Notes

Numbers regarding extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae are different within human-polluted environment as well as foodstuffs: any multicentre Western european examine.
Genome-wide association studies (GWAS) have proven effective at identifying genetic variants and genes that are associated with phenotypes in humans, animals, and plants. Since most phenotypes of plant species are complex traits regulated by many genes and their functional interactions, GWAS are increasing in popularity for genetic dissections of plant phenotypes. For the reference plant, Arabidopsis thaliana, detailed information on genetic variations became available with the completion of the 1001 Genomes Project, enabling highly resolved association mapping between chromosomal loci and complex traits. Improvements have been made in the statistical analysis methods for testing the significance of genotype-to-phenotype associations, thereby substantially reducing the confounding effects of population structures. Furthermore, there have been large efforts toward post-GWAS augmentation of signals via integration with other types of information to overcome the limited statistical power of GWAS. This chapter describes the stepwise procedure of GWAS in Arabidopsis, focusing on data analysis processes including preprocessing of genotype and phenotype data, statistical analysis to identify phenotype-associated chromosomal loci, identification of phenotype-associated genes based on the phenotype-associated loci, and finally network-based augmentation of GWAS signals to identify additional candidate genes for the phenotype.Cell suspension cultures represent a widely used experimental tool suitable to perform a variety of structural and physiological studies in a more simplified system compared to the organism in toto. In this chapter we describe the methods routinely used in our laboratory to establish and maintain Arabidopsis photosynthetic and heterotrophic cell suspension cultures, containing either chloroplasts or amyloplasts, respectively. The use of these in vitro systems may allow to obtain insights into the unique features of chloroplasts versus non-green plastids, as well as their integration in the structural and metabolic compartmentalization of the plant cell.Transient expression using protoplasts isolated from Arabidopsis suspension culture cells is a fast and useful tool for analyzing protein subcellular localization and dynamics in plant cells. Recently, super-resolution imaging techniques such as N-SIM (Nikon, Structured Illumination Microscopy) are widely used in cell biology study, allowing cell biologists to obtain unattainable details and relationships of cell structures and functions by conventional confocal imaging. To facilitate the usage of protoplasts transient expression and super-resolution imaging for protein localization and dynamic analysis in plant cell biology research, here we describe updated protocols of protoplasts isolation from Arabidopsis suspension culture cells and transient expression assay for protein trafficking and localization study. Further, using GFP-tagged ERES (Endoplasmic Reticulum Exit Site) marker proteins and RFP-tagged Golgi marker as examples, we illustrate the major tools and methods for protein localization analysis using super-resolution imaging.Transient expression in Arabidopsis thaliana seedlings allows fast expression of fluorescent markers for different subcellular compartments. This protocol describes a transient transformation assay with five-day-old seedlings using Agrobacterium tumefaciens-mediated vacuum infiltration. Three days after infiltration of the Agrobacterium containing an expression vector for a fluorescent marker of interest, cotyledon cells expressing the fluorescent protein can be imaged in a confocal microscope. This assay allows high-throughput screening of new constructs and the study of the localization of a large number of subcellular markers in Arabidopsis seedlings including wild-type, stable over-expressing and mutant lines.CRISPR/Cas9 system has emerged as a powerful genome engineering tool to study gene function and improve plant traits. Genome editing is achieved at a specific genome sequence by Cas9 endonuclease to generate double standard breaks (DSBs) directed by short guide RNAs (sgRNAs). The DSB is repaired by error-prone nonhomologous end joining (NHEJ) or error-free homology-directed repair (HDR) pathways, resulting in gene mutation or sequence replacement, respectively. These cellular DSB repair pathways can be exploited to knock out or replace genes. selleck inhibitor Also, cytidine or adenine base editors (CBEs or ABEs) fused to catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) are used to perform precise base editing without generating DSBs. In this chapter, we describe a detailed procedure to carry out single/multiple gene mutations and precise base editing in the Arabidopsis genome by using CRISPR/Cas9-based system. Specifically, the steps of target gene selection, sgRNA design, vector construction, transformation, and analysis of transgenic lines are described. The protocol is potentially adaptable to perform genome editing in other plant species such as rice.Mobile signals play pivotal roles in coordinating interorgan communication. Grafting provides an effective strategy to identify and explore the movement of the mobile signals. The mutant collection of Arabidopsis offers background-free living materials for examining the transport of mobile signals in vivo. In the past few years, many grafting methods have been developed to overcome the limitations of rosette-type growth and small size in Arabidopsis. Here we describe a non-sterile grafting method involving an insect pin to secure the scion to the rootstock. The scions can be grafted onto epicotyls or hypocotyls of soil-grown Arabidopsis rootstocks at a wide range of developmental stages. This grafting method provides a useful tool to analyze leaf-derived mobile signals in Arabidopsis.Arabidopsis has become a model plant for ecological and population genomics, owing to the substantial phenotypic and genotypic variation that exists among and within natural populations. Specially, the recent availability of large worldwide collections of accessions, together with their full genome sequences, has triggered the study of Arabidopsis natural variation. In this chapter, we describe two protocols that exploit these new resources to understand the natural variation for any trait and gene (1) the phenotypic analysis of Arabidopsis plants grown in field experiments; (2) the analysis of nucleotide diversity and environmental associations for specific genes.
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