Most cited article - PubMed ID 21599670
Allopolyploid origins of the Galeopsis tetraploids--revisiting Müntzing's classical textbook example using molecular tools
Flow cytometry has emerged as a uniquely flexible, accurate, and widely applicable technology for the analysis of plant cells. One of its most important applications centers on the measurement of nuclear DNA contents. This chapter describes the essential features of this measurement, outlining the overall methods and strategies, but going on to provide a wealth of technical details to ensure the most accurate and reproducible results. The chapter is aimed to be equally accessible to experienced plant cytometrists as well as those newly entering the field. Besides providing a step-by-step guide for estimating genome sizes and DNA-ploidy levels from fresh tissues, special attention is paid to the use of seeds and desiccated tissues for such purposes. Methodological aspects regarding field sampling, transport, and storage of plant material are also given in detail. Finally, troubleshooting information for the most common problems that may arise during the application of these methods is provided.
- Keywords
- Best practices, DAPI, DNA-ploidy level, Desiccated tissues, Flow cytometry, Genome size, Plant nuclei isolation, Plant tissues, Propidium iodide, Seeds,
- MeSH
- Cell Nucleus * genetics chemistry MeSH
- Genome Size MeSH
- DNA, Plant genetics analysis MeSH
- Genome, Plant MeSH
- Ploidies MeSH
- Flow Cytometry methods MeSH
- Plants * genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Plant MeSH
Dandelions (genus Taraxacum) comprise a group of sexual diploids and apomictic polyploids with a complicated reticular evolution. Apomixis (clonal reproduction through seeds) in this genus is considered to be obligate, and therefore represent a good model for studying the role of asexual reproduction in microevolutionary processes of apomictic genera. In our study, a total of 187 apomictic individuals composing a set of nine microspecies (sampled across wide geographic area in Europe) were genotyped for six microsatellite loci and for 162 amplified fragment length polymorphism (AFLP) markers. Our results indicated that significant genetic similarity existed within accessions with low numbers of genotypes. Genotypic variability was high among accessions but low within accessions. Clustering methods discriminated individuals into nine groups corresponding to their phenotypes. Furthermore, two groups of apomictic genotypes were observed, which suggests that they had different asexual histories. A matrix compatibility test suggests that most of the variability within accession groups was mutational in origin. However, the presence of recombination was also detected. The accumulation of mutations in asexual clones leads to the establishment of a network of clone mates. However, this study suggests that the clones primarily originated from the hybridisation between sexual and apomicts.
Flow cytometry (FCM) has been widely used in plant science to determine the amount of nuclear DNA, either in absolute units or in relative terms, as an indicator of ploidy. The requirement for fresh material in some applications, however, limits the value of FCM in field research, including plant biosystematics, ecology and population biology. Dried plant samples have proven to be a suitable alternative in some cases (large-scale ploidy screening) although tissue dehydration is often associated with a decrease in the quality of FCM analysis. The present study tested, using time-scale laboratory and in situ field experiments, the applicability of glycerol-treated nuclear suspension for DNA flow cytometry. We demonstrate that plant nuclei preserved in ice-cold buffer + glycerol solution remain intact for at least a few weeks and provide estimates of nuclear DNA content that are highly comparable and of similar quality to those obtained from fresh tissue. The protocol is compatible with both DAPI and propidium iodide staining, and allows not only the determination of ploidy level but also genome size in absolute units. Despite its higher laboriousness, glycerol-preserved nuclei apparently represent the most reliable way of sample preservation for genome size research. We assume that the protocol will provide a vital alternative to other preservation methods, especially when stringent criteria on the quality of FCM analysis are required.
- MeSH
- Cell Nucleus drug effects genetics MeSH
- Time Factors MeSH
- DNA, Plant analysis MeSH
- Glycerol pharmacology MeSH
- Cryoprotective Agents pharmacology MeSH
- Preservation, Biological methods MeSH
- Flow Cytometry * MeSH
- Plant Cells chemistry drug effects MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Plant MeSH
- Glycerol MeSH
- Cryoprotective Agents MeSH
