Cold Spring Harbor protocols最新文献

The BonnMu resource is a public transposon-tagged population designed for reverse and forward genetics studies in maize (Zea mays L.). The resource was created by crossing an active Mutator (Mu) transposon line into different inbred lines to induce insertional mutations. The resulting F₁ generation was self-pollinated to generate segregating BonnMu F₂ stocks. The Mu-tagged BonnMu F₂ stocks have insertions in 83% of all annotated maize gene models, and Mu insertion positions and photos of the seedling phenotypes of the segregating BonnMu F₂ stocks are deposited in the Maize Genetics and Genomics Database (MaizeGDB), with seeds available to the community. Here, we discuss the creation, expansion, and application of the BonnMu resource for identifying and characterizing mutations induced by Mu transposons, which represents a useful tool for functional genomics studies in maize.

Mutator (Mu) transposons facilitate untargeted insertional mutagenesis in maize by moving within the genome and disrupting genes. Such an approach has been used to generate collections such as the BonnMu resource, a Mu-tagged maize population for functional genomics studies. Mutant-Seq (Mu-Seq) is a sequencing-based method for the high-throughput identification and mapping of Mu insertion sites. The approach involves the construction of multiplexed sequencing libraries (known as Mu-Seq libraries) from Mu-tagged populations, followed by high-throughput sequencing and data processing using the Mu-Seq Workflow Utility (MuWU) tool, to determine the location of Mu insertions. Here, we provide a detailed protocol for Mu-Seq, from the generation of the maize Mu-tagged mutant population to data analysis. Researchers can use this approach to develop mutant collections customized to specific genetic backgrounds of interest, which can aid in characterizing genotype-specific mutations and identifying candidate genes linked to visible mutant phenotypes.

Maize is an important plant for both global food security and genetics research. As the importance of microorganisms to plant health is becoming clearer, there is a growing interest in understanding the relationship between maize and its associated microbiome; i.e., the collection of microorganisms living on, around, and inside the plant. The ultimate goal of this research is to use these microbial communities to support more robust and sustainable maize production. Here, we provide an overview of recent progress in the field of maize microbiome research. We discuss the major microbiome compartments (rhizosphere, phyllosphere, and endosphere) and known functions of the microbiome. We also review the methods currently available to study the maize microbiome and its functions, and discuss how to carry out maize microbiome experiments, including both a general workflow (suitable for most microbiome analyses) and maize-specific experimental considerations.

Transposable elements (TEs) are abundant and ubiquitous components of eukaryotic genomes. Since TEs were first discovered in maize (Zea mays) by Barbara McClintock in the late 1940s, these elements have been shown to be important agents in shaping genome structure and evolution. Today, maize continues to be an important model organism for molecular and quantitative genetics, and represents a particularly useful system for the study of the interplay between TEs and host genomes. While TEs constitute a significant part of the maize genome and are important drivers of genome evolution, their annotation remains a complex and challenging task. Here, we discuss genome annotation of TEs and other repetitive sequences in maize genomes. We briefly review current knowledge on the overall landscape of TE and non-TE repeats in maize, and discuss how these sequences may impact genome structure, and the genotype and phenotype within species. We also provide a summary of the main tools used to find TE polymorphisms, and briefly introduce four different bioinformatic approaches for TE and tandem repeat annotation, explaining how they can be best used by maize researchers.

One of the most common methods to survey bacterial communities is targeted amplification of the hypervariable regions of the 16s rRNA gene followed by sequencing. This protocol details Illumina library preparation of such amplicons from communities isolated from maize. We include both staggered PCR primers to improve Illumina base calling and peptide nucleic acids (PNAs) to reduce the presence of plant organelles. Primers are designed with Illumina adapter sequences for the addition of sample-specific indexes (barcodes). We also briefly discuss alternative primer sets, including ones that directly discriminate against plant organelles or that amplify different organisms (e.g., fungal internal transcribed spacer [ITS] sequences).

For most farmers, the production of maize grain is the ultimate goal of the entire field season. From the point of view of plant microbiome studies, seeds are particularly interesting in that they are the only avenue for vertical transmission of microbes from parent to offspring, though microbes can also enter maize seeds via wounds or silks. Although the presence of seed endophytes is well documented, their role, if any, in seed health and their effects on the next generation of plants are largely unknown. This protocol describes the isolation of seed endophytes. Its primary focus is properly sterilizing the seed surface, followed by grinding to release the endophytes. The end product is a cell suspension suitable for either culturing or DNA analysis.

The microbiota of maize leaves can be beneficial or detrimental to the host. Foliar diseases are the most obvious detrimental impact of the leaf microbiome, though more subtle effects of the normal (nondisease) community are an active area of research. This protocol describes two specific methodologies to sample the maize leaf microbiome: one sampling the surface (epiphyte) microbiome and one sampling the interior (endophyte) microbiome. Each method begins with collected leaf tissue and finishes with a cell suspension suitable for either isolating live microbes or extracting DNA for sequencing.

Maize (Zea mays) is a multifaceted cereal grass used globally for nutrition, animal feed, food processing, and biofuels, and a model system in genetics research. Studying the maize microbiome sometimes requires its manipulation to identify the contributions of specific taxa and ecological traits (i.e., diversity, richness, network structure) to maize growth and physiology. Due to regulatory constraints on applying engineered microorganisms in field settings, greenhouse-based experimentation is often the first step for understanding the contribution of root-associated microbiota-whether natural or engineered-to plant phenotypes. In this protocol, we describe methods to inoculate maize with a specific microbiome as a tool for understanding the microbiota's influence on its host plant. The protocol involves removal of the native seed microbiome followed by inoculation of new microorganisms; separate protocols are provided for inoculations from pure culture, from soil slurry, or by mixing in live soil. These protocols cover the most common methods for manipulating the maize microbiome in soil-grown plants in the greenhouse. The methods outlined will ultimately result in rhizosphere microbial assemblages with varying degrees of microbial diversity, ranging from low diversity (individual strain and synthetic community [SynCom] inoculation) to high diversity (percent live inoculation), with the slurry inoculation method representing an "intermediate diversity" treatment.

The soil microbiome of maize shapes its fitness, sustainability, and productivity. Accurately sampling maize's belowground microbial communities is important for identifying and characterizing these functions. Here, we describe a protocol to sample the maize rhizosphere (including the rhizoplane and endorhizosphere) and root zone (still influential but further from the root) in a form suitable for downstream analyses like culturing and DNA extractions. Although this protocol is written with Zea mays as the focus, these methods can generally be applied to any plant with similar fibrous root systems.

Transposable elements (TEs) and tandem repeat arrays are ubiquitous components of genomes across all domains of life. Many types of repetitive DNA do not appear to encode for functional proteins, and those that do, typically only code for enzymes involved in their own replication. Nevertheless, repetitive DNA sequences can significantly alter genome structure, and can have a profound impact on an organism's biology at both the molecular and organismal levels. Advances in long-read sequencing technology have enabled the resolution of previously collapsed contigs and scaffolds that are rich in repeats, which has made the accurate annotation of TEs and other repetitive sequences a crucial early step in genome analysis. Here, we provide a detailed tutorial for streamlined annotation of TEs and repeats in the genome of the model plant Zea mays (maize). Maize is ideally suited to illustrate these procedures due to its repeat-rich genome and the volume of publicly available and high-quality genomic resources. We outline four possible approaches for TE and repeat annotation, each aimed at accommodating a different set of scientific interests. Additionally, we demonstrate how to evaluate annotation quality, and provide scripts to help graphically depict TE and repeat landscapes. Although the protocol is tailored for maize, we also offer pointers for researchers working on other systems throughout and expect that these procedures will be broadly applicable to any eukaryotic genome.