Begum Rabeya, Alam Sheikh Shamimul, Menzel Gerhard, Schmidt Thomas
Department of Botany, University of Dhaka, Dhaka 1000, Bangladesh.
Ann Bot. 2009 Oct;104(5):863-72. doi: 10.1093/aob/mcp178. Epub 2009 Jul 27.
Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera.
In order to isolate highly repetitive sequences, a c(0)t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH).
Characterization of the c(0)t-1 library resulted in the detection of repetitive sequences including the (GA)(n) dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S-5.8S-25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species.
The repeated sequences isolated from D. moschatum c(0)t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal rearrangements in D. aphyllum while the number and localization of rRNA genes as well as the species-specific distribution pattern of an abundant microsatellite reflect the genomic diversity of the three Dendrobium species.
石斛属植物表现出巨大的形态多样性，且地理分布广泛。由于重复序列分析是研究染色体和基因组进化的有用工具，本研究的目的是对毛瓣石斛的重复序列进行表征，以便在相关物种石豆兰、聚石斛以及其他兰花属的代表物种中进行比较分子和细胞遗传学研究。
为了分离高度重复序列，构建了一个c(0)t-1 DNA质粒文库。对重复序列进行测序，并用作Southern杂交的探针。使用生物信息学工具分析序列差异。通过荧光原位杂交（FISH）将重复序列定位到兰花染色体上。
对c(0)t-1文库的表征导致检测到重复序列，包括(GA)(n)二核苷酸DmoO11、许多拟南芥样端粒重复序列以及高度扩增的分散重复序列DmoF14。DmoF14重复序列在六个石斛属物种中保守，但在其他三个兰花属的代表物种中有所分化。FISH分析显示DmoF14在毛瓣石斛、石豆兰和聚石斛的全基因组中分布。与端粒重复序列杂交表明石斛属物种染色体末端存在拟南芥样端粒。然而，使用端粒探针的FISH显示石豆兰中有两对染色体具有强烈的居间信号。FISH显示了三种石斛属物种中5S和18S-5.8S-25S rRNA基因的末端位置以及rDNA位点的特征数量。
从毛瓣石斛c(0)t-1 DNA中分离出的重复序列构成了毛瓣石斛、石豆兰和聚石斛基因组的主要DNA家族，其中DmoF14代表兰花基因组的一个古老成分。大型居间端粒样阵列表明石豆兰中存在染色体重排，而rRNA基因的数量和定位以及丰富微卫星的物种特异性分布模式反映了三种石斛属物种的基因组多样性。
