions as a sex switch, triggering female development when on and male development when off. Despite repeated turnover events, including a transition from the XY system to a ZW system, the sex-specific regulation of ARR17 is conserved across the poplar genus and probably beyond. Our data reveal how a single-gene-based mechanism of dioecy can enable highly dynamic sex-linked regions and contribute to maintaining recombination and integrity of sex chromosomes.

Linnaeus's very first opus, written when he was 22 years old, dealt with the analogy that exists between plants and animals in how they ‘propagate their species’, and a revised version with a plate depicting the union of male and female Mercurialis annua plants became a foundational text on the sexuality of plants. The question how systems with separate males and females have evolved in sedentary organisms that appear ancestrally bisexual has fascinated biologists ever since. The phenomenon, termed dioecy, has important consequences for plant reproductive success and is of commercial interest since it affects seed quality and fruit production. This theme issue presents a series of articles that synthesize and challenge the current understanding of how plants achieve dioecy. The articles deal with a broad set of taxa, including Coccinia , Ginkgo , Mercurialis , Populus , Rumex and Silene , as well as overarching topics, such as the field's terminology, analogies with animal sex determination systems, evolutionary pathways to dioecy, dosage compensation, and the longevity of the two sexes. In this introduction, we focus on four topics, each addressed by several articles from different angles and with different conclusions. Our highlighting of unclear or controversial issues may help future studies to build on the current understanding and to ask new questions that will expand our knowledge of plant sexual systems. This article is part of the theme issue ‘Sex determination and sex chromosome evolution in land plants’.

The number of dioecious species for which the genetic basis of sex determination has been resolved is rapidly increasing. Nevertheless, the molecular mechanisms downstream of the sex determinants remain largely elusive. Here, by RNA-sequencing early-flowering isogenic aspen (

Populus species are dioecious, but deviations from dioecy are reported in some cases. The objectives of this study were to investigate the phenotypic expression and the inheritance of subdioecy in a Populus ×canescens pedigree. The F1 progeny was monitored for sex during 14 years. Thirty per cent of individuals expressed deviations from dioecy and long-term plasticity of sex. Some plants started flowering as male, then became cosexual, and finally turned female. Two cosexual individuals were self-pollinated and generated a selfed progeny markedly impaired by inbreeding depression, but able to reproduce by outcrossing. Sex segregation of the F1 progeny statistically fitted the expected ratio 1:2:1 (female:male:cosexual). By analysis of DNA markers, the cosexual individuals were genetically clustered with the females. The segregation ratio and the genetic profile indicated that cosexual plants were female with altered sex phenotype. Linkage analysis identified a putative sex-determining region with suppressed recombination on chromosome 19 of the male Populus tremula parent. The male sex trait was linked to the pericentromeric region of the P. tremula chromosome 19, whereas the cosexual trait was linked to chromosome 19 of the female Populus alba parent. A genetic model is proposed to explain inheritance and phenotypic expression of sex.

RNA editing occurs in the endosymbiont organelles of higher plants as C-to-U conversions of defined nucleotides. The availability of large quantities of RNA sequencing data makes it possible to identify RNA editing sites and to quantify their editing extent. We have investigated RNA editing in 34 protein-coding mitochondrial transcripts of four Populus species, a genus noteworthy for its remarkably small number of RNA editing sites compared to other angiosperms. 27 of these transcripts were subject to RNA editing in at least one species. In total, 355 RNA editing sites were identified with high confidence, their editing extents ranging from 10 to 100%. The most heavily edited transcripts were ccmB with the highest density of RNA editing sites (53.7 sites / kb) and ccmFn with the highest number of sites (39 sites). Most of the editing events are at position 1 or 2 of the codons, usually altering the encoded amino acid, and are highly conserved among the species, also with regard to their editing extent. However, one SNP was found in the newly sequenced and annotated mitochondrial genome of P. alba resulting in the loss of an RNA editing site compared to P. tremula and P. davidiana. This SNP causes a C-to-T transition and an amino acid exchange from Ser to Phe, highlighting the widely discussed role of RNA editing in compensating mutations.

Even though “supermale” (YY) poplar individuals are rarely found in natural populations or breeding collections, they can arise and can be as viable as regular male and female plants. Nearly all species of the genus Populus are dioecious, i.e., form male and female flowers on separate individuals. However, bisexual poplar can occasionally be found under natural conditions or during the breeding process. According to the XX/XY model of sex determination, “supermale” (YY) individuals should only occur in the selfed progeny of a genetically male (XY) tree and not in the one of a genetically female (XX) parent. We performed detailed molecular genetic analyses of a bisexual Populus tremula XY-individual (ZP8) and its selfed progeny. The ZP8 tree was confirmed as P. tremula by chloroplast and nuclear markers, and the progeny was genotyped by microsatellite markers to verify self-pollination. In the selfed progeny but also in about 420 different poplar individuals from several breeding programmes and the wild, the X and Y chromosomes were tracked using previously reported and newly developed markers. From two self-pollination experiments of the bisexual ZP8 tree, 39 S1-individuals were obtained. Application of X- and Y-specific molecular markers revealed 15 XY, 17 YY, and 7 XX individuals. Additional investigations of about 300 different poplar clones employed in several breeding programmes as well as about 120 individuals from wild populations revealed only three trees with two Y chromosomes. Indications for inbreeding depression in the selfed progeny were obtained during germination and glasshouse cultivation, but are independent from the sex chromosome combination. Even though YY-poplar individuals are rarely found in natural populations or breeding collections, they can arise and can be as viable as regular XY- and XX-plants.

Nucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA Polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA, and a divergent non-coding (DNC) transcript with an unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast. We identified the Hda1 histone deacetylase complex (Hda1C) as a repressor of DNC in high-throughput reverse genetic screens based on quantitative single-cell fluorescence measurements. Nascent transcription profiling showed a genome-wide role of Hda1C in DNC repression. Live-cell imaging of transcription revealed that Hda1C reduced the frequency of DNC transcription. Hda1C contributed to decreased acetylation of histone H3 in DNC regions, supporting DNC repression by histone deacetylation. Our data support the interpretation that DNC results as a consequence of the NDR-based architecture of eukaryotic promoters, but that it is governed by locus-specific repression to maintain genome fidelity.

The diversity of inflorescences among flowering plants is captivating. Such charm is not only due to the variety of sizes, shapes, colors, and flowers displayed, but also to the range of reproductive systems. For instance, hermaphrodites occur abundantly throughout the plant kingdom with both stamens and carpels within the same flower. Nevertheless, 10% of flowering plants have separate unisexual flowers, either in different locations of the same individual (monoecy) or on different individuals (dioecy). Despite their rarity, dioecious plants provide an excellent opportunity to investigate the mechanisms involved in sex expression and the evolution of sex-determining regions (SDRs) and sex chromosomes. The SDRs and the evolution of dioecy have been studied in many species ranging from Ginkgo to important fruit crops. Some of these studies, for example in asparagus or kiwifruit, identified two sex-determining genes within the non-recombining SDR and may thus be consistent with the classical model for the evolution of dioecy from hermaphroditism via gynodioecy, that predicts two successive mutations, the first one affecting male and the second one female function, becoming linked in a region of suppressed recombination. On the other hand, aided by genome sequencing and gene editing, single factor sex determination has emerged in other species, such as persimmon or poplar. Despite the diversity of sex-determining mechanisms, a tentative comparative analysis of the known sex-determining genes and candidates in different species suggests that similar genes and pathways may be employed repeatedly for the evolution of dioecy. The cytokinin signaling pathway appears important for sex determination in several species regardless of the underlying genetic system. Additionally, tapetum-related genes often seem to act as male-promoting factors when sex is determined via two genes. We present a unified model that synthesizes the genetic networks of sex determination in monoecious and dioecious plants and will support the generation of hypothesis regarding candidate sex determinants in future studies.

International audience; Whole-genome sequencing of non-model organisms is now widely accessible and has allowed a range of questions in the field of molecular ecology to be investigated with greater power. However, some genomic regions that are of high biological interest remain problematic for assembly and data-handling. Three such regions are the major histocompatibility complex (MHC), sex-determining regions (SDRs) and the plant self-incompatibility locus (S-locus). Using these as examples, we illustrate the challenges of both assembling and resequencing these highly polymorphic regions and how bioinformatic and technological developments are enabling new approaches to their study. Mapping short-read sequences against multiple alternative references improves genotyping comprehensiveness at the S-locus thereby contributing to more accurate assessments of allelic frequencies. Long-read sequencing, producing reads of several tens to hundreds of kilobase pairs in length, facilitates the assembly of such regions as single sequences can span the multiple duplicated gene copies of the MHC region, and sequence through repetitive stretches and translocations in SDRs and S-locus haplotypes. These advances are adding value to short-read genome resequencing approaches by allowing, for example, more accurate haplotype phasing across longer regions. Finally, we assessed further technical improvements, such as nanopore adaptive sequencing and bioinformatic tools using pangenomes, which have the potential to further expand our knowledge of a number of genomic regions that remain challenging to study with classical resequencing approaches.

Hundreds of land plant lineages have independently evolved separate sexes in either gametophytes (dioicy) or sporophytes (dioecy), but 43% of all dioecious angiosperms are found in just 34 entirely dioecious clades, suggesting that their mode of sex determination evolved a long time ago. Here, we review recent insights on the molecular mechanisms that underlie the evolutionary change from individuals that each produce male and female gametes to individuals specializing in the production of just one type of gamete. The canonical model of sex chromosome evolution in plants predicts that two sex-determining genes will become linked in a sex-determining region (SDR), followed by expanding recombination suppression, chromosome differentiation and, ultimately, degeneration. Experimental work, however, is showing that single genes function as master regulators in model systems, such as the liverwort Marchantia and the angiosperms Diospyros and Populus. In Populus, this type of regulatory function has been demonstrated by genome editing. In other systems, including Actinidia, Asparagus and Vitis, two coinherited factors appear to independently regulate female and male function, yet sex chromosome differentiation has remained low. We discuss the best-understood systems and evolutionary pathways to dioecy, and present a meta-analysis of the sizes and ages of SDRs. We propose that limited sexual conflict explains why most SDRs are small and sex chromosomes remain homomorphic. It appears that models of increasing recombination suppression with age do not apply because selection favours mechanisms in which sex determination depends on minimal differences, keeping it surgically precise.

[EN] A dramatic evolution of fruit size has accompanied the domestication and improvement of fruit-bearing crop species. In tomato (Solanum lycopersicum), naturally occurring cis-regulatory mutations in the genes of the CLAVATA-WUSCHEL signaling pathway have led to a significant increase in fruit size generating enlarged meristems that lead to flowers with extra organs and bigger fruits. In this work, by combining mapping-by-sequencing and CRISPR/ Cas9 genome editing methods, we isolated EXCESSIVE NUMBER OF FLORAL ORGANS (ENO), an AP2/ERF transcription factor which regulates floral meristem activity. Thus, the ENO gene mutation gives rise to plants that yield larger multilocular fruits due to an increased size of the floral meristem. Genetic analyses indicate that eno exhibits synergistic effects with mutations at the LOCULE NUMBER (encoding SlWUS) and FASCIATED (encoding SlCLV3) loci, two central players in the evolution of fruit size in the domestication of cultivated tomatoes. Our findings reveal that an eno mutation causes a substantial expansion of SlWUS expression domains in a flower-specific manner. In vitro binding results show that ENO is able to interact with the GGC-box cis-regulatory element within the SlWUS promoter region, suggesting that ENO directly regulates SlWUS expression domains to maintain floral stem-cell homeostasis. Furthermore, the study of natural allelic variation of the ENO locus proved that a cis-regulatory mutation in the promoter of ENO had been targeted by positive selection during the domestication process, setting up the background for significant increases in fruit locule number and fruit size in modern tomatoes., We thank Dr. R. Fernandez-Munoz (Instituto de Hortofruticultura Subtropical y Mediterranea-Universidad de MalagaConsejo Superior de Investigaciones Cientificas) for providing seeds of wild and cultivated tomato accessions, and Dr. R. Micol-Ponce (Universidad de Almeria) for help with the EMSA experiments. This research was supported by the Spanish Ministry of Economy and Competitiveness (Grants AGL201564991-C3-1-R and AGL2015-64991-C3-3-R) and the Research and Innovation Programme of the European Union Horizon 2020 (Breeding for Resilient, Efficient and Sustainable Organic Vegetable production [BRESOV], Project ID 774244). J.M.J.-G. was funded by Agence Nationale de la Recherche (ANR) Grant tomaTE, ANR-17-CE20-0024-02 and ANR TREMPLIN-European Research Council Grant RITMO, ANR-17-ERC2-0013-01.

International audience; Circadian period and phase of cultivated tomato (Solanum lycopersicum) were changed during domestication, likely adapting the species to its new agricultural environments. Whereas the delayed circadian phase is mainly caused by allelic variation of EID1, the genetic basis of the long circadian period has remained elusive. Here we show that a partial deletion of the clock gene LNK2 is responsible for the period lengthening in cultivated tomatoes. We use resequencing data to phylogenetically classify hundreds of tomato accessions and investigate the evolution of the eid1 and lnk2 mutations along successive domestication steps. We reveal signatures of selection across the genomic region of LNK2 and different patterns of fixation of the mutant alleles. Strikingly, LNK2 and EID1 are both involved in light input to the circadian clock, indicating that domestication specifically targeted this input pathway. In line with this, we show that the clock deceleration in the cultivated tomato is light-dependent and requires the phytochrome B1 photoreceptor. Such conditional variation in circadian rhythms may be key for latitudinal adaptation in a variety of species, including crop plants and livestock.

Table 4 in the original publication reports incomplete genotype names in the column "Cross" and wrong codes in the column "Generation".

International audience; Under field conditions, drought and heat stress typically happen simultaneously and their negative impact on the agricultural production is expected to increase worldwide under the climate change scenario. In this study, we performed RNA-sequencing analysis on leaves of wild barley (Hordeum spontaneum) originated from the northern coastal region of Egypt following individual drought acclimation (DA) and heat shock (HS) treatments and their combination (CS, combined stresses) to distinguish the unique and shared differentially expressed genes (DEG). Results indicated that the number of unique genes that were differentially expressed following HS treatment exceeded the number of those expressed following DA. In addition, the number of genes that were uniquely differentially expressed in response to CS treatment exceeded the number of those of shared responses to individual DA and HS treatments. These results indicate a better adaptation of the Mediterranean wild barley to drought conditions when compared with heat stress. It also manifests that the wild barley response to CS tends to be unique rather than common. Annotation of DEG showed that metabolic processes were the most influenced biological function in response to the applied stresses.

Plants evolved so that their flowering is triggered by seasonal changes in day lengths. However, day-length sensitivity in crops limits their geographical range of cultivation, and thus modification of the photoperiod response was critical for their domestication(2-11). Here we show that loss of day-length-sensitive flowering in tomato was driven by the florigen paralog and flowering repressor SELF-PRUNING 5G (SP5G). SP5G expression is induced to high levels during long days in wild species, but not in cultivated tomato because of cis-regulatory variation. CRISPR/Cas9-engineered mutations in SP5G cause rapid flowering and enhance the compact determinate growth habit of field tomatoes, resulting in a quick burst of flower production that translates to an early yield. Our findings suggest that pre-existing variation in SP5G facilitated the expansion of cultivated tomato beyond its origin near the equator in South America, and they provide a compelling demonstration of the power of gene editing to rapidly improve yield traits in crop breeding.

The circadian clock is a molecular timekeeper that controls a wide variety of biological processes. In plants, clock outputs range from the molecular level, with rhythmic gene expression and metabolite content, to physiological processes such as stomatal conductance or leaf movements. Any of these outputs can be used as markers to monitor the state of the circadian clock. In the model plant Arabidopsis thaliana, much of the current knowledge about the clock has been gained from time course experiments profiling expression of endogenous genes or reporter constructs regulated by the circadian clock. Since these methods require labor-intensive sample preparation or transformation, monitoring leaf movements is an interesting alternative, especially in non-model species and for natural variation studies. Technological improvements both in digital photography and image analysis allow cheap and easy monitoring of circadian leaf movements. In this chapter we present a protocol that uses an autonomous point and shoot camera and free software to monitor circadian leaf movements in tomato.

The circadian clock is a molecular timekeeper that controls a wide variety of biological processes. In plants, clock outputs range from the molecular level, with rhythmic gene expression and metabolite content, to physiological processes such as stomatal conductance or leaf movements. Any of these outputs can be used as markers to monitor the state of the circadian clock. In the model plant Arabidopsis thaliana, much of the current knowledge about the clock has been gained from time course experiments profiling expression of endogenous genes or reporter constructs regulated by the circadian clock. Since these methods require labor-intensive sample preparation or transformation, monitoring leaf movements is an interesting alternative, especially in non-model species and for natural variation studies. Technological improvements both in digital photography and image analysis allow cheap and easy monitoring of circadian leaf movements. In this chapter we present a protocol that uses an autonomous point and shoot camera and free software to monitor circadian leaf movements in tomato.

Nucleosome‐depleted regions (NDRs) at gene promoters support initiation of RNA polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA and a divergent non‐coding (DNC) transcript of unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast. Using high‐throughput reverse genetic screens based on quantitative single‐cell fluorescence measurements, we identified the Hda1 histone deacetylase complex (Hda1C) as a repressor of DNC transcription. Nascent transcription profiling showed a genome‐wide role of Hda1C in repression of DNC transcription. Live‐cell imaging of transcription revealed that mutations in the Hda3 subunit increased the frequency of DNC transcription. Hda1C contributed to decreased acetylation of histone H3 in DNC transcription regions, supporting DNC transcription repression by histone deacetylation. Our data support the interpretation that DNC transcription results as a consequence of the NDR‐based architecture of eukaryotic promoters, but that it is governed by locus‐specific repression to maintain genome fidelity., A yeast reverse‐genetic screen identifies factors affecting divergent non‐coding transcription.