She C W, Liu J Y, Song Y C
Key Laboratory of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan, 430072, China.
Biotech Histochem. 2006 Jan-Feb;81(1):13-21. doi: 10.1080/10520290600661414.
Mitotic chromosome spreads of 16 plant species belonging to six families were analyzed using an improved combined PI and DAPI (CPD) staining procedure. Fluorescence in situ hybridization (FISH) with 45S rDNA probe was conducted sequentially on the same spreads to evaluate the efficiency and sensitivity of the technique. Fluorochrome staining with chromomycin A3 (CMA)-DAPI also was conducted to clarify the properties of the sequences involved in the CPD banded regions. Our results revealed that all of the NORs (rDNA sites) in the species tested were efficiently shown as red bands by CPD staining, and the number and position of the bands corresponded precisely to those of the 45S rDNA FISH signals, indicating that the detection sensitivity of CPD staining is similar to that of FISH. In 10 of the species tested including Aegilops squarrosa, Allium sativum, Oryza sativum ssp. indica, Oryza officinalis, Pisum sativum, Secale cereale, Setaria italica, Sorghum vulgare, Vicia faba and Zea mays, CPD bands were exhibited exclusively in their NORs, while in other six species including Hordeum vulgare, Allium cepa, Psophocarpus tetragonolobus, Arabidopsis thaliana, Brassica oleracea var. capitata and Lycopersicon esculentum, CPD bands appeared in chromosomal regions other than their NORs. The CPD bands were in accordance with the CMA bands in all species tested, indicating GC-rich sequences in the CPD bands and that the improved CPD staining procedure is specific for GC-rich regions in plant genomes. Our investigation not only elucidated the banding mechanisms of CPD, but also demonstrated that the CPD staining technique, which may be preferable to CMA staining, is an effective tool for detecting NORs and other GC-rich chromosomal regions in plants.
使用改进的碘化丙啶（PI）和4′,6-二脒基-2-苯基吲哚（DAPI）联合染色程序（CPD），对属于六个科的16种植物的有丝分裂染色体铺片进行了分析。在相同的铺片上依次进行45S核糖体DNA（rDNA）探针的荧光原位杂交（FISH），以评估该技术的效率和灵敏度。还进行了放线菌素A3（CMA）-DAPI荧光染色，以阐明CPD带纹区域中相关序列的特性。我们的结果表明，通过CPD染色，测试物种中的所有核仁组织区（rDNA位点）均有效地显示为红色带，且带的数量和位置与45S rDNA FISH信号的数量和位置精确对应，这表明CPD染色的检测灵敏度与FISH相似。在测试的10个物种中，包括粗山羊草、大蒜、籼稻、药用野生稻、豌豆、黑麦、粟、高粱、蚕豆和玉米，CPD带仅在其核仁组织区出现，而在其他6个物种中，包括大麦、洋葱、四棱豆、拟南芥、甘蓝型油菜和番茄，CPD带出现在其核仁组织区以外的染色体区域。在所有测试物种中，CPD带与CMA带一致，表明CPD带中富含鸟嘌呤和胞嘧啶的序列，且改进的CPD染色程序对植物基因组中富含GC的区域具有特异性。我们的研究不仅阐明了CPD的带纹机制，还证明了CPD染色技术可能比CMA染色更具优势，是检测植物核仁组织区和其他富含GC的染色体区域的有效工具。
