Spatiotemporal dynamics of the agricultural landscape mosaic drives distribution and abundance of dominant carabid beetles

Context

Agroecosystems are dynamic, with yearly changing proportions of crops. Explicit consideration of this temporal heterogeneity is required to decipher population and community patterns but remains poorly studied.

Objectives

We evaluated the impact on the activity-density of two dominant carabid species (Poecilus cupreus and Anchomenus dorsalis) of (1) local crop, current year landscape composition, and their interaction, and (2) inter-annual changes in landscape composition due to crop rotations.

Methods

Carabids were sampled using pitfall-traps in 188 fields of winter cereals and oilseed rape in three agricultural areas of western France contrasting in their spatial heterogeneity. We summarized landscape composition in the current and previous years in a multi-scale perspective, using buffers of increasing size around sampling locations.

Results

Both species were more abundant in oilseed rape, and in landscapes with a higher proportion of oilseed rape in the previous year. P. cupreus abundance was negatively influenced by oilseed rape proportion in the current year landscape in winter cereals and positively by winter cereal proportion in oilseed rape. A. dorsalis was globally impacted at finer scales than P. cupreus.

Conclusions

Resource concentration and dilution-concentration processes jointly appear to cause transient dynamics of population abundance and distribution among habitat patches. Inter-patch movements across years appear to be key drivers of carabids’ survival and distribution, in response to crop rotation. Therefore, the explicit consideration of the spatiotemporal dynamics of landscape composition can allow future studies to better evidence ecological processes behind observed species patterns and help developing new management strategies.

     

sunTILL: a TILLING resource for gene function analysis in sunflower

Background

Cultivated sunflower (Helianthus annus L.) is a globally important oilseed crop, subjected to intensive genetic and genomic studies. Although classical mutagenesis has successfully been applied to Helianthus genus in the past, we have developed the first sunflower TILLING resource.

Results

To balance the maximum mutation density with an acceptable plant survival rate, a 'kill curve' analysis was first conducted with different ethylmethanesulfonate (EMS) dosages and different exposure times. According to the germination rate, a treatment with 0.7% EMS for 6 h was chosen. An M₂ progeny of 3,651 fertile plants was obtained. Totally, 4.79% of the whole population showed clear aberrant phenotypes. A microsatellite analysis on a representative sample of the original seed stock and mutant lines confirmed the uniformity of the genetic background of plant material. The TILLING procedure was successfully applied to sunflower genome, initially by a CelI-nuclease mismatch cleavage assay coupled with a DNA-pooling level test. To investigate the efficiency of the mutagenic treatment, a pilot screening was carried out on 1,152 M₂ lines focusing on four genes, three involved in the fatty acid biosynthetic pathway and one for downy mildew resistance. A total of 9 mutant lines were identified and confirmed by sequencing; thereby, the estimated overall mutation frequency for the pilot assay resulted to be 1/475 kb.

Conclusion

A first TILLING population for a high throughput identification of EMS-induced point mutations in sunflower genome has been successfully obtained. This represents a powerful tool to a better understanding of gene function in sunflower.     

A machine vision platform for measuring imbibition of maize kernels: quantification of genetic effects and correlations with germination

Background

Imbibition (uptake of water by a dry seed) initiates the germination process. An automated method for quantifying imbibition would enable research on the genetic elements that influence the underlying hydraulic and biochemical processes. In the case of crop research, a high throughput imbibition assay could be used to investigate seed quality topics or to improve yield by selecting varieties with superior germination characteristics.

Results

An electronic force transducer measured imbibition of single maize kernels with very high resolution but low throughput. An image analysis method was devised to achieve high throughput and sufficient resolution. A transparent fixture held 90 maize kernels in contact with water on the imaging window of a flatbed document scanner that produced an image of the kernels automatically every 10 min for 22 h. Custom image analysis software measured the area A of each indexed kernel in each image to produce imbibition time courses. The ultimate change in area (ΔA) ranged from 19.3 to 23.4% in a population of 72 hybrids derived from 9 inbred parents. Kernel area as a function of time was fit well by \(A\left( t \right) = A_{f} \left( {1 - e^{ - kt} } \right)\) where A_f is the final kernel area. The swelling coefficient, k, ranged from 0.098 to 0.159 h^?1 across the genotypes. The full diallel structure of the population enabled maternal genotype effects to be assessed. In a separate experiment, measurements of kernels of the same 25 inbreds produced in three different years demonstrated that production and storage variables affected imbibition much less than genotype. In a third experiment, measurements of 30 diverse inbred lines showed that k varied inversely with germination time (r?=???0.7) and directly with germination percentage (r?=?0.7).

Conclusions

Nonspecialized imaging hardware and custom analysis software running on public cyber infrastructure form a low-cost platform for measuring seed imbibition with high resolution and throughput. We measured imbibition of thousands of kernels to determine that genotype influenced imbibition of maize kernels much more than seed production and storage environments. In some hybrids, k depended on which inbred parent was maternal. Quantitative relationships between k and germination traits were discovered.

     

A rapid,inexpensive, and semi-quantitative method for determining pollen tube extension using fluorescence

Background

To identify plant genes involved in various key traits, QTL mapping is a powerful approach. This approach is based on the use of mapped molecular markers to identify genomic regions controlling quantitative traits followed by a fine mapping and eventually positional cloning of candidate genes. Mapping technologies using SNP markers are still rather expensive and not feasible in every laboratory. In contrast, microsatellite (also called SSR for Simple Sequence Repeat) markers are technologically less demanding and less costly for any laboratory interested in genetic mapping.

Results

In this study, we present the development and the characterization of a panel of 96 highly polymorphic SSR markers along the Arabidopsis thaliana genome allowing QTL mapping among accessions of the Versailles 24 core collection that covers a high percentage of the A. thaliana genetic diversity. These markers can be used for any QTL mapping analysis involving any of these accessions. We optimized the use of these markers in order to reveal polymorphism using standard PCR conditions and agarose gel electrophoresis. In addition, we showed that the use of only three of these markers allows differentiating all 24 accessions which makes this set of markers a powerful tool to control accession identity or any cross between any of these accessions.

Conclusion

The set of SSR markers developed in this study provides a simple and efficient tool for any laboratory focusing on QTL mapping in A. thaliana and a simple means to control seed stock or crosses between accessions.     

Microhabitat conditions and landscape pattern explain nocturnal rodent activity,but not seed removal,in burned and unburned lodgepole pine forests

Context

Disturbances create spatial variation in environments that may influence animal foraging. Granivory by rodents can influence seed supply and thus plant establishment. However, effects of disturbance patterns on rodent seed removal in western North American conifer forests are generally unknown.

Objectives

We conducted a study in lodgepole pine (Pinus contorta var. latifolia) forests of Greater Yellowstone (Wyoming, USA) to answer: (1) How do seed removal and rodent activity vary between recently burned and adjacent unburned forests and with distance from fire perimeter? (2) Which microhabitat conditions explain variability in seed removal and rodent activity?

Methods

One or two years after wildfires, we established transects (n = 23) with four stations each: at 10 and 40 m from the fire perimeter in both burned and unburned forest. At stations, we deployed trays with lodgepole pine seeds and cameras pointed at trays for 28 days and quantified habitat structure and seed abundance.

Results

Seed removal, which averaged 85%, and diurnal rodent activity did not differ between burned and unburned forests or with distance from the fire perimeter; however, nocturnal rodent activity was lower in burned forests. Seed removal and diurnal rodent activity were not associated with any microhabitat conditions we measured. However, nocturnal rodent activity was associated with microhabitat in both burned and unburned forests.

Conclusions

High seed removal rates suggested that rodent foraging was not reduced by high-severity wildfire. If observed seed removal represents natural conditions, post-dispersal seed predation could influence post-fire recruitment of a widespread foundation tree species.

     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting

Background

Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T₁ progeny.

Results

An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T₁ seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T₁ progeny displayed Mendelian inheritance.

Conclusions

This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications.

     

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

Background

Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes.

Results

We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner? system we were able to detect mutations in heterozygous and homozygous states for both genes.

Conclusions

Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M₃ seed of Brassica rapa from the RevGenUK TILLING service.     

Universal endogenous gene controls for bisulphite conversion in analysis of plant DNA methylation

Background

Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once.

Results

We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca²⁺ chelator and detergent. Cell extracts prepared by this method were suitable for SDS-PAGE and immunoblot analysis. This method was also applicable to genomic DNA extraction for PCR analysis. Our method was applied to many other plant species, and worked well for some of them.

Conclusions

Our method is rapid and economical, and allows many samples to be prepared simultaneously for protein analysis. Our method is useful not only for Arabidopsis research but also research on certain other species.     

Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of switchgrass (<Emphasis Type="Italic">Panicum virgatum</Emphasis>)

Background

Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection.

Results

After evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N₆ macronutrient/B₅ micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50–100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines.

Conclusion

The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.

     

Exploration of plant genomes in the FLAGdb++ environment

Background

Efficient high throughput screening systems of useful mutants are prerequisite for study of plant functional genomics and lots of application fields. Advance in such screening tools, thanks to the development of analytic instruments. Direct analysis in real-time (DART)-mass spectrometry (MS) by ionization of complex materials at atmospheric pressure is a rapid, simple, high-resolution analytical technique. Here we describe a rapid, simple method for the genetic discrimination of intact Arabidopsis thaliana mutant seeds using metabolic profiling by DART-MS.

Results

To determine whether this DART-MS combined by multivariate analysis can perform genetic discrimination based on global metabolic profiling, intact Arabidopsis thaliana mutant seeds were subjected to DART-MS without any sample preparation. Partial least squares-discriminant analysis (PLS-DA) of DART-MS spectral data from intact seeds classified 14 different lines of seeds into two distinct groups: Columbia (Col-0) and Landsberg erecta (Ler) ecotype backgrounds. A hierarchical dendrogram based on partial least squares-discriminant analysis (PLS-DA) subdivided the Col-0 ecotype into two groups: mutant lines harboring defects in the phenylpropanoid biosynthetic pathway and mutants without these defects. These results indicated that metabolic profiling with DART-MS could discriminate intact Arabidopsis seeds at least ecotype level and metabolic pathway level within same ecotype.

Conclusion

The described DART-MS combined by multivariate analysis allows for rapid screening and metabolic characterization of lots of Arabidopsis mutant seeds without complex metabolic preparation steps. Moreover, potential novel metabolic markers can be detected and used to clarify the genetic relationship between Arabidopsis cultivars. Furthermore this technique can be applied to predict the novel gene function of metabolic mutants regardless of morphological phenotypes.     

Landscape structure shapes carnivore-mediated seed dispersal kernels

Context

Seed dispersal is recognized as having profound effects on the distribution, dynamics and structure of plant populations and communities. However, knowledge of how landscape structure shapes carnivore-mediated seed dispersal patterns is still scarce, thereby limiting our understanding of large-scale plant population processes.

Objectives

We aim to determine how the amount and spatial configuration of forest cover impacted the relative abundance of carnivorous mammals, and how these effects cascaded through the seed dispersal kernels they generated.

Methods

Camera traps activated by animal movement were used for carnivore sampling. Colour-coded seed mimics embedded in common figs were used to know the exact origin of the dispersed seed mimics later found in carnivore scats. We applied this procedure in two sites differing in landscape structure.

Results

We did not find between-site differences in the relative abundance of the principal carnivore species contributing to seed dispersal patterns, Martes foina. Mean dispersal distance and the probability of long dispersal events were higher in the site with spatially continuous and abundant forest cover, compared to the site with spatially aggregated and scarcer forest cover. Seed deposition closely matched the spatial patterning of forest cover in both study sites, suggesting behaviour-based mechanisms underpinning seed dispersal patterns generated by individual frugivore species.

Conclusions

Our results provide the first empirical evidence of the impact of landscape structure on carnivore-mediated seed dispersal kernels. They also indicate that seed dispersal kernels generated strongly depend on the effect that landscape structure exerts on carnivore populations, particularly on habitat-use preferences.

     

A high-resolution method for the localization of proanthocyanidins in plant tissues

Background

Histochemical staining of plant tissues with 4-dimethylaminocinnamaldehyde (DMACA) or vanillin-HCl is widely used to characterize spatial patterns of proanthocyanidin accumulation in plant tissues. These methods are limited in their ability to allow high-resolution imaging of proanthocyanidin deposits.

Results

Tissue embedding techniques were used in combination with DMACA staining to analyze the accumulation of proanthocyanidins in Lotus corniculatus (L.) and Trifolium repens (L.) tissues. Embedding of plant tissues in LR White or paraffin matrices, with or without DMACA staining, preserved the physical integrity of the plant tissues, allowing high-resolution imaging that facilitated cell-specific localization of proanthocyanidins. A brown coloration was seen in proanthocyanidin-producing cells when plant tissues were embedded without DMACA staining and this was likely to have been due to non-enzymatic oxidation of proanthocyanidins and the formation of colored semiquinones and quinones.

Conclusions

This paper presents a simple, high-resolution method for analysis of proanthocyanidin accumulation in organs, tissues and cells of two plant species with different patterns of proanthocyanidin accumulation, namely Lotus corniculatus (birdsfoot trefoil) and Trifolium repens (white clover). This technique was used to characterize cell type-specific patterns of proanthocyanidin accumulation in white clover flowers at different stages of development.     

Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization

Background

Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation. A very useful technique to study these processes is chromatin immunoprecipitation (ChIP). ChIP is widely used for a few model systems, including Arabidopsis, but establishment of the technique for other organisms is still remarkably challenging. Furthermore, quantitative analysis of the precipitated material and normalization of the data is often underestimated, negatively affecting data quality.

Results

We developed a robust ChIP protocol, using maize (Zea mays) as a model system, and present a general strategy to systematically optimize this protocol for any type of tissue. We propose endogenous controls for active and for repressed chromatin, and discuss various other controls that are essential for successful ChIP experiments. We experienced that the use of quantitative PCR (QPCR) is crucial for obtaining high quality ChIP data and we explain why. The method of data normalization has a major impact on the quality of ChIP analyses. Therefore, we analyzed different normalization strategies, resulting in a thorough discussion of the advantages and drawbacks of the various approaches.

Conclusion

Here we provide a robust ChIP protocol and strategy to optimize the protocol for any type of tissue; we argue that quantitative real-time PCR (QPCR) is the best method to analyze the precipitates, and present comprehensive insights into data normalization.     

A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

Background

In vivo detection of protein-bound genomic regions can be achieved by combining chromatin-immunoprecipitation with next-generation sequencing technology (ChIP-seq). The large amount of sequence data produced by this method needs to be analyzed in a statistically proper and computationally efficient manner. The generation of high copy numbers of DNA fragments as an artifact of the PCR step in ChIP-seq is an important source of bias of this methodology.

Results

We present here an R package for the statistical analysis of ChIP-seq experiments. Taking the average size of DNA fragments subjected to sequencing into account, the software calculates single-nucleotide read-enrichment values. After normalization, sample and control are compared using a test based on the ratio test or the Poisson distribution. Test statistic thresholds to control the false discovery rate are obtained through random permutations. Computational efficiency is achieved by implementing the most time-consuming functions in C++ and integrating these in the R package. An analysis of simulated and experimental ChIP-seq data is presented to demonstrate the robustness of our method against PCR-artefacts and its adequate control of the error rate.

Conclusions

The software ChIP-seq Analysis in R (CSAR) enables fast and accurate detection of protein-bound genomic regions through the analysis of ChIP-seq experiments. Compared to existing methods, we found that our package shows greater robustness against PCR-artefacts and better control of the error rate.     

Efficient virus-induced gene silencing in apple, pear and Japanese pear using Apple latent spherical virus vectors

Background

Virus-induced gene silencing (VIGS) is an effective technology for the analysis of gene functions in plants. Though there are many reports on virus vectors for VIGS in plants, no VIGS vectors available for Rosaceae fruit trees were reported so far. We present an effective VIGS system in apple, pear, and Japanese pear using Apple latent spherical virus (ALSV) vectors.

Results

Inoculation of ALSV vectors carrying a partial sequence of endogenous genes from apple [ribulose-1, 5-bisphosphate carboxylase small subunit (rbcS), alpha subunit of chloroplast chaperonin (CPN60a), elongation factor 1 alpha (EF-1a), or actin] to the cotyledons of seeds by a particle bombardment induced highly uniform knock-down phenotypes of each gene on the true leaves of seedlings from 2~3 weeks after inoculation. These silencing phenotypes continued for several months. Northern blot and RT-PCR analyses of leaves infected with ALSV containing a fragment of rbcS gene showed that the levels of rbcS-mRNA drastically decreased in the infected apple and pear leaves, and, in reverse, rbcS-siRNAs were generated in the infected leaves. In addition, some of apple seedlings inoculated with ALSV vector carrying a partial sequence of a TERMINAL FLOWER 1 gene of apple (MdTFL1) showed precocious flowering which is expected as a knock-down phenotype of the silencing of MdTFL1 gene.

Conclusions

The ALSV-based VIGS system developed have provides a valuable new addition to the tool box for functional genomics in apple, pear, and Japanese pear.     

Convergence of developmental mutants into a single tomato model system: 'Micro-Tom' as an effective toolkit for plant development research

Background

The tomato (Solanum lycopersicum L.) plant is both an economically important food crop and an ideal dicot model to investigate various physiological phenomena not possible in Arabidopsis thaliana. Due to the great diversity of tomato cultivars used by the research community, it is often difficult to reliably compare phenotypes. The lack of tomato developmental mutants in a single genetic background prevents the stacking of mutations to facilitate analysis of double and multiple mutants, often required for elucidating developmental pathways.

Results

We took advantage of the small size and rapid life cycle of the tomato cultivar Micro-Tom (MT) to create near-isogenic lines (NILs) by introgressing a suite of hormonal and photomorphogenetic mutations (altered sensitivity or endogenous levels of auxin, ethylene, abscisic acid, gibberellin, brassinosteroid, and light response) into this genetic background. To demonstrate the usefulness of this collection, we compared developmental traits between the produced NILs. All expected mutant phenotypes were expressed in the NILs. We also created NILs harboring the wild type alleles for dwarf, self-pruning and uniform fruit, which are mutations characteristic of MT. This amplified both the applications of the mutant collection presented here and of MT as a genetic model system.

Conclusions

The community resource presented here is a useful toolkit for plant research, particularly for future studies in plant development, which will require the simultaneous observation of the effect of various hormones, signaling pathways and crosstalk.     

A device for single leaf labelling with CO<Subscript>2</Subscript> isotopes to study carbon allocation and partitioning in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background

Plant biomass consists primarily of carbohydrates derived from photosynthesis. Monitoring the assimilation of carbon via the Calvin-Benson cycle and its subsequent utilisation is fundamental to understanding plant growth. The use of stable and radioactive carbon isotopes, supplied to plants as CO₂, allows the measurement of fluxes through the intermediates of primary photosynthetic metabolism, long-distance transport of sugars in the vasculature, and the synthesis of structural and storage components.

Results

Here we describe the design of a system for supplying isotopically labelled CO₂ to single leaves of Arabidopsis thaliana. We demonstrate that the system works well using short pulses of ¹⁴CO₂ and that it can be used to produce robust qualitative and quantitative data about carbon export from source leaves to the sink tissues, such as the developing leaves and the roots. Time course experiments show the dynamics of carbon partitioning between storage as starch, local production of biomass, and export of carbon to sink tissues.

Conclusion

This isotope labelling method is relatively simple to establish and inexpensive to perform. Our use of ¹⁴CO₂ helps establish the temporal and spatial allocation of assimilated carbon during plant growth, delivering data complementary to those obtained in recent studies using ¹³CO₂ and MS-based metabolomics techniques. However, we emphasise that this labelling device could also be used effectively in combination with ¹³CO₂ and MS-based techniques.

     

Dry-washes determine gene flow and genetic diversity in a common desert shrub

Context

In deserts, many plant species exhibit a patchy spatial distribution within a harsh habitat matrix, where the likelihood of propagule dispersal among patches is uncertain, but may be promoted by landscape corridors or dispersal vectors.

Objectives

We examine the connectivity of a representative desert plant species (Acacia (Senegalia) greggii), and the ability of three major factors (animal dispersal agents, water flow along dry-washes, and climate) to facilitate dispersal within four watersheds in the Mojave National Preserve.

Methods

We genotyped 323 individuals sampled across 22 one-hectare sites using ten nuclear microsatellite markers.

Results

A hierarchical AMOVA revealed no significant differentiation among watersheds (F _RT = 0.00, P > 0.10), and very little genetic structure among all sites (F _ST = 0.03, P < 0.001), indicating regional connectivity. Mantel tests indicated distance along dry-washes best explained genetic distance between sites (r = 0.47, P < 0.05) when compared to Euclidean distance (P > 0.05), a distance measure based on rodent dispersal (P > 0.05), and a distance measure avoiding inhospitable climate (P > 0.05). An AIC comparison of generalized linear models found that within site genetic diversity (H _E and allelic richness) and average relatedness were best explained by slope (which increases seed dispersal potential via water flow) and area of the upstream watershed (which determines the number of potential seed donors), rather than plant density or habitat suitability.

Conclusions

Together, these findings indicate that dry-washes are key landscape features that enhance dispersal and regional connectivity in this patchy desert plant.

     

Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

Background

We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns.

Results

The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively.

Conclusions

These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.