Brassica and Sinapis Seeds in Medieval Archaeological Sites: An Example of Multiproxy Analysis for Their Identification and Ethnobotanical Interpretation.

The genus Brassica includes some of the most important vegetable and oil crops worldwide. Many Brassica seeds (which can show diagnostic characters useful for species identification) were recovered from two archaeological sites in northern Italy, dated from between the Middle Ages and the Renaissance. We tested the combined use of archaeobotanical keys, ancient DNA barcoding, and references to ancient herbarium specimens to address the issue of diagnostic uncertainty. An unequivocal conventional diagnosis was possible for much of the material recovered, with the samples dominated by five Brassica species and Sinapis. The analysis using ancient DNA was restricted to the seeds with a Brassica-type structure and deployed a variant of multiplexed tandem PCR. The quality of diagnosis strongly depended on the molecular locus used. Nevertheless, many seeds were diagnosed down to species level, in concordance with their morphological identification, using one primer set from the core barcode site (matK). The number of specimens found in the Renaissance herbaria was not high; Brassica nigra, which is of great ethnobotanical importance, was the most common taxon. Thus, the combined use of independent means of species identification is particularly important when studying the early use of closely related crops, such as Brassicaceae.

Identification and distribution of novel badnaviral sequences integrated in the genome of cacao (Theobroma cacao).

Theobroma cacao is one of the most economically important tropical trees, being the source of chocolate. As part of an ongoing study to understand the diversity of the badnavirus complex, responsible for the cacao swollen shoot virus disease in West Africa, evidence was found recently of virus-like sequences in asymptomatic cacao plants. The present study exploited the wealth of genomic resources in this crop, and combined bioinformatic, molecular, and genetic approaches to report for the first time the presence of integrated badnaviral sequences in most of the cacao genetic groups. These sequences, which we propose to name eTcBV for endogenous T. cacao bacilliform virus, varied in type with each predominating in a specific genetic group. A diagnostic multiplex PCR method was developed to identify the homozygous or hemizygous condition of one specific insert, which was inherited as a single Mendelian trait. These data suggest that these integration events occurred before or during the species diversification in Central and South America, and prior to its cultivation in other regions. Such evidence of integrated sequences is relevant to the management of cacao quarantine facilities and may also aid novel methods to reduce the impact of such viruses in this crop.

Cacao swollen shoot virus detection and DNA barcoding of its vectors and putative vectors in Theobroma cacao L. by using polymerase chain reaction.

Cacao swollen shoot virus (CSSV) is an endemic pathogen causing significant economic losses to cacao (Theobroma cacao L.) production in West Africa. There is limited updated report on the occurrence, spread, genetic diversity and species of CSSV and its mealybug vectors, especially in Nigeria. Nigeria is presently lagging behind in the search for resistance to CSSV and its vectors in T. cacao L. The present study aimed to map and screen for the presence of CSSV and its natural vectors - female mealybugs (Pseudococcidae: Hemiptera) in cacao plantations in Nigeria. Symptomatic and asymptomatic cacao leaves and whole female mealybug samples were collected from major cacao-growing areas in Nigeria - Abia, Akwa Ibom, Cross River, Edo, Ondo and Oyo States. A total of 2568 cacao leaves from 1052 cacao trees were screened with polymerase chain reaction (PCR) using an open reading frame 1 (ORF 1) CSSV-specific primer pair. PCR screening of the mealybug species was performed using the cytochrome c oxidase subunit I (COI) gene. A combination of scanning electron microscopy (SEM) and histology for morphological identification and DNA barcoding enabled to characterise the female mealybug species. The results revealed that CSSV and its mealybug vectors are present in the major cacaogrowing areas in Nigeria. Although CSSV and its vectors have been previously reported in Cross River, Ondo and Oyo States, our results present the first documented evidence of CSSV emergence and its mealybug vectors in Abia, Akwa Ibom and Edo States. We also present the first report of Pseudococcus jackbeardsleyi (Gimpel and Miller) mealybug species on cacao in Nigeria. In conclusion, it is pertinent to re-establish coordinated routine survey and monitoring of CSSV and its mealybug vector presence in T. cacao L. in Nigeria.

Pseudomonas kirkiae sp. nov., a novel species isolated from oak in the United Kingdom, and phylogenetic considerations of the genera Pseudomonas, Azotobacter and Azomonas.

As the current episode of Acute Oak Decline (AOD) continues to affect native British oak in the United Kingdom, ongoing isolations from symptomatic and healthy oak have yielded a large Pseudomonas species population. These strains could be divided into taxa representing three potential novel species. Recently, two of these taxa were described as novel Pseudomonas species in the Pseudomonas fluorescens lineage. Here, we demonstrate using a polyphasic approach that the third taxon represents another novel Pseudomonas species. The 16S rRNA gene sequencing assigned the strains to the Pseudomonas aeruginosa lineage, while multilocus sequence analysis (based on partial gyrB, rpoB and rpoD sequences) placed the 13 strains in a single cluster on the border of the Pseudomonas stutzeri group. Whole genome intra-species comparisons (based on average nucleotide identity and in silico DNA-DNA hybridization) confirmed that the strains belong to a single taxon, while the inter-species comparisons with closest phylogenetic relatives yielded similarity values below the accepted species threshold. Therefore, we propose these strains as a novel species, namely Pseudomonas kirkiae sp. nov., with the type strain FRB 229T (P4CT=LMG 31089T=NCPPB 4674T). The phylogenetic analyses performed in this study highlighted the difficulties in assigning novel species to the genus Pseudomonas due to its polyphyletic nature and close relationship to the genus Azotobacter. We further propose that a thorough taxonomic re-evaluation of the genus Pseudomonas is essential and should be performed in the near future.

Lemurs in Cacao: Presence and Abundance within the Shade Plantations of Northern Madagascar.

The recognition that much biodiversity exists outside protected areas is driving research to understand how animals survive in anthropogenic landscapes. In Madagascar, cacao (Theobroma cacao) is grown under a mix of native and exotic shade trees, and this study sought to understand whether lemurs were present in these agroecosystems. Between November 2016 and March 2017, discussions with farmers, nocturnal reconnaissance surveys and camera traps were used to confirm the presence of lemurs in the Cokafa and Mangabe plantations near Ambanja, north-west Madagascar. Four species of lemur were encountered in nocturnal surveys: Mirza zaza, Phaner parienti, Microcebussp. and Cheirogaleussp. with encounter rates of 1.2, 0.4, 0.4 and 0.3 individuals/km, respectively. The presence of Lepilemur dorsalis was confirmed by camera trap. This is the first time lemurs have been studied in cacao plantations, and understanding how these threatened animals use anthropogenic landscapes is vital for their conservation.

Pseudomonas daroniae sp. nov. and Pseudomonas dryadis sp. nov., isolated from pedunculate oak affected by acute oak decline in the UK.

Twenty-two cream-coloured bacterial strains were isolated from oak trees affected by acute oak decline (AOD) in Southern England. Isolates were Gram-negative, motile, slightly curved rods, aerobic, non-spore-forming, catalase positive and oxidase positive. 16S rRNA gene sequence analysis placed the strains in two separate phylogenetic clusters in the Pseudomonas straminea group, with Pseudomonas flavescens as the closest phylogenetic relative. Multilocus sequence analyses of the gyrB, rpoD and rpoB genes supported the delineation of the strains into two separate taxa, which could be differentiated phenotypically and chemotaxonomically from each other, and their closest relatives. Average nucleotide identity and in silico DNA-DNA hybridization values revealed percentages of genome similarity below the species threshold (95 and 70 %, respectively) between the two taxa and the closest relatives, confirming their novel species status. Therefore, on the basis of this polyphasic approach we propose two novel Pseudomonas species, Pseudomonasdaroniae sp. nov. (type strain FRB 228T=LMG 31087T=NCPPB 4672T) and Pseudomonasdryadis sp. nov. (type strain FRB 230T=LMG 31087T=NCPPB 4673T).

Investigating ROS, RNS, and H2S-Sensitive Signaling Proteins.

The modification of proteins is a key way to alter their activity and function. Often thiols, cysteine residues, on proteins are attractive targets for such modification. Assuming that the thiol group is accessible then reactions may take place with a range of chemicals found in cells. These may include reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), reactive nitrogen species such as nitric oxide (NO), hydrogen sulfide (H2S), or glutathione. Such modifications often are instrumental to important cellular signaling processes, which ultimately result in modification of physiology of the organism. Therefore, there is a need to be able to identify such modifications. There are a variety of techniques to find proteins which may be altered in this way but here the focus is on two approaches: firstly, the use of fluorescent thiol derivatives and the subsequent use of mass spectrometry to identify the thiols involved; secondly the confirmation of such changes using biochemical assays and genetic mutants. The discussion will be based on the use of two model organisms: firstly the plant Arabidopsis thaliana (both as cell cultures and whole plants) and secondly the nematode worm Caenorhabditis elegans. However, these tools, as described, may be used in a much wider range of biological systems, including human and human tissue cultures.

Residual ground-water levels of the neonicotinoid thiacloprid perturb chemosensing of Caenorhabditis elegans.

This study investigated the neurological effects of residual ground-water levels of thiacloprid on the non-target organism Caenorhabditis elegans. Nematodes treated with thiacloprid showed a dose-dependent and significantly increased twitch response at concentrations above 50 ng mL-1 that disabled their forward locomotion in liquid culture. In comparison with untreated controls, 10 ng mL-1 thiacloprid perturbed the chemosensory ability of C. elegans such that the nematodes no longer demonstrated positive chemotaxis towards a NaCl chemo-attractant, reducing their chemotaxis index from +0.48 to near to zero. Nematodes also exhibited a locomotion characteristic of those devoid of chemo-attraction, making significantly more pirouetting turns of ≥90° than the untreated controls. Compared to the untreated controls, expression of the endocytosis-associated gene, Rab-10, was also increased in C. elegans that had developed to adulthood in the presence of 10 ng mL-1 thiacloprid, suggesting their active engagement in increased recycling of affected cellular components, such as their nAChRs. Thus, even residual, low levels of this less potent neonicotinoid that may be found in field ground-water had measurable effects on a beneficial soil organism which may have environmental and ecological implications that are currently poorly understood.

High-resolution melt and morphological analyses of mealybugs (Hemiptera: Pseudococcidae) from cacao: tools for the control of Cacao swollen shoot virus spread.

BACKGROUND: Mealybugs (Hemiptera: Coccoidea: Pseudococcidae) are key vectors of badnaviruses, including Cacao swollen shoot virus (CSSV), the most damaging virus affecting cacao (Theobroma cacao L.). The effectiveness of mealybugs as virus vectors is species dependent, and it is therefore vital that CSSV resistance breeding programmes in cacao incorporate accurate mealybug identification. In this work, the efficacy of a CO1-based DNA barcoding approach to species identification was evaluated by screening a range of mealybugs collected from cacao in seven countries. RESULTS: Morphologically similar adult females were characterised by scanning electron microscopy, and then, following DNA extraction, were screened with CO1 barcoding markers. A high degree of CO1 sequence homology was observed for all 11 individual haplotypes, including those accessions from distinct geographical regions. This has allowed the design of a high-resolution melt (HRM) assay capable of rapid identification of the commonly encountered mealybug pests of cacao. CONCLUSIONS: HRM analysis readily differentiated between mealybug pests of cacao that cannot necessarily be identified by conventional morphological analysis. This new approach, therefore, has potential to facilitate breeding for resistance to CSSV and other mealybug-transmitted diseases.

Using DNA Metabarcoding to Identify the Floral Composition of Honey: A New Tool for Investigating Honey Bee Foraging Preferences.

Identifying the floral composition of honey provides a method for investigating the plants that honey bees visit. We compared melissopalynology, where pollen grains retrieved from honey are identified morphologically, with a DNA metabarcoding approach using the rbcL DNA barcode marker and 454-pyrosequencing. We compared nine honeys supplied by beekeepers in the UK. DNA metabarcoding and melissopalynology were able to detect the most abundant floral components of honey. There was 92% correspondence for the plant taxa that had an abundance of over 20%. However, the level of similarity when all taxa were compared was lower, ranging from 22-45%, and there was little correspondence between the relative abundance of taxa found using the two techniques. DNA metabarcoding provided much greater repeatability, with a 64% taxa match compared to 28% with melissopalynology. DNA metabarcoding has the advantage over melissopalynology in that it does not require a high level of taxonomic expertise, a greater sample size can be screened and it provides greater resolution for some plant families. However, it does not provide a quantitative approach and pollen present in low levels are less likely to be detected. We investigated the plants that were frequently used by honey bees by examining the results obtained from both techniques. Plants with a broad taxonomic range were detected, covering 46 families and 25 orders, but a relatively small number of plants were consistently seen across multiple honey samples. Frequently found herbaceous species were Rubus fruticosus, Filipendula ulmaria, Taraxacum officinale, Trifolium spp., Brassica spp. and the non-native, invasive, Impatiens glandulifera. Tree pollen was frequently seen belonging to Castanea sativa, Crataegus monogyna and species of Malus, Salix and Quercus. We conclude that although honey bees are considered to be supergeneralists in their foraging choices, there are certain key species or plant groups that are particularly important in the honey bees environment. The reasons for this require further investigation in order to better understand honey bee nutritional requirements. DNA metabarcoding can be easily and widely used to investigate floral visitation in honey bees and can be adapted for use with other insects. It provides a starting point for investigating how we can better provide for the insects that we rely upon for pollination.

Environmental niche variation and evolutionary diversification of the Brachypodium distachyon grass complex species in their native circum-Mediterranean range.

UNLABELLED: • PREMISE OF THE STUDY: We conducted environmental niche modeling (ENM) of the Brachypodium distachyon s.l. complex, a model group of two diploid annual grasses (B. distachyon, B. stacei) and their derived allotetraploid (B. hybridum), native to the circum-Mediterranean region. We (1) investigated the ENMs of the three species in their native range based on present and past climate data; (2) identified potential overlapping niches of the diploids and their hybrid across four Quaternary windows; (3) tested whether speciation was associated with niche divergence/conservatism in the complex species; and (4) tested for the potential of the polyploid outperforming the diploids in the native range.• METHODS: Geo-referenced data, altitude, and 19 climatic variables were used to construct the ENMs. We used paleoclimate niche models to trace the potential existence of ancestral gene flow among the hybridizing species of the complex.• KEY RESULTS: Brachypodium distachyon grows in higher, cooler, and wetter places, B. stacei in lower, warmer, and drier places, and B. hybridum in places with intermediate climatic features. Brachypodium hybridum had the largest niche overlap with its parent niches, but a similar distribution range and niche breadth.• CONCLUSIONS: Each species had a unique environmental niche though there were multiple niche overlapping areas for the diploids across time, suggesting the potential existence of several hybrid zones during the Pleistocene and the Holocene. No evidence of niche divergence was found, suggesting that species diversification was not driven by ecological speciation but by evolutionary history, though it could be associated to distinct environmental adaptations.

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.

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.

Exploiting the Brachypodium Tool Box in cereal and grass research.

It is now a decade since Brachypodium distachyon (Brachypodium) was suggested as a model species for temperate grasses and cereals. Since then transformation protocols, large expressed sequence tag (EST) databases, tools for forward and reverse genetic screens, highly refined cytogenetic probes, germplasm collections and, recently, a complete genome sequence have been generated. In this review, we will describe the current status of the Brachypodium Tool Box and how it is beginning to be applied to study a range of biological traits. Further, as genomic analysis of larger cereals and forage grasses genomes are becoming easier, we will re-evaluate Brachypodium as a model species. We suggest that there remains an urgent need to employ reverse genetic and functional genomic approaches to identify the functionality of key genetic elements, which could be employed subsequently in plant breeding programmes; and a requirement for a Pooideae reference genome to aid assembling large pooid genomes. Brachypodium is an ideal system for functional genomic studies, because of its easy growth requirements, small physical stature, and rapid life cycle, coupled with the resources offered by the Brachypodium Tool Box.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2010-30 September 2010.

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross-tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.

Spontaneous capture of oilseed rape (Brassica napus) chloroplasts by wild B. rapa: implications for the use of chloroplast transformation for biocontainment.

Environmental concerns over the cultivation of Genetically Modified (GM) crops largely centre on the ecological consequences following gene flow to wild relatives. One attractive solution is to deploy biocontainment measures that prevent hybridization. Chloroplast transformation is the most advanced biocontainment method but is compromised by chloroplast capture (hybridization through the maternal lineage). To date, however, there is a paucity of information on the frequency of chloroplast capture in the wild. Oilseed rape (Brassica napus, AACC) frequently hybridises with wild Brassica rapa (AA, as paternal parent) and yields B. rapa-like introgressed individuals after only two generations. In this study we used chloroplast CAPS markers that differentiate between the two species to survey wild and weedy populations of B. rapa for the capture of B. napus chloroplasts. A total of 464 B. rapa plants belonging to 14 populations growing either in close proximity to B. napus (i.e. sympatric <5 m) or else were allopatric from the crop (>1 km) were assessed for chloroplast capture using PCR (trnL-F) and CAPS (trnT-L-Xba I) markers. The screen revealed that two sympatric B. rapa populations included 53 plants that possessed the chloroplast of B. napus. In order to discount these B. rapa plants as F(1) crop-wild hybrids, we used a C-genome-specific marker and found that 45 out of 53 plants lacked the C-genome and so were at least second generation introgressants. The most plausible explanation is that these individuals represent multiple cases of chloroplast capture following introgressive hybridisation through the female germ line from the crop. The abundance of such plants in sympatric sites thereby questions whether the use of chloroplast transformation would provide a sufficient biocontainment for GM oilseed rape in the United Kingdom.

Spontaneous gene flow from rapeseed (Brassica napus) to wild Brassica oleracea.

Research on the environmental risks of gene flow from genetically modified (GM) crops to wild relatives has traditionally emphasized recipients yielding most hybrids. For GM rapeseed (Brassica napus), interest has centred on the 'frequently hybridizing' Brassica rapa over relatives such as Brassica oleracea, where spontaneous hybrids are unreported in the wild. In two sites, where rapeseed and wild B. oleracea grow together, we used flow cytometry and crop-specific microsatellite markers to identify one triploid F1 hybrid, together with nine diploid and two near triploid introgressants. Given the newly discovered capacity for spontaneous introgression into B. oleracea, we then surveyed associated flora and fauna to evaluate the capacity of both recipients to harm cohabitant species with acknowledged conservational importance. Only B. oleracea occupies rich communities containing species afforded legislative protection; these include one rare micromoth species that feeds on B. oleracea and warrants further assessment. We conclude that increased attention should now focus on B. oleracea and similar species that yield few crop-hybrids, but possess scope to affect rare or endangered associates.

Genetic diversity and structure of natural and managed populations of Cedrus atlantica (Pinaceae) assessed using random amplified polymorphic DNA.

Cedrus atlantica (Pinaceae) is a large and exceptionally long-lived conifer native to the Rif and Atlas Mountains of North Africa. To assess levels and patterns of genetic diversity of this species, samples were obtained throughout the natural range in Morocco and from a forest plantation in Arbúcies, Girona (Spain) and analyzed using RAPD markers. Within-population genetic diversity was high and comparable to that revealed by isozymes. Managed populations harbored levels of genetic variation similar to those found in their natural counterparts. Genotypic analyses of molecular variance (AMOVA) found that most variation was within populations, but significant differentiation was also found between populations, particularly in Morocco. Bayesian estimates of F(ST) corroborated the AMOVA partitioning and provided evidence for population differentiation in C. atlantica. Both distance- and Bayesian-based clustering methods revealed that Moroccan populations comprise two genetically distinct groups. Within each group, estimates of population differentiation were close to those previously reported in other gymnosperms. These results are interpreted in the context of the postglacial history of the species and human impact. The high degree of among-group differentiation recorded here highlights the need for additional conservation measures for some Moroccan populations of C. atlantica.

Hybridization between Brassica napus and B. rapa on a national scale in the United Kingdom.

Measures blocking hybridization would prevent or reduce biotic or environmental change caused by gene flow from genetically modified (GM) crops to wild relatives. The efficacy of any such measure depends on hybrid numbers within the legislative region over the life-span of the GM cultivar. We present a national assessment of hybridization between rapeseed (Brassica napus) and B. rapa from a combination of sources, including population surveys, remote sensing, pollen dispersal profiles, herbarium data, local Floras, and other floristic databases. Across the United Kingdom, we estimate that 32,000 hybrids form annually in waterside B. rapa populations, whereas the less abundant weedy populations contain 17,000 hybrids. These findings set targets for strategies to eliminate hybridization and represent the first step toward quantitative risk assessment on a national scale.