Established Of GISH System For Hybrid Between Saccharum.L×E. Arundinaceus And Analysis The F₁Chromosome Composition

Development and utilization of Erianthus arundinaceum, a closely related genus of Saccharum is one of the most important practices for the germplasm innovation of sugarcane. Genomic in situ hybridization (GISH) is a useful method to identify hybrid and analyse the composition of chromosome. In this study some work was done to modify the key stage of GISH, in order to establish a GISH system for hybrid between Saccharum.L and E. arundinaceus,and analyse the chromosome composition of F1. The results were showed as follows:(1) Optimization technique of chromosome slides:The key factors for a good slide such as material selected, pretreatment and lysozyme were studied and optimized. The result of optimization as flowing:Dark, humidity and temperature fluctuation were kept for the growth of sugarcane roots. Root tips were cut when the root length reached1-2cm, and pretreatment with p-dichlorobenzene solution for2-3h. The cell wall of the root tip was removed by1.75%pectinaseand3.5cellulase for3-5h.(2) Tetechnique against chromosome missing:The new slide were cleared with bichromic acid then covered with Poly-L-ycine, baking the slide at37℃more than12h can less the chromosome missing.(3) Preparation of Probe DNA and blockade DNA:Probe DNA was prepare through nick translation method. React about1.5-2.0h can get the fragments of200bp～500bp.Blockade DNA were be preparre by boiling the genetic DNA about30min.(4) Optimization technique of hybridzation and detection:The time for pepsin trement was10min, and this step can be omitted if the chromosome slide prepare in short time and coverless.The hybridzation solution for one slide was50μl, and the Probe was3ng/μl. About20times of the proportion for Blockade DNA:Probe DNA can get the special hybrid signal. Degeneration respectively was adopted,90℃,10min was good for hybridzation solution degeneration. And chromosome on slide was Degenerated in70%formamide deionized in the condition of90℃and10min.(5) Analysis the chromosome composition of F1(Yc96-66):The GISH result of Yc96-66(E. arundinaceus×S. officinarum) show that YC96-66consist of30chromosomes from E. arundinaceus and40from S. officinarum. The chromosome transmit by the pattern of n+n.