基于改进的多色荧光原位杂交技术的染色体分析

doi:10.11924/j.issn.1000-6850.casb20191000778

中国农学通报 ›› 2020, Vol. 36 ›› Issue (12): 97-103.doi: 10.11924/j.issn.1000-6850.casb20191000778

所属专题：生物技术

基于改进的多色荧光原位杂交技术的染色体分析

孔令娜, 冯祎高, 孙冰晓, 张慧, 刘晓雪, 陈颖, 张瑞奇()

南京农业大学植物生产国家级实验教学中心,南京 210095

收稿日期:2019-10-31 修回日期:2019-12-13 出版日期:2020-04-25 发布日期:2020-04-21
通讯作者: 张瑞奇
作者简介:孔令娜,女,1979年出生,江苏江都人,高级实验师,博士,研究方向：作物分子细胞遗传。通信地址：210095 江苏南京童卫路6号南京农业大学植物生产国家级实验教学中心,Tel：025-84395800,E-mail： klnjj@njau.edu.cn
基金资助:
国家自然科学基金项目“簇毛麦5VS上不同类型抗小麦白粉病基因Pm55和Pm5V的精细定位和聚合”(31971938);中央高校基本科研业务费“提高荧光显微镜对多种荧光标记的检测能力及其实验技术研究”(KJSY201612);南京农业大学植物生产国家级实验教学中心开放性项目“Mc-FISH技术的改进及在小麦染色体工程中的应用”(ZKF201716)

Chromosome Analysis Based on an Improved Multiplex Fluorescence in Situ Hybridization (M-FISH)

Kong Lingna, Feng Yigao, Sun Bingxiao, Zhang Hui, Liu Xiaoxue, Chen Ying, Zhang Ruiqi()

National Experimental Teaching Center of Plant Production, Nanjing Agricultural University, Nanjing 210095

Received:2019-10-31 Revised:2019-12-13 Online:2020-04-25 Published:2020-04-21
Contact: Ruiqi Zhang

摘要/Abstract

摘要：

为了提高多色荧光原位杂交(M-FISH)技术的鉴定效率,简化M-FISH操作程序,探索和建立新的M-FISH实验及观察体系,本研究以‘荆辉1号’（普通小麦辉县红-荆州黑麦双二倍体）、‘南农9918’（普通小麦-簇毛麦整臂易位系T6VS.6AL）、‘扬麦22’（普通小麦-簇毛麦整臂易位系T6VS.6DL）以及‘ST5V#4S-1’（普通小麦-簇毛麦小片段易位系）等为试验材料,以2个寡核苷酸探针[(GAA)₁₀、pAs1-1]和物种基因组DNA探针作混合探针进行M-FISH分析。结果表明在一次原位杂交中同时使用3种探针,既可以识别出小麦背景中所有的外源染色体或染色体片段,又可以根据(GAA)₁₀和pAs1-1在染色体上的杂交信号分布精确鉴定出小麦和外源染色体（片段）的具体身份。这种改进的M-FISH技术可有效用于小麦及其近缘植物染色体的鉴定,并有助于快速构建物种基于FISH的分子核型。

关键词: 多色荧光原位杂交, 混合探针, 普通小麦, 近缘植物, 外源染色体（片段）

Abstract:

The aims are to improve the identification efficiency of multicolor fluorescence in situ hybridization (M-FISH), and simplify the operation procedure of M-FISH, develop and establish a new M-FISH test and observation system. In this present study, two synthesized oligonucleotide probes ((GAA)₁₀, pAs1-1) and genomic DNA probe were used as mixed probes to test four alien chromosome lines, including the amphiploid ‘Jinghui1’, the whole arm translocation lines ‘NAU9918’ and ‘Yangmai 22’ and the small fragment translocation line ‘ST5V#4S-1’. The results showed that three probes used in one in situ hybridization could not only recognize all the exogenous chromosomes or chromosome segments in the background of common wheat, but also identify the real identifications of wheat and exogenous chromosomes (segments) according to the signal distribution of (GAA)₁₀ and pAs1-1 in chromosome. Therefore, the improved M-FISH can be used to identify the chromosomes or chromosome segments of wheat and its wild relatives efficiently, and to establish the karyotype based on FISH rapidly.

Key words: multicolor fluorescence in situ hybridization (M-FISH), mixed DNA probe, common wheat, wild relatives, exogenous chromosomes (segments)

中图分类号:

S512

孔令娜, 冯祎高, 孙冰晓, 张慧, 刘晓雪, 陈颖, 张瑞奇. 基于改进的多色荧光原位杂交技术的染色体分析[J]. 中国农学通报, 2020, 36(12): 97-103.

Kong Lingna, Feng Yigao, Sun Bingxiao, Zhang Hui, Liu Xiaoxue, Chen Ying, Zhang Ruiqi. Chromosome Analysis Based on an Improved Multiplex Fluorescence in Situ Hybridization (M-FISH)[J]. Chinese Agricultural Science Bulletin, 2020, 36(12): 97-103.

图/表 3

图1. 普通小麦辉县红-荆州黑麦双二倍体‘荆辉1号’根尖细胞有丝分裂中期染色体的M-FISH分析 A：DAPI-蓝色、(GAA)10-绿色,pAs1-1-红色和Cy5-黄色各单色荧光FISH图;B：4种荧光叠加合成的多色荧光FISH图箭头所指为黑麦染色体,呈现Cy5黄色荧光信号。

图2. 小麦-簇毛麦6VS整臂易位系‘南农9918’和‘扬麦22’根尖细胞有丝分裂中期染色体的M-FISH分析 A、B：‘南农9918’有丝分裂中期染色体M-FISH分析;C、D：‘扬麦22’有丝分裂中期染色体M-FISH分析。A、C：DAPI-蓝色、(GAA)10-绿色、pAs1-1-红色和Cy5-黄色各单色荧光FISH图;B、D：4种荧光叠加合成的多色荧光FISH图。箭头所指为小麦-簇毛麦6VS整臂易位染色体,6VS呈现Cy5黄色荧光信号

图3. 小麦-簇毛麦小片段易位系‘ST5V#4S-1’根尖细胞有丝分裂中期染色体的M-FISH分析 A：DAPI-蓝色、(GAA)10-绿色、pAs1-1-红色和Cy5-黄色各单色荧光FISH图;B：4种荧光叠加合成的多色荧光FISH图。箭头所指为小麦-簇毛麦5VS小片段易位染色体T6AL.6AS-5V#4S,5VS呈现Cy5黄色荧光信号

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基于改进的多色荧光原位杂交技术的染色体分析

Chromosome Analysis Based on an Improved Multiplex Fluorescence in Situ Hybridization (M-FISH)

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