In less than a decade, the polymerase chain reaction (PCR) has evolved into the common denominator of molecular biology for nucleic acid research. The most important impact of PCR in plant molecular systematics has been rapid amplification of DNA regions, facilitating nucleotide sequencing (see Chapter 1). However, there are many other techniques aside from DNA sequencing in which PCR is appropriate and extremely useful. In this chapter we review these applications of PCR in plant systematics and highlight areas of research that have been underused in plant molecular systematics laboratories.

Contributions of PCR-Based Methods to Plant Systematics and Evolutionary Biology

Buffalograss, Buchloe dactyloides, is widely distributed throughout the Great Plains of North America, where it is an important species for rangeland forage and soil conservation. The species consists of two widespread polyploid races, with narrowly endemic diploid populations known from two regions: central Mexico and Gulf Coast Texas. We describe and compare the patterns of allozyme and RAPD variation in the two diploid races, using a set of 48 individuals from Texas and Mexico (four population samples of 12 individuals each). Twelve of 22 allozyme loci were polymorphic, exhibiting 35 alleles, while seven 10-mer RAPD primers revealed 98 polymorphic bands. Strong regional differences were detected in the extent of allozyme polymorphism: Mexican populations exhibited more internal gene diversity (He= 0.20, 0.19) than did the Texan populations (He= 0.08, 0.06), although the number of RAPD bands in Texas (n= 62) was only marginally smaller than in Mexico (n= 68). F-statistics for the allozyme data, averaged over loci, revealed strong regional differentiation (mean FRT=+ 0.30), as well as some differentiation among populations within regions (mean FPR=+ 0.09). In order to describe and compare the partitioning of genetic variation for multiple allozyme and RAPD loci, we performed an Analysis of Molecular Variance (AMOVA). AMOVA for both allozyme and RAPD data revealed similar qualitative patterns: large regional differences and smaller (but significant) population differences within regions. RAPDs revealed greater variation among regions (58.4% of total variance) than allozymes (45.2%), but less variation among individuals within populations (31.9% for RAPDs vs. 45.2% for allozymes); the proportion of genetic variance among populations within regions was similar (9.7% for RAPDs vs. 9.6% for allozymes). Despite this large-scale concordance of allozyme and RAPD variation patterns, multiple correlation Mantel techniques revealed that the correlations were low on an individual by individual basis. Our findings of strong regional differences among the diploid races will facilitate further study of polyploid evolution in buffalograss.

Evolutionary implications of allozyme and RAPD variation in diploid populations of dioecious buffalograss Buchloë dactyloides

More than 9000 papers using the random amplified polymorphic DNA (RAPD) or related techniques (e.g. the arbitrarily primed polymerase chain reaction (AP-PCR)) have been published from 1990 to 2005. The RAPD method has been initially used to detect polymorphism in genetic mapping, taxonomy and phylogenetic studies and later in genotoxicity and carcinogenesis studies. Despite their extensive use, these techniques have also attracted some criticisms, mainly for lack of reproducibility. In the light of their widespread applications, the objectives of this review are to (1) identify the potential factors affecting the optimisation of the RAPD and AP-PCR assays, (2) critically describe and analyse these techniques in genotoxicity and carcinogenesis studies, (3) compare the RAPD assay with other well used methodologies, (4) further elucidate the impact of DNA damage and mutations on the RAPD profiles, and finally (5) provide some recommendations/guidelines to further improve the applications of the assays and to help the identification of the factors responsible for the RAPD changes. It is suggested that after proper optimisation, the RAPD is a reliable, sensitive and reproducible assay, has the potential to detect a wide range of DNA damage (e.g. DNA adducts, DNA breakage) as well as mutations (point mutations and large rearrangements) and therefore can be applied to genotoxicity and carcinogenesis studies. Nevertheless, the interpretation of the changes in RAPD profiles is difficult since many factors can affect the generation of RAPD profiles. It is therefore important that these factors are identified and taken into account while using these assays. On the other hand, further analyses of the relevant bands generated in RAPD profile allow not only to identify some of the molecular events implicated in the genomic instability but also to discover genes playing key roles, particularly in the initiation and development of malignancy. Finally, to elucidate the potential genotoxic effects of environmental contaminants, a powerful strategy could be firstly to use the RAPD assay as a screening method and secondly to apply more specific methods measuring for instance DNA adducts, gene mutations or cytogenetic effects. It is also envisaged that these assays (i.e. RAPD and related techniques), which reflect effects at whole genome level, would continue to complement the use of emerging technologies (e.g. microarrays which aim to quantify expression of individual genes).

The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review.

As part of the development of a molecular toolkit for the study of diversity within large plant germplasm collections, RAPD technology has been applied to accessions of rice (Oryza sativa) obtained from the major world collection held at IRRI (the International Rice Research Institute) which supplies germplasm to breeders. Methods for the speedy extraction of DNA representative of a rice accession, its amplification by PCR to reveal reproducible products, and the analysis of the banding data using numerical techniques have been established. The biological meaningfulness of RAPD data has also been demonstrated by reference to previous work on classification and crossability.

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Use of RAPD for the study of diversity within plant germplasm collections

Finger millet (Eleusine coracana), an allotetraploid cereal, is widely cultivated in the arid and semiarid regions of the world. Three DNA marker techniques, restriction fragment length polymorphis...

Assessment of genome origins and genetic diversity in the genus Eleusine with DNA markers.

RAPD analysis was applied to onion (Allium cepa) and otherAllium species in order to assess the degree of polymorphism within the genus and to investigate if this approach was suitable for genetic studies of onion. Seven cultivars ofA. cepa, including shallot, and single cultivars of Japanese bunching onion (A. fistulosum), chive (A. schoenoprasum), leek (A. ampeloprasum), and a wild relative of onion (A. roylei), were evaluated for variability using a set of 20 random 10-mer primers. Seven out of the twenty primers revealed scorable polymorphisms between cultivars ofA. cepa and these will be further evaluated for use in genetic mapping. Wide variations in banding profiles between species were observed with nearly every primer tested. These were assessed for use in systematic studies within the genus. Ninety-one band positions were scored (+/-) for all the cultivars studied. Genetic distances between each of the cultivars were calculated and cluster analysis was used to generate a dendrogram showing phylogenetic relationships between them. The resulting analysis was in broad agreement with previous classifications of the species studied, confirming the validity of the method. However, amongst the species studied, it placedA. roylei as the closest relative ofA. cepa, questioning the current classification of the former species in the section Rhizideum.

Random amplified polymorphic DNA (RAPD) markers for genetic analysis in Allium

METHOD FOR THE PRODUCTION OF PROTEINS IN PLANT FLUIDS A method of producing a genetically transformed fluid-producing plant comprises: i) inserting into the plant tissue a gene or gene fragment controlling the expression of a target product, and ii) regenerating a plant from said tissue, the genetically transformed plant being capable of expressing the target product in the fluid that it produces. There are also provided clones of genetically transformed fluid-producing plants which contain in their cells a chromosomal insert such that the target product is expressed in the fluids that the plants produce. There is further described a method of producing a protein or other target product which comprises:- i) harvesting the fluid from a genetically transformed fluid-producing tree or plant, or a clone thereof, and ii) recovering the target protein or other product from said fluid. Most preferably, the plants are rubber (Hevea) plants and the genes are foreign genes that code for pharmaceutically valuable protein products which can be harvested in the latex produced by the plants.

Method for the production of proteins in plant fluids.

The original publication is available at www.springerlink.com Copyright Humana Press [Full text of this article is not available in the UHRA]

Robert J. Slater

Papers

Random amplified polymorphic DNA (RAPD) markers for genetic analysis in Allium

Method for the production of proteins in plant fluids.

Mung-bean nuclease 1 (EC 3.1.30.1).

The Timing of Referred Examinations.

Eukaryotic nuclear RNA polymerases (EC 2.7.7.6).