Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (5): 103-111.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1085

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Enhancing Hybridization Signal of Sugarcane Chromosome Oligonucleotide Probe via Multiple Fluorescence Labeled Primers

LI Xin-yi1,3(), JIANG Chun-xiu1,3, XUE Li1,3, JIANG Hong-tao1,3, YAO Wei1,3, DENG Zu-hu1,2,3, ZHANG Mu-qing1,3, YU Fan1,3()   

  1. 1. State Key Laboratory for Protection and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004
    2. National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou 350002
    3. Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning 530004
  • Received:2022-09-02 Online:2023-05-26 Published:2023-06-08
  • Contact: YU Fan E-mail:lixinyi1776@163.com;yufanky@163.com

Abstract:

Fluorescence in situ hybridization(FISH)is one of the most important methods in plant molecular cytogenetics. In recent years, low copy oligonucleotide probes based on reference genome design have been widely used in FISH. However, the resolution of oligo-FISH is limited due to the large number of repeats distributed in plant genomes. The sugarcane(Sacchanum spp.)chromosome specific oligo probes were amplified by using fluorescent PCR primers containing multiple fluorophore groups, and the sugarcane fluorescence in situ hybridization system was further optimized to improve the efficiency of sugarcane oligo probes in recognizing chromosomes of related species. By developing a new method of multi-fluorescent labeled oligo probes in sugarcane and optimizing the sugarcane fluorescence hybridization system, the minimum resolution of fluorescence signal was effectively broadened,the signal-to-noise ratio(SNR)increased, and sugarcane oligo probes were used to successfully identify sorghum chromosomes 1-10. The new method of multi-fluorescent labeled primers enhanced the signal of oligo probe and the optimization of FISH system provide a reference for improving oligo-FISH of identifying chromosomes and capturing weak fluorescence signals in other species in the future.

Key words: sugarcane, fluorescence in situ hybridization, oligonucleotide probes, chromosome identification, oligo-FISH