Abstract
BackgroundPreparation of good chromosome spreads without cytoplasmic contamination is the crucial step in cytogenetic mapping. To date, cytogenetic research in the Orchidaceae family has been carried out solely on mitotic metaphase chromosomes. Well-spread meiotic pachytene chromosomes can provide higher resolution and fine detail for analysis of chromosomal structure and are also beneficial for chromosomal FISH (fluorescence in situ hybridization) mapping. However, an adequate method for the preparation of meiotic pachytene chromosomes in orchid species has not yet been reported.ResultsTwo Taiwanese native Phalaenopsis species were selected to test the modified drop method for preparation of meiotic pachytene chromosomes from pollinia. In this modified method, pollinia were ground and treated with an enzyme mixture to completely remove cell walls. Protoplasts were resuspended in ethanol/glacial acetic acid and dropped onto a wet inclined slide of 30° from a height of 0.5 m. The sample was then flowed down the inclined plane to spread the chromosomes. Hundreds of pachytene chromosomes with little to no cytoplasmic contamination were well spread on each slide. We also showed that the resolution of 45S rDNA-containing chromosomes at the pachytene stage was up to 20 times higher than that at metaphase. Slides prepared following this modified drop method were amenable to FISH mapping of both 45S and 5S rDNA on pachytene chromosomes and, after FISH, the chromosomal structure remained intact for further analysis.ConclusionThis modified drop method is suitable for pachytene spreads from pollinia of Phalaenopsis orchids. The large number and high-resolution pachytene spreads, with little or no cytoplasmic contamination, prepared by the modified drop method could be used for FISH mapping of DNA fragments to accelerate the integration of cytogenetic and molecular research in Phalaenopsis orchids.
Highlights
Preparation of good chromosome spreads without cytoplasmic contamination is the crucial step in cytogenetic mapping
Preparation of meiotic chromosome spreads using the modified drop method Owing to the loss of some pollen mother cell (PMC) during chromosome preparation, in protoplast preparation, pollinia from one P. aphrodite subsp. formosana flower bud or 2–3 pollinia of P. equestris were used to prepare 15– 25 slides with similar quality
The samples flow down freely along the inclined plane to spread chromosomes, instead of blowing or spraying bursting medium, which is relatively more technical expertise demanded. The advantages of this method over the previous methods include: (1) the cell walls of tightly compacted PMCs can be uniformly eliminated; (2) no technical expertise is needed for dropping protoplasts onto inclined slides using a dropper, or spreading chromosomes; (3) most chromosome spreads are free from contamination by wall fragments and cytoplasm; (4) several slides with similar quality can be prepared in a single experiment; (5) up to 100 meiotic pachytene spreads can be obtained on each slide; (6) the chromosomal structure remains intact even after Fluorescence in situ hybridization (FISH) when samples are dropped from the optimized dropping height
Summary
Preparation of good chromosome spreads without cytoplasmic contamination is the crucial step in cytogenetic mapping. Cytogenetic research in the Orchidaceae family has been carried out solely on mitotic metaphase chromosomes. Preparation of chromosome spreads is the foundation of cytogenetic research. High-resolution FISH mapping on pachytene chromosomes is a powerful technique to help integrate physical and genetic maps and to evaluate genome assembly quality. This strategy has been successfully demonstrated in plant species, for example tomato [1, 2], potato [3], cucumber [4], Rosa [5] and Amborella [6].
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