A DNA-based method for studying root responses to drought in field-grown wheat genotypes

Root systems are critical for water and nutrient acquisition by crops. Current methods measuring root biomass and length are slow and labour-intensive for studying root responses to environmental stresses in the field. Here, we report the development of a method that measures changes in the root DNA concentration in soil and detects root responses to drought in controlled environment and field trials. To allow comparison of soil DNA concentrations from different wheat genotypes, we also developed a procedure for correcting genotypic differences in the copy number of the target DNA sequence. The new method eliminates the need for separation of roots from soil and permits large-scale phenotyping of root responses to drought or other environmental and disease stresses in the field.Chun Y. Huang, Haydn Kuchel, James Edwards, Sharla Hall, Boris Parent, Paul Eckermann, Herdina, Diana M. Hartley, Peter Langridge & Alan C. McKa

Population genomics of marine zooplankton

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here for personal use, not for redistribution. The definitive version was published in Bucklin, Ann et al. "Population Genomics of Marine Zooplankton." Population Genomics: Marine Organisms. Ed. Om P. Rajora and Marjorie Oleksiak. Springer, 2018. doi:10.1007/13836_2017_9.The exceptionally large population size and cosmopolitan biogeographic distribution that distinguish many – but not all – marine zooplankton species generate similarly exceptional patterns of population genetic and genomic diversity and structure. The phylogenetic diversity of zooplankton has slowed the application of population genomic approaches, due to lack of genomic resources for closelyrelated species and diversity of genomic architecture, including highly-replicated genomes of many crustaceans. Use of numerous genomic markers, especially single nucleotide polymorphisms (SNPs), is transforming our ability to analyze population genetics and connectivity of marine zooplankton, and providing new understanding and different answers than earlier analyses, which typically used mitochondrial DNA and microsatellite markers. Population genomic approaches have confirmed that, despite high dispersal potential, many zooplankton species exhibit genetic structuring among geographic populations, especially at large ocean-basin scales, and have revealed patterns and pathways of population connectivity that do not always track ocean circulation. Genomic and transcriptomic resources are critically needed to allow further examination of micro-evolution and local adaptation, including identification of genes that show evidence of selection. These new tools will also enable further examination of the significance of small-scale genetic heterogeneity of marine zooplankton, to discriminate genetic “noise” in large and patchy populations from local adaptation to environmental conditions and change.Support was provided by the US National Science Foundation to AB and RJO (PLR-1044982) and to RJO (MCB-1613856); support to IS and MC was provided by Nord University (Norway)

Ribosomal DNA Deletions Modulate Genome-Wide Gene Expression: “rDNA–Sensitive” Genes and Natural Variation

The ribosomal rDNA gene array is an epigenetically-regulated repeated gene locus. While rDNA copy number varies widely between and within species, the functional consequences of subtle copy number polymorphisms have been largely unknown. Deletions in the Drosophila Y-linked rDNA modifies heterochromatin-induced position effect variegation (PEV), but it has been unknown if the euchromatic component of the genome is affected by rDNA copy number. Polymorphisms of naturally occurring Y chromosomes affect both euchromatin and heterochromatin, although the elements responsible for these effects are unknown. Here we show that copy number of the Y-linked rDNA array is a source of genome-wide variation in gene expression. Induced deletions in the rDNA affect the expression of hundreds to thousands of euchromatic genes throughout the genome of males and females. Although the affected genes are not physically clustered, we observed functional enrichments for genes whose protein products are located in the mitochondria and are involved in electron transport. The affected genes significantly overlap with genes affected by natural polymorphisms on Y chromosomes, suggesting that polymorphic rDNA copy number is an important determinant of gene expression diversity in natural populations. Altogether, our results indicate that subtle changes to rDNA copy number between individuals may contribute to biologically relevant phenotypic variation

Development of a TaqMan qPCR assay for detection of Alexandrium spp and application to harmful algal bloom monitoring

The Genus Alexandrium is a widespread dinoflagellate marine phytoplankton that is the primary causative organism causing Paralytic Shellfish Poisoning (PSP) intoxications in European waters. EU food safety directives specify that EU Member States must implement a routine monitoring programme to mitigate risks associated with bio-accumulation of biotoxins by bivalve shellfish, such as those produced by Alexandrium. This strategic drive comprises of both direct testing of bivalve flesh for the presence of regulated toxins and an early warning phytoplankton monitoring programme. In the UK the flesh testing moved away from animal bio-assays to analytical chemistry techniques, whereas phytoplankton monitoring methods have seen little technological advancement since implementation. Methods currently utilize light microscopy and manual enumeration of different algal species. These methods although proven are time consuming, reliant on highly trained staff, have high limits of detection (LOD) with low specificity, unable to reliably identify Alexandrium to species level. The implications of these limitations of the techniques mean that in the case of Alexandrium the LOD is also the action limit and as such it is easy to miss positive samples affecting the efficacy of any early warning strategy. This study outlines the development, preliminary method characterisation, validation and trial implementation of an alternative early warning technique, utilizing quantitative PCR to identify water samples containing Alexandrium cells. The approach outlined in this document, showed an improved correlation with flesh toxicity, improved sensitivity, improved throughput compared to traditional light microscopy methods and there was also good correlation with higher cell abundance samples when compared to the light microscopy results. The application of this approach to routine water samples was explored and was found to demonstrate potential as a corroborative method for use during flesh intoxication episodes. This study offers potential for future improvements in the accuracy and sensitivity of phytoplankton monitoring whilst ensuring continuity of public safety, providing cost savings and offering new research opportunities

Effect of polyhydroxy compounds on the thermal and mechanical properties of ageing starch gels

Starch based products, such as cakes and cookies, are formulated with relatively high amounts of sugar. Relatively little research has been done on the effects of sugars upon starch retrogradation and the anti-retrogradation mechanism of sugars is not yet understood. This study was conducted to provide further information on the effect of polyols (polyhydroxy compounds) on the thermal and mechanical properties of concentrated starch gels and provide further insight into the anti-retrogradation mechanism(s) of polyols. The development of ordered structures in ageing starch, in the presence of polyols, was probed by small strain dynamic rheometry and differential scanning calorimetry (DSC). Polyols were added at a ratio of 1:0.5:1.5 (w/w) for starch:polyol:water mixtures. The effect of polyols on the formation of "ordered" structures within ageing amylopectin networks was studied by DSC. The addition of glucose oligosaccharides of DP 1 to 3 retarded retrogradation with increasing DP, oligomers with a DP 4 to 7 exerted little effect, while maltooctaose promoted this process. The effect of glucose-based disaccharides with different glucosidic linkages on the reorganization of amylopectin short DP chains was also examined. Disaccharides with more extended rigid structures (i.e. cellobiose, B (1->4) glucosidic linkage) retarded retrogradation of amylopectin to a greater extent than disaccharides with more flexible glucosidic linkages (i.e. maltose, a (1->4) linkage); however, differences among the disaccharides were rather minor over longer storage periods. Minor differences were found when the anti-retrogradation behaviour of sugars and their respective sugar alcohols was compared. However, the addition of glyceraldehyde completely inhibited the formation of "ordered" structures in the ageing waxy maize starch network over the storage period examined. The effect of pentoses and hexoses on the retrogradation of waxy maize starch gels was studied. Pentoses (i.e. ribose and xylose) were found to retard the reorganization of amylopectin chains more effectively than hexoses (i.e. fructose and glucose). In fact, fructose was shown to accelerate the rate of retrogradation as compared to the control (starch-water). Finally, it was shown that retrogradation of amylopectin was strongly influenced by the concentration of polyol added. Ribose continuously retarded the formation of "ordered" structures within ageing waxy maize starch gels with increasing concentration (0-37.5% w/w), whereas fructose promoted this process at concentrations greater than 7.5% (w/w). A comparative study was undertaken to examine the effect of polyols on the thermal and mechanical properties of waxy maize, wheat, potato and pea starch gels. The addition of polyols, at a ratio of 1:0.5:1.5 (w/w) for starch:polyol:water mixtures inhibited chain reorganization of starch gels, as followed by DSC and dynamic rheology, in the following order: ribose > sucrose > maltotriose > water alone, glucose > fructose. The effects of polyols on the development of the retrogradation endotherm (AH) and gel rigidity (increase in G') were found to be less pronounced for potato and pea starches than waxy maize or wheat starches. Significant correlations (p <_ 0.05) were observed between the effect of monosaccharides on the retrogradation endotherm (AH) and their hydration properties (n H and n DHN). An anti-retrogradation mechanism of polyols was proposed on the basis of their effect on the three-dimensional hydrogen-bonded structure of water

Distribution of the uncultured protist MAST-4 in the Indian Ocean, Drake Passage and Mediterranean Sea assessed by real-time quantitative PCR

12 pages, 5 figures, 1 tableMolecular surveys of marine picoeukaryotes have revealed a large number of sequences unrelated to cultured organisms, such as those forming the marine stramenopile (MAST)-4 clade. Recent FISH (fluorescent in situ hybridization) data have shown that MAST-4 cells are uncultured heterotrophic flagellates of 2–3 μm in size that have a global distribution in non-polar marine waters. However, FISH is time-consuming and hard to apply to the many samples generated during oceanographic cruises, so we developed a real-time quantitative polymerase chain reaction (Q-PCR) protocol to determine rapidly the abundance of this group using environmental DNA. We designed a primer set targeting the 18S rRNA genes (rDNA) of MAST-4 and optimized and calibrated the Q-PCR protocol using a plasmid with the target sequence as insert. The Q-PCR was then applied to quantify MAST-4 rDNA molecules along three marine transects, longitudinal in the Indian Ocean, latitudinal in the Drake Passage and coastal–offshore in the Mediterranean Sea, and to a temporal study in a Mediterranean Sea coastal station. MAST-4 was detected in all samples processed (averaged abundances between 500 and 1000 rDNA molecules ml−1) except in mesopelagic and Antarctic samples, where it was virtually absent. In general, it was more abundant in the coast than offshore and in the deep chlorophyll maximum than at surface. A comparison of Q-PCR and FISH signals in well-controlled microbial incubations indicated that MAST-4 cells have around 30 copies of the rDNA operon. This Q-PCR assay quickly yielded quantitative data of uncultured MAST-4 cells and confirmed their wide distribution and putative ecological importanceWe thank the captains and crews of Research Vessels Hespérides and Melville and the chief scientists (T. Calafat, C. Pedrós-Alió and D. Blackman) for providing an optimal environment for sampling. This study was supported by projects TRANSINDICO (REN2000-1471-CO2-01/MAR), ESTRAMAR (CTM2004-12631/MAR, MEC) and PROTAL (HA2004-088, MCyT) to R.M. and by an F.P.I. fellowship from the Spanish Ministry of Education and Science to R.R.M.Peer reviewe

High-throughput amplicon sequencing of rRNA genes requires a copy number correction to accurately reflect the effects of management practices on soil nematode community structure

Nematodes are abundant consumers in grassland soils, but more sensitive and specific methods of enumeration are needed to improve our understanding of how different nematode species affect, and are affected by, ecosystem processes. High‐throughput amplicon sequencing is used to enumerate microbial and invertebrate communities at a high level of taxonomic resolution, but the method requires validation against traditional specimen‐based morphological identifications. To investigate the consistency between these approaches, we enumerated nematodes from a 25‐year field experiment using both morphological and molecular identification techniques in order to determine the long‐term effects of annual burning and nitrogen enrichment on soil nematode communities. Family‐level frequencies based on amplicon‐sequencing were not initially consistent with specimen‐based counts, but correction for differences in rRNA gene copy number using a genetic algorithm improved quantitative accuracy. Multivariate analysis of corrected sequence‐based abundances of nematode families was consistent with, but not identical to, analysis of specimen‐based counts. In both cases, herbivores, fungivores, and predator/omnivores generally were more abundant in burned than non‐burned plots, while bacterivores generally were more abundant in non-burned or nitrogen enriched plots. Discriminate analysis of sequence‐based abundances identified putative indicator species representing each trophic group. We conclude that high-throughput amplicon sequencing can be a valuable method for characterizing nematode communities at high taxonomic resolution as long as rRNA gene copy number variation is accounted for and accurate sequence databases are available

Diversity patterns and activity of uncultured marine heterotrophic flagellates unveiled with pyrosequencing

11 pages, 7 figures, 2 tablesFlagellated heterotrophic microeukaryotes have key roles for the functioning of marine ecosystems as they channel large amounts of organic carbon to the upper trophic levels and control the population sizes of bacteria and archaea. Still, we know very little on the diversity patterns of most groups constituting this evolutionary heterogeneous assemblage. Here, we investigate 11 groups of uncultured flagellates known as MArine STramenopiles (MASTs). MASTs are ecologically very important and branch at the base of stramenopiles. We explored the diversity patterns of MASTs using pyrosequencing (18S rDNA) in coastal European waters. We found that MAST groups range from highly to lowly diversified. Pyrosequencing (hereafter ‘454’) allowed us to approach to the limits of taxonomic diversity for all MAST groups, which varied in one order of magnitude (tens to hundreds) in terms of operational taxonomic units (98% similarity). We did not evidence large differences in activity, as indicated by ratios of DNA:RNA-reads. Most groups were strictly planktonic, although we found some groups that were active in sediments and even in anoxic waters. The proportion of reads per size fraction indicated that most groups were composed of very small cells (~2–5 μm). In addition, phylogenetically different assemblages appeared to be present in different size fractions, depths and geographic zones. Thus, MAST diversity seems to be highly partitioned in spatial scales. Altogether, our results shed light on these ecologically very important but poorly known groups of uncultured marine flagellatesFinancial support for this work has been provided by a Marie Curie Intra-European Fellowship grant (PIEF-GA-2009-235365) to RL and by projects BioMarKs (2008-6530, ERA-net Biodiversa, EU) and FLAME (CGL2010-16304, MICINN, Spain) to RM. Large-scale computing resources were provided by the Canarian Institute of Astrophysics (www.iac.es), through the Barcelona Supercomputer Center and the Spanish Network of Supercomputing (grants BCV-2010-3-0003 and 2011-2-0003/3-0005 to RL and RM). We thank the BioMarKs consortium for undertaking the sampling and performing the initial laboratory processing of the samples, in particular Sarah Romac. We thank Hiroyuki Ogata and Jean-Michel Claverie for the implementation of bioinformatics tools through a BioMarKs grant and a project from the French National Research Agency (ANR-08-BDVA-003) to Jean-Michel Claverie. Javier del Campo is thanked for providing curated Sanger sequences of Ochrophyta. Berit Kaasa at the University of Oslo is thanked for running the nutrient analyses. We thank the three reviewers and the editor who helped to improve this workPeer reviewe