Chinese Bulletin of Botany ›› 2023, Vol. 58 ›› Issue (3): 433-439.DOI: 10.11983/CBB22055

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Double-stranded Labelled Oligo-FISH in Rice Chromosomes

Shang Sun1, Yingying Hu1, Yangshuo Han2, Chao Xue2, Zhiyun Gong1,2()   

  1. 1Jiangsu Key Laboratory of Crop Genetics and Physiology/Key Laboratory of Plant Functional Genomics of the Ministry of Education/Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Agricultural College of Yangzhou University, Yangzhou 225009, China
    2Jiangsu Co-innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China
  • Received:2022-03-24 Accepted:2022-08-04 Online:2023-05-01 Published:2023-05-17
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Abstract: The technique of identification and preparation of chromosome(s) are important tools in genetic research. Oligonucleotide fluorescence in situ hybridization (oligo-FISH) is an emerging chromosome identification technique in recent years. Flexible and efficient probes are the key factors in the process of oligo-FISH. The labelling process of traditional single-stranded oligo probes (ss-oligos) is complicated and the cost of obtaining individual probes is high. By improving the ss-oligo probes labelling process, we obtain the probes by PCR amplification with specific primers targeting the whole chromosome (fragment), which simplifies the probe labelling process, reduces the cost and improves the labelling efficiency. In this study, we describe in detail the synthesis and labelling of a modified double-stranded labelled oligo probes (ds-oligos) library in rice (Oryza sativa), the preparation of mitotic chromosomes and the hybridization process of ds-oligo probes. By designing gradient experiments, the optimal denaturation time and temperature of chromosome and oligo probe in rice were found to be 85°C for 3.5 min and 90°C for 6 min, respectively. This is the first study to establish a chromosomal double-stranded labelled oligo-FISH system in rice, which provides a powerful tool for the preparation and precise identification of chromosomes in a variety of plants.

Key words: rice, oligo-FISH, mitotic