The value of twinned pollinator-pollen metabarcoding: bumblebee pollination service is weakly partitioned within a UK grassland community.

Predicting ecological impact of declining bumblebee (Bombus) populations requires better understanding of interactions between pollinator partitioning of floral resources and plant partitioning of pollinator resources. Here, we combine Cytochrome Oxidase 1 (CO1) barcoding for bumblebee identification and rbcL metabarcoding of pollen carried by bees in three species-rich UK pastures. CO1 barcoding assigned 272 bees to eight species, with 33 individuals belonging to the cryptic Bombus lucorum complex (16 B. lucorum and 17 B. cryptarum). Seasonal bias in capture rates varied by species, with B. pratorum found exclusively in June/July and B. pascuorum more abundant in August. Pollen metabarcoding coupled with PERMANOVA and NMDS analyses revealed all bees carried several local pollen species and evidence of pollen resource partitioning between some species pairings, with Bombus pratorum carrying the most divergent pollen load. There was no evidence of resource partitioning between the two cryptic species present, but significantly divergent capture rates concorded with previous suggestions of separation on the basis of foraging behaviour being shaped by local/temporal differences in climatic conditions. Considering the bee carriage profile of pollen species revealed no significant difference between the nine most widely carried plant species. However, there was a sharp, tipping point change in community pollen carriage across all three sites that occurred during the transition between late July and early August. This transition resulted in a strong divergence in community pollen carriage between the two seasonal periods in both years. We conclude that the combined use of pollen and bee barcoding offers several benefits for further study of plant-pollinator interactions at the landscape scale.

Active and adaptive plasticity in a changing climate.

Better understanding of the mechanistic basis of plant plasticity will enhance efforts to breed crops resilient to predicted climate change. However, complexity in plasticity's conceptualisation and measurement may hinder fruitful crossover of concepts between disciplines that would enable such advances. We argue active adaptive plasticity is particularly important in shaping the fitness of wild plants, representing the first line of a plant's defence to environmental change. Here, we define how this concept may be applied to crop breeding, suggest appropriate approaches to measure it in crops, and propose a refocussing on active adaptive plasticity to enhance crop resilience. We also discuss how the same concept may have wider utility, such as in ex situ plant conservation and reintroductions.

Dual-locus DNA metabarcoding reveals southern hairy-nosed wombats (Lasiorhinus latifrons Owen) have a summer diet dominated by toxic invasive plants.

Habitat degradation and summer droughts severely restrict feeding options for the endangered southern hairy-nosed wombat (SHNW; Lasiorhinus latifrons). We reconstructed SHNW summer diets by DNA metabarcoding from feces. We initially validated rbcL and ndhJ diet reconstructions using autopsied and captive animals. Subsequent diet reconstructions of wild wombats broadly reflected vegetative ground cover, implying local rather than long-range foraging. Diets were all dominated by alien invasives. Chemical analysis of alien food revealed Carrichtera annua contains high levels of glucosinolates. Clinical examination (7 animals) and autopsy (12 animals) revealed that the most degraded site also contained most individuals showing signs of glucosinolate poisoning. We infer that dietary poisoning through the ingestion of alien invasives may have contributed to the recent population crashes in the region. In floristically diverse sites, individuals appear to be able to manage glucosinolate intake by avoidance or episodic feeding but this strategy is less tractable in the most degraded sites. We conclude that recovery of the most affected populations may require effective Carrichtera management and interim supplementary feeding. More generally, we argue that protection against population decline by poisoning in territorial herbivores requires knowledge of their diet and of those food plants containing toxic principles.

The potential of aerosol eDNA sampling for the characterisation of commercial seed lots.

Seed shipments, silos and storage houses often contain weed seeds or seeds of restricted crops such as undeclared genetically modified (GM) varieties. Random sub-sampling is the favoured approach to detect unwanted biological materials in seed lots but is prohibitively expensive or else ineffective for the huge volumes of seeds moved in commercial operations. This study uses maize and cowpea seed admixtures as an exemplar to evaluate the feasibility of using aerosol sampling of "seed dust" as an alternative to seed sub-sampling. In an initial calibration phase, qPCR of the rbcL barcode followed by high-resolution melting (HRM) of a DNA titration series revealed a strong linear relationship between mix composition and HRM profiles. However, the relationship became skewed when flour mixes were used to build the titration, implying a DNA extraction bias favouring cowpea. Aerosol samples of seed dust above a titration of mixed seed samples were then collected along vertical and lateral axes. Aerosols were characterised by light microscopy, qPCR-HRM and next-generation DNA sequencing (Illumina MiSeq). Both molecular approaches again showed bias but this time in a reverse direction to flour samples. Microscopic examination of the aerosol sample suggested this divergence could be attributed to differences in abundance of airborne starch particles. Despite the bias, it was nevertheless possible to estimate relative abundance of each species using the abundance of minibarcodes. In light of these results we explore the feasibility of aerosol sampling for commercial seed lot characterisation.

Replacing Sanger with Next Generation Sequencing to improve coverage and quality of reference DNA barcodes for plants.

We estimate the global BOLD Systems database holds core DNA barcodes (rbcL + matK) for about 15% of land plant species and that comprehensive species coverage is still many decades away. Interim performance of the resource is compromised by variable sequence overlap and modest information content within each barcode. Our model predicts that the proportion of species-unique barcodes reduces as the database grows and that 'false' species-unique barcodes remain >5% until the database is almost complete. We conclude the current rbcL + matK barcode is unfit for purpose. Genome skimming and supplementary barcodes could improve diagnostic power but would slow new barcode acquisition. We therefore present two novel Next Generation Sequencing protocols (with freeware) capable of accurate, massively parallel de novo assembly of high quality DNA barcodes of >1400 bp. We explore how these capabilities could enhance species diagnosis in the coming decades.

Meristem micropropagation of cassava (Manihot esculenta) evokes genome-wide changes in DNA methylation.

There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta) cultivars. Principal Co-ordinate Analysis of profiles generated by methylation-sensitive amplified polymorphism revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype by sequencing identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologs have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops.

Epi-fingerprinting and epi-interventions for improved crop production and food quality.

Increasing crop production at a time of rapid climate change represents the greatest challenge facing contemporary agricultural research. Our understanding of the genetic control of yield derives from controlled field experiments designed to minimize environmental variance. In spite of these efforts there is substantial residual variability among plants attributable to Genotype × Environment interactions. Recent advances in the field of epigenetics have revealed a plethora of gene control mechanisms that could account for much of this unassigned variation. These systems act as a regulatory interface between the perception of the environment and associated alterations in gene expression. Direct intervention of epigenetic control systems hold the enticing promise of creating new sources of variability that could enhance crop performance. Equally, understanding the relationship between various epigenetic states and responses of the crop to specific aspects of the growing environment (epigenetic fingerprinting) could allow for a more tailored approach to plant agronomy. In this review, we explore the many ways in which epigenetic interventions and epigenetic fingerprinting can be deployed for the improvement of crop production and quality.

Assessing the value of imperfect biocontainment nationally: rapeseed in the United Kingdom as an exemplar.

Paternal biocontainment methods (PBMs) act by preventing pollen-mediated transgene flow. They are compromised by transgene escape via the crop-maternal line. We therefore assess the efficacy of PBMs for transgenic rapeseed (Brassica napus) biocontainment across the United Kingdom by estimating crop-maternal hybridization with its two progenitor species. We used remote sensing, field surveys, agricultural statistics, and meta-analysis to determine the extent of sympatry between the crop and populations of riparian and weedy B. rapa and B. oleracea. We then estimated the incidence of crop-maternal hybridization across all settings to predict the efficacy of PBMs. Evidence of crop chloroplast capture by the progenitors was expanded to a national scale, revealing that crop-maternal gene flow occurs at widely variable rates and is dependent on both the recipient and setting. We use these data to explore the value that this kind of biocontainment can bring to genetic modification (GM) risk management in terms of reducing the impact that hybrids have on the environment rather than preventing or reducing hybrid abundance per se.

Transgenerational, dynamic methylation of stomata genes in response to low relative humidity.

Transgenerational inheritance of abiotic stress-induced epigenetic modifications in plants has potential adaptive significance and might condition the offspring to improve the response to the same stress, but this is at least partly dependent on the potency, penetrance and persistence of the transmitted epigenetic marks. We examined transgenerational inheritance of low Relative Humidity-induced DNA methylation for two gene loci in the stomatal developmental pathway in Arabidopsis thaliana and the abundance of associated short-interfering RNAs (siRNAs). Heritability of low humidity-induced methylation was more predictable and penetrative at one locus (SPEECHLESS, entropy ≤ 0.02; χ2 < 0.001) than the other (FAMA, entropy ≤ 0.17; χ2 ns). Methylation at SPEECHLESS correlated positively with the continued presence of local siRNAs (r2 = 0.87; p = 0.013) which, however, could be disrupted globally in the progeny under repeated stress. Transgenerational methylation and a parental low humidity-induced stomatal phenotype were heritable, but this was reversed in the progeny under repeated treatment in a previously unsuspected manner.

Differential effect of three base modifications on DNA thermostability revealed by high resolution melting.

High resolution melting (HRM) can detect and quantify the presence of 5-methylcytosine (5mC) in DNA samples, but the ability of HRM to diagnose other DNA modifications remains unexplored. The DNA bases N6-methyladenine and 5-hydroxymethylcytosine occur across almost all phyla. While their function remains controversial, their presence perturbs DNA structure. Such modifications could affect gene regulation, chromatin condensation and DNA packaging. Here, we reveal that DNA containing N6-methyladenine or 5-hydroxymethylcytosine exhibits reduced thermal stability compared to cytosine-methylated DNA. These thermostability changes are sufficiently divergent to allow detection and quantification by HRM analysis. Thus, we report that HRM distinguishes between sequence-identical DNA differing only in the modification type of one base. This approach is also able to distinguish between two DNA fragments carrying both N6-methyladenine and 5-methylcytosine but differing only in the distance separating the modified bases. This finding provides scope for the development of new methods to characterize DNA chemically and to allow for low cost screening of mutant populations of genes involved in base modification. More fundamentally, contrast between the thermostabilizing effects of 5mC on dsDNA compared with the destabilizing effects of N6-methyladenine (m6A) and 5-hydroxymethylcytosine (5hmC) raises the intriguing possibility of an antagonistic relationship between modification types with functional significance.

DNA barcoding the native flowering plants and conifers of Wales.

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification.

Confocal observations of late-acting self-incompatibility in Theobroma cacao L.

Cocoa (Theobroma cacao) has an idiosyncratic form of late-acting self-incompatibility that operates through the non-fusion of incompatible gametes. Here, we used high-resolution confocal microscopy to define fine level changes to the embryo sac of the strongly self-incompatible cocoa genotype SCA 24 in the absence of pollination, and following compatible and incompatible pollination. All sperm nuclei had fused with the female nuclei by 48 h following compatible pollinations. However, following incompatible pollinations, we observed divergence in the behaviour of sperm nuclei following release into the embryo sac. Incomplete sperm nucleus migration occurred in approximately half of the embryo sacs, where the sperm nuclei had so far failed to reach the female gamete nuclei. Sperm nuclei reached but did not fuse with the female gamete nuclei in the residual cases. We argue that the cellular mechanisms governing sperm nucleus migration to the egg nucleus and those controlling subsequent nuclear fusion are likely to differ and should be considered independently. Accordingly, we recommend that future efforts to characterise the genetic basis of LSI in cocoa should take care to differentiate between these two events, both of which contribute to failed karyogamy. Implications of these results for continuing efforts to gain better understanding of the genetic control of LSI in cocoa are discussed.

DNA barcoding simplifies environmental risk assessment of genetically modified crops in biodiverse regions.

Transgenes encoding for insecticidal crystal (Cry) proteins from the soil-dwelling bacterium Bacillus Thuringiensis have been widely introduced into Genetically Modified (GM) crops to confer protection against insect pests. Concern that these transgenes may also harm beneficial or otherwise valued insects (so-called Non Target Organisms, NTOs) represents a major element of the Environmental Risk Assessments (ERAs) used by all countries prior to commercial release. Compiling a comprehensive list of potentially susceptible NTOs is therefore a necessary part of an ERA for any Cry toxin-containing GM crop. In partly-characterised and biodiverse countries, NTO identification is slowed by the need for taxonomic expertise and time to enable morphological identifications. This limitation represents a potentially serious barrier to timely adoption of GM technology in some developing countries. We consider Bt Cry1A cowpea (Vigna unguiculata) in Nigeria as an exemplar to demonstrate how COI barcoding can provide a simple and cost-effective means of addressing this problem. Over a period of eight weeks, we collected 163 insects from cowpea flowers across the agroecological and geographic range of the crop in Nigeria. These individuals included 32 Operational Taxonomic Units (OTUs) spanning four Orders and that could mostly be assigned to genus or species level. They included 12 Lepidopterans and two Coleopterans (both potentially sensitive to different groups of Cry proteins). Thus, barcode-assisted diagnoses were highly harmonised across groups (typically to genus or species level) and so were insensitive to expertise or knowledge gaps. Decisively, the entire study was completed within four months at a cost of less than 10,000 US$. The broader implications of the findings for food security and the capacity for safe adoption of GM technology are briefly explored.

Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development.

Environmental cues influence the development of stomata on the leaf epidermis, and allow plants to exert plasticity in leaf stomatal abundance in response to the prevailing growing conditions. It is reported that Arabidopsis thaliana 'Landsberg erecta' plants grown under low relative humidity have a reduced stomatal index and that two genes in the stomatal development pathway, SPEECHLESS and FAMA, become de novo cytosine methylated and transcriptionally repressed. These environmentally-induced epigenetic responses were abolished in mutants lacking the capacity for de novo DNA methylation, for the maintenance of CG methylation, and in mutants for the production of short-interfering non-coding RNAs (siRNAs) in the RNA-directed DNA methylation pathway. Induction of methylation was quantitatively related to the induction of local siRNAs under low relative humidity. Our results indicate the involvement of both transcriptional and post-transcriptional gene suppression at these loci in response to environmental stress. Thus, in a physiologically important pathway, a targeted epigenetic response to a specific environmental stress is reported and several of its molecular, mechanistic components are described, providing a tractable platform for future epigenetics experiments. Our findings suggest epigenetic regulation of stomatal development that allows for anatomical and phenotypic plasticity, and may help to explain at least some of the plant's resilience to fluctuating relative humidity.

Cytosine methylation regulates oviposition in the pathogenic blood fluke Schistosoma mansoni.

Similar to other metazoan pathogens, Schistosoma mansoni undergoes transcriptional and developmental regulation during its complex lifecycle and host interactions. DNA methylation as a mechanism to control these processes has, to date, been discounted in this parasite. Here we show the first evidence for cytosine methylation in the S. mansoni genome. Transcriptional coregulation of novel DNA methyltransferase (SmDnmt2) and methyl-CpG-binding domain proteins mirrors the detection of cytosine methylation abundance and implicates the presence of a functional DNA methylation machinery. Genome losses in cytosine methylation upon SmDnmt2 silencing and the identification of a hypermethylated, repetitive intron within a predicted forkhead gene confirm this assertion. Importantly, disruption of egg production and egg maturation by 5-azacytidine establishes an essential role for 5-methylcytosine in this parasite. These findings provide the first functional confirmation for this epigenetic modification in any worm species and link the cytosine methylation machinery to platyhelminth oviposition processes.

Direct detection and quantification of methylation in nucleic acid sequences using high-resolution melting analysis.

High-resolution melting (HRM) analysis exploits the reduced thermal stability of DNA fragments that contain base mismatches to detect single nucleotide polymorphisms (SNPs). However, the capacity of HRM to reveal other features of DNA chemistry remains unexplored. DNA methylation plays a key role in regulating gene expression and is essential for normal development in many higher organisms. The presence of methylated bases perturbs the double-stranded DNA structure, although its effect on DNA thermal stability is largely unknown. Here, we reveal that methylated DNA has enhanced thermal stability and is sufficiently divergent from nonmethylated DNA to allow detection and quantification by HRM analysis. This approach reliably distinguishes between sequence-identical DNA differing only in the methylation of one base. The method also provides accurate discrimination between mixes of methylated and nonmethylated DNAs, allowing discrimination between DNA that is 1% and 0% methylated and also between 97.5% and 100% methylated. Thus, the method provides a new means of adjusting thermal optima for DNA hybridization and PCR-based techniques and to empirically measure the impact of DNA methylation marks on the thermostability of regulatory regions. In the longer term, it could enable the development of new techniques to quantify methylated DNA.

Production of haploids and doubled haploids in oil palm.

BACKGROUND: Oil palm is the world's most productive oil-food crop despite yielding well below its theoretical maximum. This maximum could be approached with the introduction of elite F1 varieties. The development of such elite lines has thus far been prevented by difficulties in generating homozygous parental types for F1 generation. RESULTS: Here we present the first high-throughput screen to identify spontaneously-formed haploid (H) and doubled haploid (DH) palms. We secured over 1,000 Hs and one DH from genetically diverse material and derived further DH/mixoploid palms from Hs using colchicine. We demonstrated viability of pollen from H plants and expect to generate 100% homogeneous F1 seed from intercrosses between DH/mixoploids once they develop female inflorescences. CONCLUSIONS: This study has generated genetically diverse H/DH palms from which parental clones can be selected in sufficient numbers to enable the commercial-scale breeding of F1 varieties. The anticipated step increase in productivity may help to relieve pressure to extend palm cultivation, and limit further expansion into biodiverse rainforest.

A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis.

BACKGROUND: The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR. RESULTS: Here, we report the first combined use of Multiplex Tandem PCR (MT-PCR) and High Resolution Melt (HRM) analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker. CONCLUSIONS: A pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.