植物寡核苷酸荧光原位杂交技术方法

doi:10.11983/CBB22265

植物学报 ›› 2023, Vol. 58 ›› Issue (2): 274-284.DOI: 10.11983/CBB22265

植物寡核苷酸荧光原位杂交技术方法

张凡凡¹, 邢新滢¹, 石文清², 沈懿², 程祝宽²^,³^,^*()

¹北京师范大学生命科学学院, 细胞增殖与调控生物学教育部重点实验室, 北京 100875
²中国科学院遗传与发育生物学研究所, 植物基因组学国家重点实验室及植物基因研究中心, 北京 100101
³扬州大学农学院, 江苏省作物基因组学与分子育种重点实验室/植物功能基因组学教育部重点实验室, 江苏省粮食作物现代生产技术协同创新中心, 扬州 225009

收稿日期:2022-11-20 接受日期:2023-01-10 出版日期:2023-03-01 发布日期:2023-03-15
通讯作者: ^*E-mail: zkcheng@genetics.ac.cn
基金资助:
北京市自然科学基金(6214043);国家自然科学基金(32001522);国家自然科学基金(32000368);中央高校基本科研业务费专项资金(2021NTST20)

Protocals for Oligonucleotide Fluorescence in situ Hybridization in Plants

Fanfan Zhang¹, Xinying Xing¹, Wenqing Shi², Yi Shen², Zhukuan Cheng²^,³^,^*()

¹Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
²State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
³Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding/Key Laboratory of Plant Functional Genomics of the Ministry of Education, College of Agriculture, Yangzhou University, Yangzhou 225009, China

Received:2022-11-20 Accepted:2023-01-10 Online:2023-03-01 Published:2023-03-15
Contact: ^*E-mail: zkcheng@genetics.ac.cn

摘要/Abstract

摘要：

寡核苷酸荧光原位杂交技术是一种整合了生物信息学分析、DNA高通量合成以及荧光原位杂交实验的新兴技术。该技术适用于任何具有参考基因组的植物物种, 并可根据研究需求设计靶向某个染色体区域、整条染色体或一组染色体的寡核苷酸探针, 目前已成功应用于数种植物的核型分析、染色体变异、种群演化、同源染色体配对、单倍型分析和异源多倍体识别等研究。该文详述了植物寡核苷酸探针的设计、扩增和标记、染色体制备以及荧光原位杂交的具体操作流程, 以期促进寡核苷酸荧光原位杂交技术在细胞遗传学研究中的应用。

关键词: 荧光原位杂交, 寡核苷酸探针, 染色体, 植物

Abstract:

Oligonucleotide fluorescence in situ hybridization (Oligo-FISH) is a new technology that integrates bioinformatics analysis, high throughput DNA synthesis and fluorescence in situ hybridization experiments. It is applicable to plant species with a reference genome. Oligo probes are able design to a chromosomal region, an entire chromosome, and a set of chromosomes according to researchers’ requirements. Oligo-FISH has been successfully applied to studies on karyotyping, chromosome variation, population evolution, homologous chromosome pairing, haplotype analysis, and heteropolyploid identification in several plant species. Here, we introduce the concrete operational processes of oligonucleotide-based probe design, amplification and labeling, chromosomes preparation, and fluorescence in situ hybridization in plants, so as to facilitate Oligo-FISH application in the cytogenetics research in the future.

Key words: fluorescence in situ hybridization, oligonucleotide probe, chromosome, plant

张凡凡, 邢新滢, 石文清, 沈懿, 程祝宽. 植物寡核苷酸荧光原位杂交技术方法. 植物学报, 2023, 58(2): 274-284.

Fanfan Zhang, Xinying Xing, Wenqing Shi, Yi Shen, Zhukuan Cheng. Protocals for Oligonucleotide Fluorescence in situ Hybridization in Plants. Chinese Bulletin of Botany, 2023, 58(2): 274-284.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22265

https://www.chinbullbotany.com/CN/Y2023/V58/I2/274

图/表 2

图1 寡核苷酸荧光原位杂交实验流程图 (A) 寡核苷酸探针的扩增和标记; (B) 荧光原位杂交(FISH)实验

Figure 1 Outlines of oligonucleotide fluorescence in situ hybridization (A) Amplification and labeling of oligonucleotide-based probes; (B) Procedures of fluorescence in situ hybridization (FISH)

图2 水稻花粉母细胞减数分裂粗线期染色体的寡核苷酸荧光原位杂交鉴定 (A) 粗线期染色体; (B) 特异性靶向水稻11号染色体短臂的寡核苷酸探针(11 S)信号; (C) 特异性靶向水稻11号染色体长臂的寡核苷酸探针(11 L)信号; (D) 三通道合并图(蓝色为染色体, 绿色为11 S, 红色为11 L)。Bars=5 μm

Figure 2 Characterization of rice meiotic pachytene chromosomes by oligonucleotide fluorescence in situ hybridization (A) Pachytene chromosomes; (B) The signal of oligonucleotide probe specific to the short arm of chromosome 11 in rice (11 S); (C) The signal of oligonucleotide probe specific to the long arm of chromosome 11 in rice (11 L); (D) Three-channel combined panel (Blue: Chromosomes; Green: 11 S; Red: 11 L). Bars=5 μm

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植物寡核苷酸荧光原位杂交技术方法

Protocals for Oligonucleotide Fluorescence in situ Hybridization in Plants

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