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.