Cold Spring Harbor protocols最新文献

The BonnMu resource represents a tagged collection of maize (Zea mays L.) Mutator (Mu) transposon-induced mutants, designed for functional genomics studies. Here, we describe the use of the BonnMu collection for identifying and characterizing mutations. Specifically, we describe workflows for use in both reverse and forward genetics strategies in maize. For reverse genetics, users first acquire a BonnMu F₂ stock of interest based on data accessible at the Maize Genetics and Genomics Database (MaizeGDB). We provide details here for their subsequent propagation and for the confirmation of Mu insertions by genotyping via PCR, with the ultimate goal of establishing genotype-phenotype relationships of interest. For forward genetics studies, we describe a workflow that involves a combined approach of Mutant-Seq (Mu-Seq) and bulked segregant RNA-seq (BSR-Seq), to identify the causal gene underlying a mutant phenotype of interest.

Root anatomy plays a crucial role in regulating essential processes such as the absorption and movement of water and nutrients in plants. Root anatomy also impacts the energy costs of building and sustaining root tissues, tissue mechanics, and interactions with other organisms. Although several studies in maize have confirmed the functional utility of numerous root anatomical traits, such as that of cortical cell size and number for stress adaptation, there have been significant obstacles in measuring and analyzing root anatomical characteristics. This has resulted in gaps in our understanding of the genetic control and range of phenotypic variations among different cultivars, and how this diversity relates to overall fitness. Here, we review root anatomical phenotypes in maize and their function in stress adaptation, and briefly discuss phenotyping methods available for root anatomy. We further introduce a simple and accessible phenotyping approach that enables a comprehensive investigation of maize root anatomy. Detailed characterization of root traits and the implementation of robust methods for root anatomical phenotyping could have wide-ranging benefits across various areas of plant science, from fundamental research to enhancing crop breeding efforts.

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.

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.

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.

Root anatomy plays a critical structural and functional role in the maize root system, and regulates edaphic stress tolerance. The function and genetic basis of several maize root anatomical traits for stress tolerance have been demonstrated. Leveraging root anatomical traits in maize thus holds great potential for developing cultivars with greater nutrient and water efficiency. Key for such approaches is the ability to characterize the root anatomy of plants of interest. Here, we outline a systematic method for preparing, imaging, and analyzing maize root cross-sections. The protocol describes root sectioning (by hand or using a vibratome), preparation of microscope slides and toluidine blue staining, imaging under a light microscope, and both manual and semiautomated methods for anatomical feature extraction from images. The protocol enables the visualization and quantification of various anatomical tissues and traits, and its simplicity, adaptability, and accessibility make it an ideal choice for both small- and large-scale phenotyping studies in maize and other plant species. This standardized protocol provides researchers with a comprehensive methodology to accurately dissect root structures, enabling in-depth analyses that are essential for understanding plant growth, development, and adaptive value for stress tolerance.

Olfactory classical conditioning paradigms have been extensively used since the early 1970s to apply genetic approaches to the study of memory in Drosophila. Over the intervening years, advances in genetics have largely changed the focus of studies from the role of single genes in memory to investigation of memory-relevant neuronal circuits. However, the original behavioral paradigms have remained largely unaltered, besides investigators making a few useful tweaks to the training and testing apparatus and modifications to the operating procedures. In this protocol, we provide the reader with a detailed description of the manufacture and assembly of a typical T-maze apparatus, where populations of adult flies can be trained and their odor memory tested later, by giving them a binary choice between the two trained odors. We describe how variations of the training apparatus permit both aversive (odor-shock) and appetitive (odor-sugar) memories to be studied. In addition, we describe a recent modification of the apparatus and protocol that permits study of multisensory (color and odor) aversive and appetitive learning. Control assays for sensory acuity and locomotion are also included.

Memory has been extensively studied in Drosophila since the early 1970s. Straightforward aversive and appetitive conditioning paradigms train populations of flies to associate the pairing of one of two odors with either punishment or reward. After training, the flies show either preferential avoidance or approach behavior, to the appropriate odor, when given a choice between the two odors in a simple T-maze apparatus. These basic experimental approaches have proven useful in understanding the genetic, molecular, cellular, and neuronal network bases of various valence-specific memories in the fly brain. In addition, numerous modifications to these assays have permitted the study of a broad range of memory-related phenomena. Labile short-term avoidance and approach memories can be readily distinguished from more stable "consolidated" long-term memory equivalents. Prior or subsequent experience of the training cues, and manipulations of the flies' condition, have revealed how parallel competing memories and incompatible states can temporarily interfere with memory retrieval, providing insight into mechanisms of forgetting. Recent studies have also modified the training and testing apparatus to allow simultaneous presentation of odors and colors, providing insight into mechanisms of multisensory learning.

Phage-displayed antibody libraries can be constructed using any species that is easily immunized. The pComb3XSS phagemid vector is commonly used for library cloning and phage display. This phagemid encodes the origin of replication of the filamentous bacteriophage f1 but lacks all the genes required for replication and assembly of phage particles. The replication and the assembly of phage from these phagemids thus requires a "helper" phage that provides the genes essential for those steps during library production and bio-panning. One of those helper phages is VCSM13. In this protocol, we describe the preparation of VCSM13 helper phage. Users should prepare VCSM13 helper phage for library reamplification and for bio-panning.