Fluorescence in situ hybridization and karyotyping analysis of Phalaenopsis equestris using pachytene chromosomes
Date Issued
2012
Date
2012
Author(s)
Hsueh, Hao-Yen
Abstract
Phalaenopsis orchid is the important floral industry in Taiwan. It however has a long period of life cycle and deficiency of genetic map as well as cytogenetic research. P. equestris is a native and representive orchid in Taiwan, and the kind of its relatively small genome is suitable for analysis in Phalaenopsis genome sequencing project in the future. The size and morphology among somatic metaphase chromosomes were close, and it is difficult to identify each with conventional staining. Ninteen pachytene chromosomes of meiotic stage were studied in this thesis showing their lower contration and high resolution that could contribute to construct a karyotype. Single chromosome morphology can be described by analyzing average length, distribution of chromatin, centromere position and presence of secondary constriction of chromosomes. Based on the results of karyotype, the average length of each pachytene chromosome was 21.95 µm and a 2.35-fold difference between the largest and smallest ones was found. Regarding each chromosome morphology, all heterochromatin were restricted around centromere, but the amount and distribution had significant differences that could distinguish these chromosomes clearly with some heterochromatin knobs. Almost all P. equestris chromosomes are metacentric or submetacentric while only one chromosome which includes a secondary constriction on the end of short arm belonging to subtelocentric. In comparison with mitotic metaphase, pachytene chromosomes possessed more than 12-fold higher resolution and thus they can facilitate the construction of chromosome maps. Furthermore, 5S rDNA,45S rDNA, Arabidopsis-type telomeric sequences and SOC1 gene were mapped to chromosomes using fluorescence in situ hybridization. The results showed that chromosome 17 had a 5S rDNA locus and chromosome 13 had a 45S rDNA locus on the ends of their short arms. Furthermore, SOC1 was located on the short arm of chromosome 1, and telomeric sequences located on both ends of every chromosomes. Based on the variation in the spatial and quantitative distribution of DNA sequences, a complete karyotype could be constructed combined with molecular markers to distinguish similar chromosomes.
In this study we first developed P. equestris high-resolution karyotype better than previous studies which focus on mitotic metaphase chromosomes, and we hope it will become a model plant used for other orchids. In addition, in the future with the research of chromosome rearrangements of Phalaenopsis species, we may understand how Phalaenopsis chromosomes evolved.
Subjects
cytogenetics
P. equestris
pachytene chromosomes
karyotyping
fluorescence in situ hybridization
Type
thesis
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