Phylogenetic analysis of DNA sequence data.

The goal of phylogenetics is to construct relationships that are true representations of the evolutionary history of a group of organisms or genes. The history inferred from phylogenetic analysis is usually depicted as branching in tree-like diagrams or networks. In nematology, phylogenetic studies have been applied to resolve a wide range of questions dealing with improving classifications and testing evolution processes, such as co-evolution, biogeography and many others. There are several main steps involved in a phylogenetic study: (i) selection of ingroup and outgroup taxa for a study; (ii) selection of one or several gene fragments for a study; (iii) sample collection, obtaining PCR products and sequencing of gene fragments; (iv) visualization, editing raw sequence data and sequence assembling; (v) search for sequence similarity in a public database; (vi) making and editing multiple alignment of sequences; (vii) selecting appropriate DNA model for a dataset; (viii) phylogenetic reconstruction using minimum evolution, maximum parsimony, maximum likelihood and Bayesian inference; (ix) visualization of tree files and preparation of tree for a publication; and (x) sequence submission to a public database. Molecular phylogenetic study requires particularly careful planning because it is usually relatively expensive in terms of the cost in reagents and time.

Characterization of nematode mitochondrial genomes.

This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.

Staining chromosomes.

In this chapter, the basic information for staining chromosomes of Meloidogyne spp. and free-living nematodes is presented, together with information on staining giant cells and syncytia.

Behavioural and physiological assays.

This chapter focuses on some of the principal techniques for analysing nematode behaviour and physiology, such as attraction/repulsion plate assays, pluronic gel assays, movement assays, electrophysiology, stylet activity, water content changes, oxygen consumption assays, collection of female sex pheromone, virgin females and males and viability tests, among others.

Handling, fixing, staining and mounting nematodes.

Numerous techniques for handling, fixing, staining and mounting nematodes have been published during the development of the science of nematology over the last 60 years. This chapter includes those techniques that have been shown to be widely adapted and least hazardous to the user and the environment.

Methods for nematode extraction.

Nematodes can be present in different matrices. This chapter describes several methods to extract nematodes from soil and plant parts. It is crucial that an appropriate method is chosen for the purpose of the research as different types of nematodes, and even different nematode stages, are extracted depending on the method. Factors to consider for choosing the optimal extraction method are the extraction efficiency of the method, the maximum sample size that can be analysed and costs of the extraction equipment. In addition, water consumption, labour and the time needed before nematodes can be examined can be important factors.

Isolation and characterization of tandem repeats in nematode genomes.

This chapter provides an overview of the practical methodologies that can be used to identify and characterize the tandem repeats that are most frequently used as genetic markers in nematodes (including plant-parasitic and entomopathogenic nematodes), namely satellite DNA and microsatellites. The objective is not to provide turnkey protocols, but rather to return to the main principles that govern these protocols. Case studies on nematodes will serve to illustrate the point. In that respect, two well-defined situations are to be considered, depending on whether genomic resources for the species under investigation are available or not.

Sampling.

Nematodes can be present in various parts of plants or at various depths in the soil, depending on the circumstances and the life stage. This means that one should be aware of these possibilities when collecting samples. Also, samples should be taken from the proper matrix at the appropriate time, taking into account the developmental stages the nematodes might be in. In this chapter, the purpose of sampling, sampling techniques and, related to this, the sampling tools, and the handling and storage of the samples before processing of plant-parasitic and entomopathogenic nematodes are discussed. Some protocols are described in detail as examples.

Handling, fixing, staining and mounting nematodes.

Numerous techniques for handling, fixing, staining and mounting nematodes have been published during the development of the science of nematology over the last 60 years. This chapter includes those techniques that have been shown to be widely adapted and least hazardous to the user and the environment.

Isoelectric focusing of proteins.

This chapter focuses on the efficacy of isoelectric focusing, which is a technique used for separating charged molecules by differences in their isoelectric point, in the examination of general proteins and specific enzymatic differences among plant parasitic nematodes, particularly the potato cyst and root knot nematodes.