Application of a modified drop method for high-resolution pachytene chromosome spreads in two Phalaenopsis species

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.

Induction of Polyploidy Using Colchicine in Flower Buds from Phalaenopsis Hybrids

“…1). This was in accordance with the opinion of Kuo et al [9] that the basic chromosome number of Phalaenopsis reaches x = 19. This study proves that the chromosomes in the three types of orchids are polyploid.…”

Chromosome‐level assembly, genetic and physical mapping of Phalaenopsis aphrodite genome provides new insights into species adaptation and resources for orchid breeding

“…Chromosome spreads were prepared mainly according to a previously published protocol (Kuo et al ., ). The fixed pollinia (glacial acetic acid: ethanol, 1:3 (v/v)) with chromosomes at the pachytene stage were first rinsed for 5 min each in distilled water and 10 m M citrate buffer (4 m M citric acid and 6 m M tri‐sodium citrate, pH 4.5).…”

Segmental and tandem chromosome duplications led to divergent evolution of thechalcone synthasegene family inPhalaenopsisorchids

“…Flower buds of P. aphrodite (2 n = 2 x = 38) with a size range of 8.50–9.20 mm were collected to obtain developing pollinia with chromosomes at the meiotic pachytene stage. Pachytene chromosomal spreads were prepared following the modified drop method, as previously described (Kuo et al , 2016). Meiotic pachytene spreads with little to no cytoplasm and good pachytene chromosome spreading were selected and stored at 4 °C for later use.…”