Current protocols in plant biology最新文献

Maize (Zea mays) has a long history as a model organism for genetic analysis due to its ease of crossing, relatively large number of progeny, and ability to determine many genotypic traits through phenotypic kernel markers. Fast-Flowering Mini-Maize A and B (FFMM) are two recently developed lines of maize selected for traits that make them more conducive to research. FFMM flowers more quickly than standard maize and is much smaller; thus it requires a slightly modified protocol for care and crossing. The following protocol describes how to plant, care for, and cross FFMM in greenhouse, growth chamber, and field conditions. © 2017 by John Wiley & Sons, Inc.

Phenotypic measurements and images of crops grown under controlled-environment conditions can be analyzed to compare plant growth and other phenotypes from diverse varieties. Those demonstrating the most favorable phenotypic traits can then be used for crop improvement strategies. This article details a protocol for image-based root and shoot phenotyping of plants grown in the greenhouse to compare traits among different varieties. Diverse maize lines were grown in the greenhouse in large 8-gallon treepots in a clay granule substrate. Replicates of each line were harvested at 4 weeks, 6 weeks, and 8 weeks after planting to capture developmental information. Whole-plant phenotypes include biomass accumulation, ontogeny, architecture, and photosynthetic efficiency of leaves. Image analysis was used to measure leaf surface area and tassel size and to extract shape variance information from complex 3D root architectures. Notably, this framework is extensible to any number of above- or below-ground phenotypes, both morphological and physiological. © 2017 by John Wiley & Sons, Inc.

Genome-wide association studies (GWAS) have developed into a valuable approach for identifying the genetic basis of phenotypic variation. In this article, we provide an overview of the design, analysis, and interpretation of GWAS. First, we present results from simulations that explore key elements of experimental design as well as considerations for collecting the relevant genomic and phenotypic data. Next, we outline current statistical methods and tools used for GWA analyses and discuss the inclusion of covariates to account for population structure and the interpretation of results. Given that many false positive associations will occur in any GWA analysis, we highlight strategies for prioritizing GWA candidates for further statistical and empirical validation. While focused on plants, the material we cover is also applicable to other systems. © 2017 by John Wiley & Sons, Inc.

Plant small RNAs are ∼20 to 24 nucleotide noncoding RNAs that typically have repressive regulatory roles in gene expression, functioning at the transcriptional or post-transcriptional level. This influence on regulation of developmental and physiological processes has direct effects on phenotype. High-throughput sequencing technologies have enabled the sequencing of millions of small RNAs. Along with decreased sequencing costs, recent improvements in small RNA library construction have facilitated the ability to use minimal amounts of input RNA for analysis. This unit describes steps to isolate total RNA from limited amounts of plant tissue to construct small RNA libraries and perform small RNA data processing. © 2017 by John Wiley & Sons, Inc.

Genotyping-by-sequencing (GBS) refers to a suite of related methods that obtain genotype data from samples by using restriction enzyme digestion followed by high-throughput sequencing. GBS is a refinement of restriction site–associated DNA sequencing (RADseq) methods, with a goal of being able to perform library preparations quickly, cost-effectively, and in a high-throughput manner. This protocol contains the steps necessary to go from purified DNA to Illumina-ready libraries. It also covers the considerations that go into planning a GBS experiment. © 2017 by John Wiley & Sons, Inc.

This unit presents a highly reliable protocol to produce and screen metaphase chromosome spreads from root tip cell suspensions of soybean (Glycine max), or other legumes. The procedures represent soybean-optimized versions of protocols developed for maize. The use of pressurized nitrous oxide to reliably generate metaphase-arrested chromosomes is crucial to overcoming one of the challenges of working with tiny and numerous soybean chromosomes. © 2017 by John Wiley & Sons, Inc.

This article presents protocols for fluorescence in situ hybridization (FISH) in the cultivated soybean, Glycine max. The protocols represent soybean-optimized versions developed for maize. We describe the use of two different probes types: genomic-repeat-based fluorescently-tagged oligonucleotides and bacterial artificial chromosomes (BACs). The two probe types can be used either individually or together, depending on the experimental questions. The article also includes starting points for executing FISH in additional legume species. © 2017 by John Wiley & Sons, Inc.

A technique that produces large numbers of good quality pachytene chromosome preparations has been developed for Populus. Anthers at the pachytene stage of meiosis are used as materials. There are two main modifications in our method relative to the traditional squashing method that address the challenges the thick cytoplasm observed during the pachytene phase of meiosis presents. One is the temperature during squashing, i.e., the slide is placed on a 52°C heater for squashing. The other is the removal of the cover slip using 45% acetic acid. © 2016 by John Wiley & Sons, Inc.

Higher plants are composed of a multitude of tissues with particular functions, reflected by distinct profiles of transcripts, proteins, and metabolites. Although the rapid development of “omics” technologies has advanced plant science tremendously within recent years, analysis is frequently performed on whole organ or whole plant extracts, causing the loss of spatial information. Mass spectrometry–based imaging (MSI) approaches have become a powerful tool to decipher spatially resolved molecular information. Matrix-assisted laser desorption/ionization (MALDI) is the most widespread ionization method utilized for MSI and has recently been applied to plant science. A range of different plant organs and tissues has been successfully analyzed by MSI, and patterns of various classes of metabolites from primary and secondary metabolism have been obtained. This protocol describes a method for analysis of spatial metabolite distributions in cryosections of developing barley grains. Detailed procedures for sample preparation, mass spectrometry measurement, and data analysis are provided. © 2016 by John Wiley & Sons, Inc.

Live-cell imaging is a powerful tool that allows investigators to directly observe the dynamics of cellular processes. Live imaging has proven particularly useful in studying mitotic and meiotic chromosome segregation, where the assembly of spindles and movement of chromosomes can be quantified in ways not possible with fixed cells. This protocol describes how to image live meiosis in the agriculturally important plant, maize. The creation of fluorescently tagged tubulin allows visualization of maize spindles, and nucleic acid dyestain chromosomes. This protocol describes all steps required for live imaging, including how to grow plants, screen for relevant genotypes, harvest meiotic cells, and collect live movies of meiosis. While this protocol was developed for imaging fluorescently tagged tubulin, it can be easily modified to observe the meiotic dynamics of any fluorescently labeled protein of interest. © 2016 by John Wiley & Sons, Inc.