A simple method to score single nucleotide polymorphisms based on allele‐specific PCR and primer‐induced fragment‐length variation

We describe a simple protocol to genotype single nucleotide polymorphisms (SNPs), which combines allele‐specific polymerase chain reaction (PCR) with fragment‐length analysis. Three primers are used in the PCR: two allele‐specific forward primers with a length‐difference and one reverse primer. The forward primers induce a length‐difference between the SNP‐variants, which can be assessed with standard fragment‐length analyses. We designed primers for 21 SNPs, and codominance was achieved for 76% of these SNPs. An inexpensive and flexible laser‐detection scoring protocol can be achieved with multiplex scoring and by incorporating the M13(‐21) genotyping method.     

Leaf trait associations with environmental variation in the wide‐ranging shrub Dodonaea viscosa subsp. angustissima (Sapindaceae)

Intra‐species variation in specific leaf area (SLA) and leaf area (LA) provides mechanistic insight into the persistence and function of plants, including their likely success under climate change and their suitability for revegetation. We measured SLA and LA in 101 Australian populations of the perennial shrub Dodonaea viscosa (L.) Jacq. subsp. angustissima (narrow‐leaf hop‐bush) (Sapindaceae). Populations were located across about a 1000 km north–south gradient, with climate grading from arid desert to mesic Mediterranean. We also measured leaves from 11 populations across an elevational gradient (300–800 m asl), where aridity and temperature decrease with elevation. We used regression and principal component analyses to relate leaf traits to the abiotic environment. SLA displayed clinal variation, increasing from north to south and correlated with latitude and the first principal component of joint environmental variables. Both SLA and LA correlated positively with most climatic and edaphic variables. Across latitude, LA showed more variability than SLA. Changes in leaf density and thickness may have caused the relative stability of SLA. Only LA decreased with elevation. The absence of a SLA response to elevation could be a consequence of abiotic conditions that favour low SLA at both ends of the elevational gradient. We demonstrated that the widely distributed narrow‐leaf hop‐bush shows considerable variability in LA and SLA, which allows it to persist in a broad environmental envelope. As this shrub is widely used for revegetation in Australia, South America and the Asia‐Pacific region, our results are consistent with the notion that seed used to revegetate mesic environments could be sourced from more arid areas to increase seed suitability to future climate change.     

Within‐species patterns challenge our understanding of the leaf economics spectrum

The utility of plant functional traits for predictive ecology relies on our ability to interpret trait variation across multiple taxonomic and ecological scales. Using extensive data sets of trait variation within species, across species and across communities, we analysed whether and at what scales leaf economics spectrum (LES) traits show predicted trait–trait covariation. We found that most variation in LES traits is often, but not universally, at high taxonomic levels (between families or genera in a family). However, we found that trait covariation shows distinct taxonomic scale dependence, with some trait correlations showing opposite signs within vs. across species. LES traits responded independently to environmental gradients within species, with few shared environmental responses across traits or across scales. We conclude that, at small taxonomic scales, plasticity may obscure or reverse the broad evolutionary linkages between leaf traits, meaning that variation in LES traits cannot always be interpreted as differences in resource use strategy.     

A tropical epiphytic orchid uses a low‐light interception strategy in a spatially heterogeneous light environment

Light is considered a non‐limiting factor for vascular epiphytes. Nevertheless, an epiphyte's access to light may be limited by phorophyte shading and the spatio‐temporal environmental patchiness characteristic of epiphytic habitats. We assessed the extent to which potential light interception in Rodriguezia granadensis, an epiphytic orchid, is determined by individual factors (plant size traits and leaf traits), or environmental heterogeneity (light patchiness) within the crown of the phorophyte, or both. We studied 104 adult plants growing on Psidium guajava trees in two habitats with contrasting canopy cover: a dry tropical forest edge, and isolated trees in a pasture. We recorded the number of leaves and the leaf area, the leaf position angles, and the potential exposure of the leaf surface to direct irradiance (silhouette area of the leaf blade), and the potential irradiance incident on each plant. We found the epiphytes experience a highly heterogeneous light environment in the crowns of P. guajava. Nonetheless, R. granadensis plants displayed a common light interception strategy typical of low‐light environments, resembling terrestrial, forest understory plants. Potential exposure of the total leaf surface to direct irradiance correlated positively with plant size and within‐plant variation in leaf orientation. In many‐leaved individuals, within‐plant variation in leaf angles produced complementary leaf positions that enhanced potential light interception. This light interception strategy suggests that, in contrast to current wisdom, enhancing light capture is important for vascular epiphytes in canopies with high spatio‐temporal heterogeneity in light environments.     

Plant responses to climatic extremes: within‐species variation equals among‐species variation

Within‐species and among‐species differences in growth responses to a changing climate have been well documented, yet the relative magnitude of within‐species vs. among‐species variation has remained largely unexplored. This missing comparison impedes our ability to make general predictions of biodiversity change and to project future species distributions using models. We present a direct comparison of among‐ versus within‐species variation in response to three of the main stresses anticipated with climate change: drought, warming, and frost. Two earlier experiments had experimentally induced (i) summer drought and (ii) spring frost for four common European grass species and their ecotypes from across Europe. To supplement existing data, a third experiment was carried out, to compare variation among species from different functional groups to within‐species variation. Here, we simulated (iii) winter warming plus frost for four grasses, two nonleguminous, and two leguminous forbs, in addition to eleven European ecotypes of the widespread grass Arrhenatherum elatius. For each experiment, we measured: (i) C/N ratio and biomass, (ii) chlorophyll content and biomass, and (iii) plant greenness, root ¹⁵N uptake, and live and dead tissue mass. Using coefficients of variation (CVs) for each experiment and response parameter, a total of 156 within‐ vs. among‐species comparisons were conducted, comparing within‐species variation in each of four species with among‐species variation for each seed origin (five countries). Of the six significant differences, within‐species CVs were higher than among‐species CVs in four cases. Partitioning of variance within each treatment in two of the three experiments showed that within‐species variability (ecotypes) could explain an additional 9% of response variation after accounting for the among‐species variation. Our observation that within‐species variation was generally as high as among‐species variation emphasizes the importance of including both within‐ and among‐species variability in ecological theory (e.g., the insurance hypothesis) and for practical applications (e.g., biodiversity conservation).     

A global meta‐analysis of the relative extent of intraspecific trait variation in plant communities

Recent studies have shown that accounting for intraspecific trait variation (ITV) may better address major questions in community ecology. However, a general picture of the relative extent of ITV compared to interspecific trait variation in plant communities is still missing. Here, we conducted a meta‐analysis of the relative extent of ITV within and among plant communities worldwide, using a data set encompassing 629 communities (plots) and 36 functional traits. Overall, ITV accounted for 25% of the total trait variation within communities and 32% of the total trait variation among communities on average. The relative extent of ITV tended to be greater for whole‐plant (e.g. plant height) vs. organ‐level traits and for leaf chemical (e.g. leaf N and P concentration) vs. leaf morphological (e.g. leaf area and thickness) traits. The relative amount of ITV decreased with increasing species richness and spatial extent, but did not vary with plant growth form or climate. These results highlight global patterns in the relative importance of ITV in plant communities, providing practical guidelines for when researchers should include ITV in trait‐based community and ecosystem studies.     

Combining taxon‐by‐trait and taxon‐by‐site matrices for analysing trait patterns of macroinvertebrate communities: a rejoinder to Monaghan & Soares ()

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The 1st step initiation essential for allergen‐specific IgE antibody production upon the 2nd step: Induction of non‐specific IgE+ small B cells containing secondly‐sensitized allergen‐specific ones in mice firstly‐sensitized with an allergen

There was a significant amount of non‐specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)‐specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain‐, but not mite feces‐ or pollen‐specific IgE⁺ cells and an i.n. injection of papain induced papain‐specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces‐specific IgE⁺ cells in the lymph nodes nor mite feces‐specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen‐specific IgE⁺ small B cells in the lymph nodes on Day 10, when non‐specific IgE Ab titers reached a peak in the serum, implying induction of related allergen‐specific IgE⁺ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen‐specific IgE Abs in the serum. These results indicate that when firstly‐sensitized non‐specific IgE⁺ small B cells in mouse lymph nodes include some secondly‐sensitized allergen‐specific ones, mice produce IgE Abs specific for the secondly‐injected allergen.

     

Intraspecific trait variation increases species diversity in a trait‐based grassland model

Intraspecific trait variation (ITV) is thought to play a significant role in community assembly, but the magnitude and direction of its influence are not well understood. Although it may be critical to better explain population persistence, species interactions, and therefore biodiversity patterns, manipulating ITV in experiments is challenging. We therefore incorporated ITV into a trait‐ and individual‐based model of grassland community assembly by adding variation to the plants’ functional traits, which then drive life‐history tradeoffs. Varying the amount of ITV in the simulation, we examine its influence on pairwise‐coexistence and then on the species diversity in communities of different initial sizes. We find that ITV increases the ability of the weakest species to invade most, but that this effect does not scale to the community level, where the primary effect of ITV is to increase the persistence and abundance of the competitively‐average species. Diversity of the initial community is also of critical importance in determining ITV's efficacy; above a threshold of interspecific diversity, ITV does not increase diversity further. For communities below this threshold, ITV mainly helps to increase diversity in those communities that would otherwise be low‐diversity. These findings suggest that ITV actively maintains diversity by helping the species on the margins of persistence, but mostly in habitats of relatively low alpha and beta diversity.     

Genotyping‐by‐sequencing‐based genome‐wide association studies on Verticillium wilt resistance in autotetraploid alfalfa (Medicago sativa L.)

Verticillium wilt (VW) is a fungal disease that causes severe yield losses in alfalfa. The most effective method to control the disease is through the development and use of resistant varieties. The identification of marker loci linked to VW resistance can facilitate breeding for disease‐resistant alfalfa. In the present investigation, we applied an integrated framework of genome‐wide association with genotyping‐by‐sequencing (GBS) to identify VW resistance loci in a panel of elite alfalfa breeding lines. Phenotyping was performed by manual inoculation of the pathogen to healthy seedlings, and scoring for disease resistance was carried out according to the standard test of the North America Alfalfa Improvement Conference (NAAIC). Marker–trait association by linkage disequilibrium identified 10 single nucleotide polymorphism (SNP) markers significantly associated with VW resistance. Alignment of the SNP marker sequences to the M. truncatula genome revealed multiple quantitative trait loci (QTLs). Three, two, one and five markers were located on chromosomes 5, 6, 7 and 8, respectively. Resistance loci found on chromosomes 7 and 8 in the present study co‐localized with the QTLs reported previously. A pairwise alignment (blastn ) using the flanking sequences of the resistance loci against the M. truncatula genome identified potential candidate genes with putative disease resistance function. With further investigation, these markers may be implemented into breeding programmes using marker‐assisted selection, ultimately leading to improved VW resistance in alfalfa.     

Complex patterns of cis‐regulatory polymorphisms in ebony underlie standing pigmentation variation in Drosophila melanogaster

Pigmentation traits in adult Drosophila melanogaster were used in this study to investigate how phenotypic variations in continuous ecological traits can be maintained in a natural population. First, pigmentation variation in the adult female was measured at seven different body positions in 20 strains from the Drosophila melanogaster Genetic Reference Panel (DGRP) originating from a natural population in North Carolina. Next, to assess the contributions of cis‐regulatory polymorphisms of the genes involved in the melanin biosynthesis pathway, allele‐specific expression levels of four genes were quantified by amplicon sequencing using a 454 GS Junior. Among those genes, ebony was significantly associated with pigmentation intensity of the thoracic segment. Detailed sequence analysis of the gene regulatory regions of this gene indicated that many different functional cis‐regulatory alleles are segregating in the population and that variations outside the core enhancer element could potentially play important roles in the regulation of gene expression. In addition, a slight enrichment of distantly associated SNP pairs was observed in the ~10 kb cis‐regulatory region of ebony, which suggested the presence of interacting elements scattered across the region. In contrast, sequence analysis in the core cis‐regulatory region of tan indicated that SNPs within the region are significantly associated with allele‐specific expression level of this gene. Collectively, the data suggest that the underlying genetic differences in the cis‐regulatory regions that control intraspecific pigmentation variation can be more complex than those of interspecific pigmentation trait differences, where causal genetic changes are typically confined to modular enhancer elements.     

Expression of scFv‐Mel‐Gal4 triple fusion protein as a targeted DNA‐carrier in Escherichia Coli

Liver‐directed gene therapy has become a promising treatment for many liver diseases. In this study, we constructed a multi‐functional targeting molecule, which maintains targeting, endosome‐escaping, and DNA‐binding abilities for gene delivery. Two single oligonucleotide chains of Melittin (M) were synthesized. The full‐length cDNA encoding anti‐hepatic asialoglycoprotein receptor scFv C1 (C1) was purified from C1/pIT2. The GAL4 (G) gene was amplified from pSW50‐Gal4 by polymerase chain reaction. M, C1 and G were inserted into plasmid pGC4C26H to product the recombinant plasmid pGC‐C1MG. The fused gene C1MG was subsequently subcloned into plasmid pET32c to product the recombinant plasmid C1MG/pET32c and expressed in Escherichia coli BL21. The scFv‐Mel‐Gal4 triple fusion protein (C1MG) was purified with a Ni²⁺ chelating HiTrap HP column. The fusion protein C1MG of roughly 64 kD was expressed in inclusion bodies; 4.5 mg/ml C1MG was prepared with Ni²⁺ column purification. Western blot and immunohistochemistry showed the antigen‐binding ability of C1MG to the cell surface of the liver‐derived cell line and liver tissue slices. Hemolysis testing showed that C1MG maintained membrane‐disrupting activity. DNA‐binding capacity was substantiated by luciferase assay, suggesting that C1MG could deliver the DNA into cells efficiently on the basis of C1MG. Successful expression of C1MG was achieved in E. coli, and C1MG recombinant protein confers targeting, endosome‐escaping and DNA‐binding capacity, which makes it probable to further study its liver‐specific DNA delivery efficacy in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Inter‐individual variation in colour patterns in noctuid moths characterizes long‐distance dispersers and agricultural pests

A high capacity for long‐distance dispersal is a key to success for species confronted with environmental heterogeneity, habitat modification, fragmentation and loss. However, dispersal capacity is difficult to quantify and therefore poorly known in most taxa. Here, we report on a test for an association of variation in dispersal capacity with variable colouration of noctuid moths. First, using data from 12 experienced lepidopterologists, we showed that despite variation among experts in average assessments, different species are consistently classified as having non‐variable, variable or highly variable colour patterns when assessed by different experts. We then compared the incidence of non‐resident species with high inter‐individual variation in colour patterns recorded on the isolated island Utklippan (n = 47), with that in a species pool of potential long‐distance dispersers from the nearest mainland (n = 295). Species with high inter‐individual colour pattern variation were over‐represented on the island compared with species having non‐variable colouration. This finding constitutes rare evidence from the wild of long‐distance dispersal, measured on a spatial scale relevant for moths when tracking habitats in fragmented and changing landscapes or when keeping pace with environmental challenges associated with climate change. Finally, we showed that Swedish noctuid moths classified as agricultural pests (n = 28) had more variable colour patterns compared with non‐pests (n = 368). The majority of agricultural pests were also recorded on the isolated island, an outcome that is indicative of pest species having high dispersal capacity. Data on colour pattern variation may thus offer a simple and cost‐effective proxy to estimate dispersal capacity and can also help identify potential pest species. Our findings are potentially useful when modelling and predicting population and range dynamics of species in spatiotemporally heterogeneous environments, with direct implications for conservation biology and pest management.     

The leaf economics spectrum’s morning coffee: plant size-dependent changes in leaf traits and reproductive onset in a perennial tree crop

Background and AimsSize-dependent changes in plant traits are an important source of intraspecific trait variation. However, there are few studies that have tested if leaf trait co-variation and/or trade-offs follow a within-genotype leaf economics spectrum (LES) related to plant size and reproductive onset. To our knowledge, there are no studies on any plant species that have tested whether or not the shape of a within-genotype LES that describes how traits covary across whole plant sizes, is the same as the shape of a within-genotype LES that represents environmentally driven trait plasticity.MethodsWe quantified size-dependent variation in eight leaf traits in a single coffee genotype (Coffea arabica var. Caturra) in managed agroecosystems with different environmental conditions (light and fertilization treatments), and evaluated these patterns with respect to reproductive onset. We also evaluated if trait covariation along a within-genotype plant-size LES differed from a within-genotype environmental LES defined with trait data from coffee growing in different environmental conditions.Key ResultsLeaf economics traits related to resource acquisition – maximum photosynthetic rates (A) and mass-based leaf nitrogen (N) concentrations – declined linearly with plant size. Structural traits – leaf mass, leaf thickness, and leaf mass per unit area (LMA) – and leaf area increased with plant size beyond reproductive onset, then declined in larger plants. Three primary LES traits (mass-based A, leaf N and LMA) covaried across a within-genotype plant-size LES, with plants moving towards the ‘resource-conserving’ end of the LES as they grow larger; in coffee these patterns were nearly identical to a within-genotype environmental LES.ConclusionsOur results demonstrate that a plant-size LES exists within a single genotype. Our findings indicate that in managed agroecosystems where resource availability is high the role of reproductive onset in driving within-genotype trait variability, and the strength of covariation and trade-offs among LES traits, are less pronounced compared with plants in natural systems. The consistency in trait covariation in coffee along both plant-size and environmental LES axes indicates strong constraints on leaf form and function that exist within plant genotypes.