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| Optimization of Fluorescent in Situ Hybridization System in Hybrid Progeny of Saccharum officinarum and Erianthus arundinaceus |
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Abstract Optimizing an efficient system is the basis of related research by using fluorescent in situ hybridization. In this study, the key factors which effect on fluorescence in situ hybridization results, such as chromosomal pretreatment, denaturing conditions and denaturing ways, were screened in order to optimize a suitable system for fluorescent in situ hybridization in sugarcane(Saccharum). Sugarcane root tips of YC96-66 which is F1 between Erianthus arundinaceus and Saccharum officinarum were pretreated with three following ways, p-dichlorobenzene under the condition of in vitro, α-bromidenaphthalene under the condition of in vitro and 4℃ low temperature water under the condition of non in vitro to screen chromosome morphology. The chromosome was denatured in two different ways with the optimal temperature on simultaneous denaturation at 70, 80, 90 and 98℃ and separate denaturation at 60, 70, 80 and 90℃ to compare the effects of hybridization signal. Results showed that chromosome morphology was stubby when using p-dichlorobenzene under the condition of in vitro which was suitable for sugarcane genomic in situ hybridization study on the count of chromosomal inheritance patterns research. Using the methods of α-bromonaphthalene under the condition of in vitro and 4℃ low temperature water under the condition of non in vitro, chromosome morphology was long and its centromere constriction was more obvious, so they both were suitable for chromosome mapping, karyotype analysis or other research. The effects of hybridization signal were better in the treatment of simultaneous denaturation at 80℃ and separate denaturation at temperature ranged from 70 to 80℃. Base on the screening results, separate denaturation at 80℃ was adopted to optimize an fluorescent in situ hybridization system. The effect of the system was detected using different pretreatment according the different aims. Pretreated with α-bromidenaphthalene, got the effect of longer, scattering chromosome, strong signal of 45S rDNA and high-resolution. Pretreated with p-dichlorobenzene, got the effect of shorter, scattering chromosome and uniform fluorescence signal. This study creates an important platform to following fluorescent in situ hybridization research, provides the technical support for utilization of closely relatives wild germplasm and the basic data for further cytogenetics research in the distant hybridization progeny materials.
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Received: 12 September 2013
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