Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species

Background  

Genotype analysis using multiple single nucleotide polymorphisms (SNPs) is a useful but labor-intensive or high-cost procedure in plant research. Here we describe an alternative genotyping method that is suited to multi-sample or multi-locus SNP genotyping and does not require electrophoresis or specialized equipment.     

Transient expression vectors for functional genomics,quantification of promoter activity and RNA silencing in plants

Background  

We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.     

Development and evaluation of a high-throughput,low-cost genotyping platform based on oligonucleotide microarrays in rice

Background  

We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features.     

Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0)

Background  

We previously developed a high-throughput system called 'Ice-Cap' for growing Arabidopsis seedlings in a 96-well format and rapidly collecting tissue for subsequent DNA extraction and genotyping. While the originally described Ice-Cap method is an effective tool for high-throughput genotyping, one shortcoming of the first version of Ice-Cap is that optimal seedling growth is highly dependent on specific environmental conditions. Here we describe several technical improvements to the Ice-Cap method that make it much more robust and provide a detailed protocol for implementing the method.     

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.     

Occupancy and detectability of African crowned eagles in an urban mosaic landscape: The importance of natural forest areas

Diminishing forests as a result of anthropogenic activities continues to impact the persistence of terrestrial species negatively. Raptors are particularly susceptible to human activities because of their low population densities and large home range sizes. In this study, we investigated the occupancy and detection probabilities of the Near-Threatened African crowned eagle (Stephanoaetus coronatus) in Durban, EThekwini Municipality, South Africa. Using point count surveys, we documented the presence/absence of African crowned eagles in 42 sampling sites in the urban mosaic landscape of Durban. We used the presence/absence data to model their occupancy and detection probability in our surveyed sites. The naïve occupancy of African crowned eagles was 0.6, and the estimated occupancy and detection probability were 0.78 + 0.061 and 0.40 + 0.085, respectively. Based on the best models, the occupancy of African crowned eagles was positively influenced by forests (β = 1.24 + 0.69) and negatively influenced by disturbance (β = −1.69 + 0.78) and roads. The detection probability of these eagles was positively influenced by disturbance (β = 0.34 + 0.29) and the presence of exotic tree plantations (β = 0.33 + 0.47) and negatively influenced by the type of surrounding settlements (i.e., urban or rural) (β = −0.59 + 0.43). Based on the averaged models, occupancy is positively influenced by forests (β = 0.15 + 1.48) and negatively influenced by roads (β = −0.05 + 0.34), building density (β = −0.07 + 0.79) and disturbance (β = −0.24 + 3.36). Detection probability is negatively influenced by surrounding settlement (β = −0.12 + 0.41) and positively influenced by disturbance (β = 0.04 + 3.21) and plantations (β = 0.01 + 0.12). Overall, our results showed the persistence of African crowned eagles in the urban mosaic landscape as a consequence of natural and managed green spaces, especially forests. This highlighted the importance of natural forests and exotic tree plantations in ensuring the survival and thriving of African crowned eagles and the key role these green spaces play in species conservation in this urban mosaic landscape.     

An R package for SNP marker-based parent-offspring tests

Background

With the advancement of genotyping technologies, whole genome and high-density SNP markers have been widely used for genotyping of mapping populations and for characterization of germplasm lines in many crops. Before conducting SNP data analysis, it is necessary to check the individuals to ensure the integrity of lines for further data analysis.

Results

We have developed an R package to conduct a parent-offspring test of individuals which are genotyped with a fixed set of SNP markers for further genetic studies. The program uses monomorphic SNP loci between parents and their progeny genotypes to calculate the similarity between each offspring and their parents. Based on the similarity of parents and individual offspring, the users can determine the threshold level for the individuals to be included for further data analysis. We used an F₅-derived soybean population of ‘5601T’ x PI 157440 that was genotyped with 1,536 SNPs to illustrate the procedure and its application.

Conclusions

The R package ‘ParentOffspring’ coupled with the available SNP genotyping platforms could be used to detect the possible variants in a specific cross, as well as the potential errors in sample handling and genotyping processes. It can be used in any crop which is genotyped with a fixed set of SNP markers.

     

Applying fire connectivity and centrality measures to mitigate the cheatgrass-fire cycle in the arid West,USA

Context

Strategic placement of fuel treatments across large landscapes is an important step to mitigate the collective effects of fires interacting over broad spatial and temporal extents. On landscapes where highly invasive cheatgrass (Bromus tectorum) is increasing fire activity, such an approach could help maintain landscape resilience.

Objectives

Our objectives are to 1) model and map fire connectivity on a cheatgrass-invaded landscape, as well as the centrality of large cheatgrass patches, in order to inform a landscape fuel treatment (i.e., a network of greenstrips); and 2) evaluate the modeled greenstrip network based on changes to cheatgrass patch centrality.

Methods

Our analysis covers 485-km² on the Kaibab National Forest in Northern Arizona. We apply a circuit-theoretic model of fire connectivity between all pairs of large cheatgrass patches. Based on these results, we calculate a measure of centrality for each patch to inform fuel treatment placement. We evaluate the modeled greenstrip network by comparing the pre- and post-treatment centrality of each patch.

Results

After modeling fire connectivity across the landscape, we identify 25 of 68 large cheatgrass patches with relatively high centrality. When we simulate greenstrips around these focal patches, model results suggest that they are effective in reducing the centrality for at least 19 of the 25 patches.

Conclusions

Fire connectivity models provide robust network centrality measures, which can help generate multiple, landscape fuel treatment alternatives and facilitate on-the-ground decisions. The extension of these methods is well suited for landscape fuels management in other vegetation communities and ecosystems.

     

A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts

Background  

Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking.     

Genome-wide identification of microsatellites in white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) using FIASCO and phpSSRMiner

Background  

Allotetraploid white clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in white clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for white clover.     

Landscape makers and landscape takers: links between farming systems and landscape patterns along an intensification gradient

Context

Agricultural intensification is a leading cause of landscape homogenization, with negative consequences for biodiversity and ecosystem services. Conserving or promoting heterogeneity requires a detailed understanding of how farm management affects, and is affected by, landscape characteristics.

Objectives

We assessed relationships between farming systems and landscape characteristics, hypothesising that less-intensive systems act as landscape takers, by adapting management to landscape constraints, whereas more intensive systems act as landscape makers, by changing the landscape to suit farming needs.

Methods

We mapped dominant farming systems in a region of southern Portugal: traditional cereal-grazed fallow rotations; specialization on annual crops; and specialization on either cattle or sheep. We estimated landscape metrics in 241 1-km² buffers representing the farming systems, and analysed variation among and within systems using multivariate statistics and beta diversity metrics.

Results

Landscape composition varied among systems, with dominance by either annual crops (Crop system) or pastures (Sheep), or a mixture between the two (Traditional and Cattle). There was a marked regional gradient of local landscape heterogeneity, but this contributed little to variation among systems. Landscape beta diversity declined from the Sheep to the Crop system, and it was inversely related to agriculture intensity.

Conclusions

Less intensive farming systems appeared compatible with a range of landscape characteristics (landscape takers), and may thus be particularly suited to agri-environmental management. More intensive systems appeared less flexible in terms of landscape characteristics (landscape makers), likely promoting regional homogenization. Farming systems may provide a useful standpoint to address the design of agri-environment schemes.

     

<Emphasis Type="Italic">De novo</Emphasis> assembly of potential linear artificial chromosome constructs capped with expansive telomeric repeats

Background  

Artificial chromosomes (ACs) are a promising next-generation vector for genetic engineering. The most common methods for developing AC constructs are to clone and combine centromeric DNA and telomeric DNA fragments into a single large DNA construct. The AC constructs developed from such methods will contain very short telomeric DNA fragments because telomeric repeats can not be stably maintained in Escherichia coli.     

A novel fluorescent pH probe for expression in plants

Background  

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants.     

An online database for plant image analysis software tools

Background

Recent years have seen an increase in methods for plant phenotyping using image analyses. These methods require new software solutions for data extraction and treatment. These solutions are instrumental in supporting various research pipelines, ranging from the localisation of cellular compounds to the quantification of tree canopies. However, due to the variety of existing tools and the lack of central repository, it is challenging for researchers to identify the software that is best suited for their research.

Results

We present an online, manually curated, database referencing more than 90 plant image analysis software solutions. The website, plant-image-analysis.org, presents each software in a uniform and concise manner enabling users to identify the available solutions for their experimental needs. The website also enables user feedback, evaluations and new software submissions.

Conclusions

The plant-image-analysis.org database provides an overview of existing plant image analysis software. The aim of such a toolbox is to help users to find solutions, and to provide developers a way to exchange and communicate about their work.

     

Towards a better mechanistic understanding of edge effects

Context

Predicting and managing edge effects requires an understanding of the mechanisms that drive them. However, analytical methods that dominate edge effects research are not well suited to discriminating mechanisms, because they do not measure ‘indirect’ edge effects: effects that are mediated by covariates in statistical models.

Objective

To discuss the value of indirect effects for improving mechanistic understanding of edge effects.

Methods

We explain how measuring indirect effects improves mechanistic understanding, and provide guidance on how to do so. We also conduct a literature review to examine awareness of indirect effects in empirical studies of mechanisms underpinning edge effects. Finally, we use a recent paper in Villaseñor et al. (Landscape Ecol 30:229–245, 2015) as a case study to discuss how failure to measure indirect edge effects may limit mechanistic understanding.

Results

Indirect effects provide a means to translate conceptual models of edge effects into mechanistic pathways that are testable and quantifiable. Moreover, failure to measure indirect edge effects can result in impacts of habitat edges being underestimated. However, few studies that we identified in our literature review quantified indirect effects (7 %, n = 72). Worryingly, 11 % of studies did not account for indirect effects despite using statistical models that potentially contained them, possibly resulting in incorrect inference.

Conclusions

A better awareness of indirect effects will help researchers to understand the mechanisms that underpin edge effects, while ensuring that impacts of habitat edges are not underestimated.

     

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> and oat

Background  

Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.     

High-throughput <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated barley transformation

Background  

Plant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare) is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low (<10%) transformation efficiencies.     

3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei

Background  

Fluorescent hybridization techniques are widely used to study the functional organization of different compartments within the mammalian nucleus. However, few examples of such studies are known in the plant kingdom. Indeed, preservation of nuclei 3D structure, which is required for nuclear organization studies, is difficult to fulfill.     

UCE: A uracil excision (USER™)-based toolbox for transformation of cereals

Background  

Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs. These problems are obviated by "The Uracil Specific Excision Reagent (USER™)" technology (New England Biolabs) which thus offers a new and very time-efficient method for engineering of big and complex plasmids.