Double-stranded Labelled Oligo-FISH in Rice Chromosomes

doi:10.11983/CBB22055

Abstract

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

Shang Sun, Yingying Hu, Yangshuo Han, Chao Xue, Zhiyun Gong. Double-stranded Labelled Oligo-FISH in Rice Chromosomes[J]. Chinese Bulletin of Botany, 2023, 58(3): 433-439.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22055

https://www.chinbullbotany.com/EN/Y2023/V58/I3/433

Figures/Tables 3

Figure 1 Establishment of double-stranded oligo-painting probes based on multiplex PCR in rice The blue rectangles are chromosomes; the small yellow triangles are centromeres; the red rectangles represent TAMRA-labelled chromosome 8 oligo signals; the green rectangles represent FAM-labelled chromosome 10 oligo signals. Specific forward and reverse primers for the amplification of different chromosomal segments represented by F and R, respectively. The dashed line represents the selected specific oligo set with connectors at both ends.

Figure 2 Exploring the denaturation time of rice chromosomes in double-stranded oligo-FISH (A)-(C) During the experiment, the effects of oligo signals labelled by TAMRA on chromosome 8 while the denaturation time at 1 min 30 s, 2 min 30 s and 3 min 30 s, respectively. (D)-(F) During the experiment, the effects of oligo signals labelled by FAM on chromosome 10 while the denaturation time at 1 min 30 s, 2 min 30 s and 3 min 30 s, respectively. The white arrow points to the painting-chromosomes after magnification. Chromosomes were counterstained with DAPI. Bars=5 μm

Figure 3 Double-stranded labelled oligo-FISH based on chromosome painting targets the mitosis of chromosome 8 and 10 analysis of Nipponbare (A) Oligo-painting analysis on mitotic interphase chromoso-me 8 of Nipponbare; (B) Oligo-painting analysis on mitotic interphase chromosome 10 of Nipponbare; (C) Oligo-painting analysis on mitotic prometaphase chromosome 8 of Nippon-bare; (D) Oligo-painting analysis on mitotic prometaphase chromosome 10 of Nipponbare. Red: TAMRA-labelled probe signals of whole chromosome 8; Green: FAM-labelled probe signals of whole chromosome 10. Chromosomes were coun-terstained with DAPI. Bars=5 μm

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