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A range of molecular methods can be employed for the characterization of natural and induced nucleotide variation in plants. These facilitate a better understanding of gene function and allow a reduction in the time needed to breed new mutant varieties. Molecular biology, however, can be difficult to master, and while efficient, many protocols rely on expensive pre-made kits. The FAO/IAEA Plant Breeding and Genetics Laboratory (PBGL) has developed a series of low-cost and easy to use approaches for the molecular characterization of mutant plant materials. The protocols are designed specifically to avoid complicated procedures, expensive equipment, and the use of hazardous chemicals. Furthermore, these protocols have been validated by research fellows from many developing countries.

All laboratories should have standardized health and safety rules and practices. These can vary from region to region due to differences in legislation. Before beginning new experiments, please consult your local safety guidelines. Failure to follow these rules could result in accidents, fines, or a closure of the laboratory. Consider the following guidelines in this chapter applicable to all laboratories.

Of importance to the successful extraction of genomic DNA from plant tissues is the collection of the suitable material and proper storage of the tissues before DNA isolation. If the samples are not properly treated, DNA can be degraded prior to isolation. The rate of sample degradation can vary dramatically from species to species depending on the method of sample collection. Mechanisms of genomic DNA degradation include exposure to endogenous nucleases due to organellar and cellular lysis. To prevent this from occurring, leaf or root tissues are commonly flash frozen in liquid nitrogen and then stored at −80 °C. At these temperatures, nucleases remain inactive and DNA is stable. Thawing of tissue in some species can lead to rapid degradation. Therefore, during the extraction procedure, it may be necessary to grind the tissue to a fine powder in the presence of liquid nitrogen and expose frozen tissue immediately to a lysis buffer containing EDTA, which inhibits nuclease activity. This chapter provides an alternative method for sample collection and storage. Silica gel is used to desiccate tissues at room temperature. This avoids the use of liquid nitrogen and storage at −80 °C.

The methods described in this chapter were developed to avoid toxic organic phase separation utilized in many low-cost DNA extraction protocols such as the CTAB method. The steps involve: (1) lysis of the plant material, (2) binding of DNA to silica powder under chaotropic conditions, (3) washing the bound DNA, and (4) elution of DNA from the silica powder. This method has been tested in several plant species and the applicability of such DNA preparations for molecular marker studies in barley is shown in Chap. .

PCR is used to amplify regions to be interrogated for the presence of mutations (SNP and small indel polymorphisms). While PCR is a common practice and many protocols exist, reaction conditions are provided here that are optimized for TILLING and Ecotilling assays utilizing native agarose gel electrophoresis.

Denaturation and annealing of PCR products allows DNA strands with small sequence differences to hybridize together. The result is heteroduplexed molecules that are single stranded in polymorphic sequence locations, but double stranded elsewhere. These molecules are the substrates for cleavage by single-strand-specific nucleases such as CEL I, crude Celery Juice Extract (CJE) containing CEL I, and other plant extracts containing single-strand-specific nucleases [Till et al. (Nucleic Acids Res, 32:2632–2641, 2004)]. Enzymatic cleavage initiates on a single strand and can result in double strand breaks. The products of cleavage can therefore be observed using native gel electrophoresis.

A crude celery extract containing the single-strand-specific nuclease CEL I, has been widely used in TILLING and Ecotilling projects around the world. Yet, celery is hard to come by in some countries. Sequences homologous to CEL I can be found in different plant species. Previous work showed that similar mismatch cleavage activities could be found in crude extracts of mung bean (Till BJ, Burtner C, Comai L, Henikoff S. Nucleic Acids Res 32:2632–2641, 2004). It is likely that the same activity can be recovered in many different plant species. Therefore, a protocol for the extraction of active enzyme was developed that uses plants common across the world, namely weeds. Monocotyledenous and dicotyledenous weedy plants from the grassland, field and waste grounds around crop fields are suitable for this protocol. Due to lower recovery of enzymatic activity compared to celery-based extractions, a centrifuge-based filter method is applied to concentrate the enzyme extract.

Standard agarose gel electrophoresis is a quick method for the evaluation of the quality and quantity of DNA. This chapter provides examples of genomic DNA produced using the low-cost extraction protocol, PCR amplification using the extracted genomic DNA, and enzymatic mismatch cleavage of PCR products with crude celery juice extract and weed juice extract to detect mutations.

The approaches described here provide rapid and low-cost alternatives for sample preparation, genomic DNA extraction, and mutation discovery. When evaluating the methods, it is important to remember that protocol adaptations may be necessary to compensate for sample differences (species and genotype), environmental conditions in the laboratory, and quality of the water and chemicals used. Cost savings in DNA preparation must be balanced with the shelf-life and suitability of the samples for use in downstream applications. With the appropriate validation of sample quality and longevity, the protocols described here can provide sufficient DNA for a variety of molecular applications such as marker studies and TILLING, at approximately one tenth of the cost per sample when compared to commercial kits.