关键词: 普通小麦, 端体, 细胞周期同步化, 流式细胞术, 染色体分选

Abstract: 【Objective】The study aims to optimize chromosome sorting and construction of chromosome-specific library to simplify the wheat global genome research.【Method】Using double-block method to synchronize root tip meristem cells of common wheat ditelosomic line 5DL, the chromosomes were released for flow karyotypic analysis after fixed in 2% polyformaldehyde in Tris-HCl buffer followed by mechanical homogenization. Flow sorting was performed on chromosomes from the putative single peak and composite peaks. PCR method detected flow sorted chromosome 5DL and chromosomes from composite peaks were subjected to 1 U BamHI digestion. In the ligation reaction, the ratio, 10:1 (high-molecular-weight (HMW) DNA:BIBAC vector, w/w) was applied and then 1:20 (ligation:electro-competent cell, v/v) performed in electroporation. Random 100 recombinators were selected for full digestion with NotI to determine the insert sizes.【Result】The mitotic index can reach over 60% after the following treatment with 1.25 mmol•L-1 hydroxyurea for 18 h, 6 h in hydroxyurea (HU)-free Hoagland’s solution for recovery and 2.5-h 1 µmol•L-1 trifluralin. Based on fluorescence microscope and PCR detection, histograms of fluorescence intensity acquired after the analysis of DAPI-stained common wheat ditelosomic line 5DL chromosomes comprised three composite peaks and two single peaks representing chromosome 5DL and 3B, respectively. Partial enzyme digestion of high-molecular-weight (HMW) DNA demonstrated that 50～300 kb fragments can be acquired with an average insert size of 100 kb or so.【Conclusion】The study presents a useful method in constructing chromosome- and chromosome arm-specific library.

Key words: Common wheat ditelosomic line, Cell cycle synchronization, Flow cytometry, Chromosome sorting