Global range expansion history of pepper (Capsicum spp.) revealed by over 10,000 genebank accessions.

Genebanks collect and preserve vast collections of plants and detailed passport information, with the aim of preserving genetic diversity for conservation and breeding. Genetic characterization of such collections has the potential to elucidate the genetic histories of important crops, use marker-trait associations to identify loci controlling traits of interest, search for loci undergoing selection, and contribute to genebank management by identifying taxonomic misassignments and duplicates. We conducted a genomic scan with genotyping by sequencing (GBS) derived single nucleotide polymorphisms (SNPs) of 10,038 pepper (Capsicum spp.) accessions from worldwide genebanks and investigated the recent history of this iconic staple. Genomic data detected up to 1,618 duplicate accessions within and between genebanks and showed that taxonomic ambiguity and misclassification often involve interspecific hybrids that are difficult to classify morphologically. We deeply interrogated the genetic diversity of the commonly consumed Capsicum annuum to investigate its history, finding that the kinds of peppers collected in broad regions across the globe overlap considerably. The method ReMIXTURE-using genetic data to quantify the similarity between the complement of peppers from a focal region and those from other regions-was developed to supplement traditional population genetic analyses. The results reflect a vision of pepper as a highly desirable and tradable cultural commodity, spreading rapidly throughout the globe along major maritime and terrestrial trade routes. Marker associations and possible selective sweeps affecting traits such as pungency were observed, and these traits were shown to be distributed nonuniformly across the globe, suggesting that human preferences exerted a primary influence over domesticated pepper genetic structure.

Combining next-generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals.

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M2 ) family derived from a heterozygous (M1 ) parent were identified using whole-genome shotgun (WGS) sequencing of a small number of (M2 ) individuals with mutant and wild-type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self-pollinated (M3 ) offspring. Finally, we filtered for segregating EMS-induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M2 ) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non-synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.

MicroProtein-Mediated Recruitment of CONSTANS into a TOPLESS Trimeric Complex Represses Flowering in Arabidopsis.

MicroProteins are short, single domain proteins that act by sequestering larger, multi-domain proteins into non-functional complexes. MicroProteins have been identified in plants and animals, where they are mostly involved in the regulation of developmental processes. Here we show that two Arabidopsis thaliana microProteins, miP1a and miP1b, physically interact with CONSTANS (CO) a potent regulator of flowering time. The miP1a/b-type microProteins evolved in dicotyledonous plants and have an additional carboxy-terminal PF(V/L)FL motif. This motif enables miP1a/b microProteins to interact with TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins. Interaction of CO with miP1a/b/TPL causes late flowering due to a failure in the induction of FLOWERING LOCUS T (FT) expression under inductive long day conditions. Both miP1a and miP1b are expressed in vascular tissue, where CO and FT are active. Genetically, miP1a/b act upstream of CO thus our findings unravel a novel layer of flowering time regulation via microProtein-inhibition.

Transcriptomic basis for reinforcement of elm antiherbivore defence mediated by insect egg deposition.

Plant responses to insect egg depositions are known to shape subsequent defensive responses to larvae hatching from the eggs. Elm (Ulmus minor) leaves, on which elm leaf beetles laid their eggs, mount a more efficient defence against larvae hatching from the eggs. However, the molecular mechanisms of this egg-mediated, improved defence are insufficiently understood and have so far only been studied in annual plants. We analysed the dynamics of transcriptomic changes in larval feeding-damaged elm leaves with and without prior egg deposition using de novo assembled RNA-seq data. Compared to egg-free leaves, egg deposition-treated leaves showed earlier and/or faster transcriptional regulations, as well as slightly enhanced differential transcriptional regulation after the onset of larval feeding. These early responding transcripts were overrepresented in gene ontology terms associated with post-translational protein modification, signalling and stress (defence) responses. We found evidence of transcriptional memory in initially egg deposition-induced transcripts whose differential expression was reset prior to larval hatching, but was more rapidly induced again by subsequent larval feeding. This potential memory effect of prior egg deposition, as well as the earlier/faster and enhanced feeding-induced differential regulation of transcripts in egg deposition-treated leaves, may contribute to the egg-mediated reinforcing effect on the elm's defence against larvae. Hence, our study shows that a plant's experience of a stress-indicating environmental cue (here: insect eggs) can push the dynamics of the plant's transcriptomic response to subsequent stress (here: larval feeding). Such experience-mediated acceleration of a stress-induced plant response may result in improved stress resistance.

Holocentromeres in Rhynchospora are associated with genome-wide centromere-specific repeat arrays interspersed among euchromatin.

Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinetochores distributed along almost the entire poleward surface of the chromatids, to which spindle fibers attach. No centromere-specific DNA sequence has been found for any holocentric organism studied so far. It was proposed that centromeric repeats, typical for many monocentric species, could not occur in holocentrics, most likely because of differences in the centromere organization. Here we show that the holokinetic centromeres of the Cyperaceae Rhynchospora pubera are highly enriched by a centromeric histone H3 variant-interacting centromere-specific satellite family designated "Tyba" and by centromeric retrotransposons (i.e., CRRh) occurring as genome-wide interspersed arrays. Centromeric arrays vary in length from 3 to 16 kb and are intermingled with gene-coding sequences and transposable elements. We show that holocentromeres of metaphase chromosomes are composed of multiple centromeric units rather than possessing a diffuse organization, thus favoring the polycentric model. A cell-cycle-dependent shuffling of multiple centromeric units results in the formation of functional (poly)centromeres during mitosis. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromeric chromatin organization among eukaryotes. Thus, different types of holocentromeres exist in different species, namely with and without centromeric repetitive sequences.

REVOLUTA and WRKY53 connect early and late leaf development in Arabidopsis.

As sessile organisms, plants have to continuously adjust growth and development to ever-changing environmental conditions. At the end of the growing season, annual plants induce leaf senescence to reallocate nutrients and energy-rich substances from the leaves to the maturing seeds. Thus, leaf senescence is a means with which to increase reproductive success and is therefore tightly coupled to the developmental age of the plant. However, senescence can also be induced in response to sub-optimal growth conditions as an exit strategy, which is accompanied by severely reduced yield. Here, we show that class III homeodomain leucine zipper (HD-ZIPIII) transcription factors, which are known to be involved in basic pattern formation, have an additional role in controlling the onset of leaf senescence in Arabidopsis. Several potential direct downstream genes of the HD-ZIPIII protein REVOLUTA (REV) have known roles in environment-controlled physiological processes. We report that REV acts as a redox-sensitive transcription factor, and directly and positively regulates the expression of WRKY53, a master regulator of age-induced leaf senescence. HD-ZIPIII proteins are required for the full induction of WRKY53 in response to oxidative stress, and mutations in HD-ZIPIII genes strongly delay the onset of senescence. Thus, a crosstalk between early and late stages of leaf development appears to contribute to reproductive success.

Meta-Analysis of Arabidopsis KANADI1 Direct Target Genes Identifies a Basic Growth-Promoting Module Acting Upstream of Hormonal Signaling Pathways.

An intricate network of antagonistically acting transcription factors mediates the formation of a flat leaf lamina of Arabidopsis (Arabidopsis thaliana) plants. In this context, members of the class III homeodomain leucine zipper (HD-ZIPIII) transcription factor family specify the adaxial domain (future upper side) of the leaf, while antagonistically acting KANADI transcription factors determine the abaxial domain (future lower side). Here, we used a messenger RNA sequencing approach to identify genes regulated by KANADI1 (KAN1) and subsequently performed a meta-analysis combining our data sets with published genome-wide data sets. Our analysis revealed that KAN1 acts upstream of several genes encoding auxin biosynthetic enzymes. When exposed to shade, we found three YUCCA genes, YUC2, YUC5, and YUC8, to be transcriptionally up-regulated, which correlates with an increase in the levels of free auxin. When ectopically expressed, KAN1 is able to transcriptionally repress these three YUC genes and thereby block shade-induced auxin biosynthesis. Consequently, KAN1 is able to strongly suppress shade-avoidance responses. Taken together, we hypothesize that HD-ZIPIII/KAN form the basis of a basic growth-promoting module. Hypocotyl extension in the shade and outgrowth of new leaves both involve auxin synthesis and signaling, which are under the direct control of HD-ZIPIII/KAN.

Extensive tissue-specific transcriptomic plasticity in maize primary roots upon water deficit.

Water deficit is the most important environmental constraint severely limiting global crop growth and productivity. This study investigated early transcriptome changes in maize (Zea mays L.) primary root tissues in response to moderate water deficit conditions by RNA-Sequencing. Differential gene expression analyses revealed a high degree of plasticity of the water deficit response. The activity status of genes (active/inactive) was determined by a Bayesian hierarchical model. In total, 70% of expressed genes were constitutively active in all tissues. In contrast, <3% (50 genes) of water deficit-responsive genes (1915) were consistently regulated in all tissues, while >75% (1501 genes) were specifically regulated in a single root tissue. Water deficit-responsive genes were most numerous in the cortex of the mature root zone and in the elongation zone. The most prominent functional categories among differentially expressed genes in all tissues were 'transcriptional regulation' and 'hormone metabolism', indicating global reprogramming of cellular metabolism as an adaptation to water deficit. Additionally, the most significant transcriptomic changes in the root tip were associated with cell wall reorganization, leading to continued root growth despite water deficit conditions. This study provides insight into tissue-specific water deficit responses and will be a resource for future genetic analyses and breeding strategies to develop more drought-tolerant maize cultivars.

Homeodomain leucine-zipper proteins and their role in synchronizing growth and development with the environment.

The Arabidopsis (Arabidopsis thaliana L.) genome encodes for four distinct classes of homeodomain leucine-zipper (HD-ZIP) transcription factors (HD-ZIPI to HD-ZIPIV), which are all organized in multi-gene families. HD-ZIP transcription factors act as sequence-specific DNA-binding proteins that are able to control the expression level of target genes. While HD-ZIPI and HD-ZIPII proteins are mainly associated with environmental responses, HD-ZIPIII and HD-ZIPIV are primarily known to act as patterning factors. Recent studies have challenged this view. It appears that several of the different HD-ZIP families interact genetically to align both morphogenesis and environmental responses, most likely by modulating phytohormone-signaling networks.

Genome-wide binding-site analysis of REVOLUTA reveals a link between leaf patterning and light-mediated growth responses.

Unlike the situation in animals, the final morphology of the plant body is highly modulated by the environment. During Arabidopsis development, intrinsic factors provide the framework for basic patterning processes. CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) transcription factors are involved in embryo, shoot and root patterning. During vegetative growth HD-ZIPIII proteins control several polarity set-up processes such as in leaves and the vascular system. We have identified several direct target genes of the HD-ZIPIII transcription factor REVOLUTA (REV) using a chromatin immunoprecipitation/DNA sequencing (ChIP-Seq) approach. This analysis revealed that REV acts upstream of auxin biosynthesis and affects directly the expression of several class II HD-ZIP transcription factors that have been shown to act in the shade-avoidance response pathway. We show that, as well as involvement in basic patterning, HD-ZIPIII transcription factors have a critical role in the control of the elongation growth that is induced when plants experience shade. Leaf polarity is established by the opposed actions of HD-ZIPIII and KANADI transcription factors. Finally, our study reveals that the module that consists of HD-ZIPIII/KANADI transcription factors controls shade growth antagonistically and that this antagonism is manifested in the opposed regulation of shared target genes.

COMPOSITUM 1 contributes to the architectural simplification of barley inflorescence via meristem identity signals.

Grasses have varying inflorescence shapes; however, little is known about the genetic mechanisms specifying such shapes among tribes. Here, we identify the grass-specific TCP transcription factor COMPOSITUM 1 (COM1) expressing in inflorescence meristematic boundaries of different grasses. COM1 specifies branch-inhibition in barley (Triticeae) versus branch-formation in non-Triticeae grasses. Analyses of cell size, cell walls and transcripts reveal barley COM1 regulates cell growth, thereby affecting cell wall properties and signaling specifically in meristematic boundaries to establish identity of adjacent meristems. COM1 acts upstream of the boundary gene Liguleless1 and confers meristem identity partially independent of the COM2 pathway. Furthermore, COM1 is subject to purifying natural selection, thereby contributing to specification of the spike inflorescence shape. This meristem identity pathway has conceptual implications for both inflorescence evolution and molecular breeding in Triticeae.

Linked-read sequencing of gametes allows efficient genome-wide analysis of meiotic recombination.

Meiotic crossovers (COs) ensure proper chromosome segregation and redistribute the genetic variation that is transmitted to the next generation. Large populations and the demand for genome-wide, fine-scale resolution challenge existing methods for CO identification. Taking advantage of linked-read sequencing, we develop a highly efficient method for genome-wide identification of COs at kilobase resolution in pooled recombinants. We first test this method using a pool of Arabidopsis F2 recombinants, and recapitulate results obtained from the same plants using individual whole-genome sequencing. By applying this method to a pool of pollen DNA from an F1 plant, we establish a highly accurate CO landscape without generating or sequencing a single recombinant plant. The simplicity of this approach enables the simultaneous generation and analysis of multiple CO landscapes, accelerating the pace at which mechanisms for the regulation of recombination can be elucidated through efficient comparisons of genotypic and environmental effects on recombination.

Laser Capture Microdissection-Based RNA-Seq of Barley Grain Tissues.

Spatiotemporal patterning throughout the plant body depends to a large degree on cell- and tissue-specific expression of genes. Subsequently, for a better understanding of cell and tissue differentiation processes during plant development it is important to conduct transcript analyses in individual cells or tissue types rather than in bulk tissues. Laser capture microdissection (LCM) provides a useful method for isolating specific cell types from complex tissue structures for downstream applications. Contrasting to mammalian cells, the texture of plant cells is more critical due to hard, cellulose-rich cell walls, large vacuoles, and air spaces which complicates tissue preparation and extraction of macromolecules, like DNA and RNA. In particular, developing barley seeds (i.e. grains) depict cell types with differences in osmomolarity (meristematic, differentiating and degenerating tissues) and contain high amounts of the main storage product starch. In this study, we report about methods allowing tissue-specific transcriptome profiling by RNA-seq of developing barley grain tissues from low-input RNA amounts. Details on tissue preparation, laser capture microdissection, RNA isolation, and linear mRNA amplification to produce high-quality samples for Illumina sequencing are provided. Particular emphasis was placed on the influence of the mRNA amplification step on the transcriptome data and the fidelity of deduced expression levels obtained by the developed methods. Analysis of RNA-seq data confirmed sample processing as a highly reliable and reproducible procedure that was also used for transcriptome analyses of different tissue types from barley plants.

SSR-seq: Genotyping of microsatellites using next-generation sequencing reveals higher level of polymorphism as compared to traditional fragment size scoring.

Microsatellites (or simple sequence repeats, SSR) are widely used markers in population genetics. Traditionally, genotyping was and still is carried out through recording fragment length. Now, next-generation sequencing (NGS) makes it easy to obtain also sequence information for the loci of interest. This avoids misinterpretations that otherwise could arise due to size homoplasy. Here, an NGS strategy is described that allows to genotype hundreds of individuals at many custom-designed SSR loci simultaneously, combining multiplex PCR, barcoding, and Illumina sequencing. We created three different datasets for which alleles were coded according to (a) length of the repetitive region, (b) total fragment length, and (c) sequence identity, in order to evaluate the eventual benefits from having sequence data at hand, not only fragment length data. For each dataset, genetic diversity statistics, as well as F ST and R ST values, were calculated. The number of alleles per locus, as well as observed and expected heterozygosity, was highest in the sequence identity dataset, because of single-nucleotide polymorphisms and insertions/deletions in the flanking regions of the SSR motif. Size homoplasy was found to be very common, amounting to 44.7%-63.5% (mean over all loci) in the three study species. Thus, the information obtained by next-generation sequencing offers a better resolution than the traditional way of SSR genotyping and allows for more accurate evolutionary interpretations.

Development of microsatellite markers and assembly of the plastid genome in Cistanthe longiscapa (Montiaceae) based on low-coverage whole genome sequencing.

Cistanthe longiscapa is an endemic annual herb and characteristic element of the Chilean Atacama Desert. Principal threats are the destruction of its seed deposits by human activities and reduced germination rates due to the decreasing occurrence of precipitation events. To enable population genetic and phylogeographic analyses in this species we performed paired-end shotgun sequencing (2x100 bp) of genomic DNA on the Illumina HiSeq platform and identified microsatellite (SSR) loci in the resulting sequences. From 29 million quality-filtered read pairs we obtained 549,174 contigs (average length 614 bp; N50 = 904). Searching for SSRs revealed 10,336 loci with microsatellite motifs. Initially, we designed primers for 96 loci, which were tested for PCR amplification on three C. longiscapa individuals. Successfully amplifying loci were further tested on eight individuals to screen for length variation in the resulting amplicons, and the alleles were exemplarily sequenced to infer the basis for the observed length variation. Finally we arrived at 26 validated SSR loci for population studies in C. longiscapa, which resulted in 146 bi-allelic SSR markers in our test sample of eight individuals. The genomic sequences were also used to assemble the plastid genome of C. longiscapa, which provides an additional set of maternally inherited genetic markers.

Phytotoxicity of cobalt ions on the duckweed Lemna minor - Morphology, ion uptake, and starch accumulation.

Cobalt (Co2+) inhibits vegetative growth of Lemna minor gradually from 1 µM to 100 µM. Fronds accumulated up to 21 mg Co2+ g(-1) dry weight at 10 µM external Co2+ indicating hyperaccumulation. Interestingly, accumulation of Co2+ did not decrease the iron (Fe) content in fronds, highlighting L. minor as a suitable system for studying effects of Co2+ undisturbed by Fe deficiency symptoms unlike most other plants. Digital image analysis revealed the size distribution of fronds after Co2+ treatment and also a reduction in pigmentation of newly formed daughter fronds unlike the mother fronds during the 7-day treatment. Neither chlorophyll nor photosystem II fluorescence changed significantly during the initial 4d, indicating effective photosynthesis. During the later phase of the 7-day treatment, however, chlorophyll content and photosynthetic efficiency decreased in the Co2+-treated daughter fronds, indicating that Co2+ inhibits the biosynthesis of chlorophyll rather than leading to the destruction of pre-existing pigment molecules. In addition, during the first 4d of Co2+ treatment starch accumulated in the fronds and led to the transition of chloroplasts to chloro-amyloplasts and amylo-chloroplasts, while starch levels strongly decreased thereafter.

Overexpression of Arabidopsis thaliana ERI, the homolog of C. elegans Enhancer of RNAinterference, leads to enhanced growth.

Organisms adopt a wide range of strategies to adapt to change. Gene silencing describes the ability of organisms to modulate the expression of susceptible genes at certain times at the transcriptional or the translational level. In all known eukaryotic organisms 21-nt long short interfering RNAs (siRNAs) are the effector molecules of post-transcriptional gene silencing (PTGS), while 24-nt long siRNAs are involved in PTGS in plants. Mutant studies in Caenorhabditis elegans lead to the identification of the enzyme ERI (Enhancer of RNAinterference) with enhanced PTGS. Although the genes involved in growth vigor and growth rate are still unknown, it becomes clearer that the population of small RNAs plays a role in the very early phase of plant development. To pinpoint the link between growth and siRNAs, the expression of Arabidopsis uni-gene Enhancer of RNAi (ERI) homolog from C. elegans was modulated. Increased degradation of small RNAs was achieved by ectopic AtERI overexpression in planta. Based on global small RNA analysis, AtERI overexpression affects mainly the population of 21 mers, excluding miRNAs. To identify target genes, AtERI gain-of-function mutants were analyzed, and differentially abundant small RNAs were identified. Plants with an elevated level of AtERI were bigger in all three light intensities analyzed, indicating an inhibitory function of particular small RNAs in plant growth, with differences in relative growth rates depending on developmental stage and light intensity. Understanding the role of these siRNAs could open new avenues for enhancing plant growth.

Control of stem cell homeostasis via interlocking microRNA and microProtein feedback loops.

Stem cells in the shoot apex of plants produce cells required for the formation of new leaves. Adult leaves are composed of multiple tissue layers arranged along the dorso-ventral (adaxial/abaxial) axis. Class III homeodomain leucine zipper (HD-ZIPIII) transcription factors play an important role in the set-up of leaf polarity in plants. Loss of HD-ZIPIII function results in strongly misshapen leaves and in severe cases fosters the consumption of the apical stem cells, thus causing a growth arrest in mutant plants. HD-ZIPIII mRNA is under tight control by microRNAs 165/166. In addition to the microRNA-action a second layer of regulation is established by LITTLE ZIPPER (ZPR)-type microProteins, which can interact with HD-ZIPIII proteins, forming attenuated protein complexes. Here we show that REVOLUTA (REV, a member of the HD-ZIPIII family) directly regulates the expression of ARGONAUTE10 (AGO10), ZPR1 and ZPR3. Because AGO10 was shown to dampen microRNA165/6 function, REV establishes a positive feedback loop on its own activity. Since ZPR-type microProteins are known to reduce HD-ZIPIII protein activity, REV concomitantly establishes a negative feedback loop. We propose that the interconnection of these microRNA/microProtein feedback loops regulates polarity set-up and stem cell activity in plants.

ATHB4 and HAT3, two class II HD-ZIP transcription factors, control leaf development in Arabidopsis.

In response to plant proximity or canopy shade, plants can react by altering elongation growth and development. Several members of the class II homeodomain-leucine zipper (HD-ZIPII) transcription factor family have been shown to play an instrumental role in the responses to shade. HD-ZIP members of the class III (HD-ZIPIII), by contrast, are involved in basic patterning processes. We recently showed that REVOLUTA (REV), a member of the HD-ZIPIII family, directly and positively regulates the expression of several genes involved in shade-induced growth, such as those encoding HD-ZIPII factors HAT2, HAT3, ATHB2/HAT4 and ATHB4, and of the components of the auxin biosynthesis pathway YUCCA5 and TAA1. Furthermore, we could demonstrate a novel role for HD-ZIPIII in shade-induced promotion of growth. Here we show that besides responding to shade, ATHB4 and HAT3 have a critical role in establishing the dorso-ventral axis in cotyledons and developing leaves. Loss-of-function mutations in these two HD-ZIPII genes (athb4 hat3) results in severely abaxialized, entirely radialized leaves. Conversely, overexpression of HAT3 results in adaxialized leaf development. Taken together, our findings unravel a so far unappreciated role for an HD-ZIPII/HD-ZIPIII module required for dorso-ventral patterning of leaves. The finding that HD-ZIPII/HD-ZIPIII also function in shade avoidance suggests that this module is at the nexus of patterning and growth promotion.