Phylogenetics is a discipline reconstructing evolutionary relationships of organisms. With improvements in sequencing technique, analytic methods, and computation power, the molecular data have been used widely and have promoted greatly the rapid development of molecular phylogenetics. The phylogenetic tree has become a powerful tool in many areas of biology, such as ecology and comparative biology. Currently, phylogenetic studies mainly focus on phylogenetic tree reconstructions by using various software, however, some fundamental principles or matters that should be paid attention when performing phylogenetic analyses are sometimes weakened or even ignored. Here, we present the workflow and methods in details for phylogenetic tree reconstruction based on molecular data, including taxon sampling, molecular marker selection, sequence alignment, partitioning and model selection, combined analysis of multiple markers, and topological test. Currently, the widely used methods of phylogenetic reconstructions are maximum parsimony, maximum likelihood, and Bayesian inference. We thereby provide the detailed operating flows and corresponding commands for these three methods, respectively. We expect this paper will provide a reference for relevant researches.

Oligonucleotide fluorescence in situ hybridization (Oligo-FISH) is a new technology that integrates bioinformatics analysis, high throughput DNA synthesis and fluorescence in situ hybridization experiments. It is applicable to plant species with a reference genome. Oligo probes are able design to a chromosomal region, an entire chromosome, and a set of chromosomes according to researchers’ requirements. Oligo-FISH has been successfully applied to studies on karyotyping, chromosome variation, population evolution, homologous chromosome pairing, haplotype analysis, and heteropolyploid identification in several plant species. Here, we introduce the concrete operational processes of oligonucleotide-based probe design, amplification and labeling, chromosomes preparation, and fluorescence in situ hybridization in plants, so as to facilitate Oligo-FISH application in the cytogenetics research in the future.

CRISPR/Cas9-based genome editing technology has been an important tool to study the gene function and genomic modification. Directed by a guide RNA, Cas9 protein can cleavage the genomic DNA at the target site, and produce mutations, including deletion, insertion, substitution and fragment deletion, by DNA double strand break (DSB) repair mechanism. In this protocol, we introduce the method to use CRISPR/Cas9 system to increase the efficiency of genomic DNA fragment deletion with microhomology-mediated end joining, especially the details in target design and detection of mutant plants.

Because the genome of an organism determines its primary phenotype, evolutionary principles suggest that genetic variations enhance phenotypic diversity towards increased fitness. Targeted saturation mutagenesis of crop genes could be used to screen for genetic variants with improved agronomic traits. Compared to traditional mutational breeding or directed evolution in heterologous organisms, targeted mutagenesis via dual cytosine and adenine base editors effectively generates endogenous mutagenesis and facilitates in vivo directed evolution of plant genes. In this protocol, we detail the process towards using saturated targeted endogenous mutagenesis editors (STEMEs) to generate targeted, random mutagenesis of plant genes. In particular, we focus on the process of designing targets, screening and genotyping the resulting evolved variants.

Successful pathogen inoculation and following accurate rating are the basis of barley disease resistance studies. Here, we summarized selected barley leaf rust inoculation methods including spraying and smearing, and two evaluation indexes which are widely used in barley slow rusting evaluation. Key issues worthy affecting the assays were also discussed.

Rice is the most important crop in the world. However, rice blast caused by Magnaporthe oryzae and sheath blight caused by Rhizoctonia solani are two of diseases, which threaten both yield and quality of rice most severely. To ensure food security, it is very important to identify disease-resistant rice germplasm, clone disease resistant genes, uncover the molecular basis and apply them in rice breeding program. Accurate evaluation of the disease resistance of rice is fundamental to both uncover disease resistance mechanism and improve resistance in rice breeding. Here, we describe the common methods for evaluating rice blast disease resistance by spraying inoculation of seedlings with M. oryzae, injection inoculation at rice tillering and booting stage, and punch inoculation of detached rice leaves. We also describe the methods for evaluating rice sheath blight disease resistance by field inoculation with R. solani at rice tillering stage, greenhouse inoculation at rice booting stage, and inoculation of rice detached-stems in growth chamber. We believe these methods could provide useful protocols for colleagues who aim to identify rice disease-resistant resources, dissect the underlying molecular mechanism and breed elite rice varieties with improved disease resistance.

Transcription affects the growth and development of plants through regulating the spatio-temporal expression of downstream genes. The interaction between transcription factors and DNA is a key section in the process of exploring transcriptional regulatory networks. In the past few years, researchers utilize yeast one hybrid (Y1H) and electrophoresis mobility shift assay (EMSA) to examine whether a transcription factor directly interacts with target DNA. In addition, transient luciferase activity assay provides a convenient method for researchers to test the regulation of transcription factors on downstream gene expression. In this paper, we elaborate the principles, methods, and advantages and limitations of Y1H, EMSA and transient luciferase activity assay, to provide technical references for exploring the transcription factor-DNA interactions.

Chromatin immunoprecipitation (ChIP) is a teschnique used to investigate protein-DNA interaction. Briefly, protein and associated DNA are crosslinked, which is optional. Then the DNA-protein complexes are fragmented. By using an appropriate antibody, cross-linked DNA fragments associated with the protein of interest are selectively immunoprecipitated. ChIP is commonly used to identify binding regions of DNA-binding proteins, such as transcription factors. ChIP is also used to analyze histone modification profiles in combination with antibodies against specific histone modifications. Here we describe protocols and related tips for cross-linked ChIP and ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP), which can be applied to rare cell populations at the 10³ cell level.