Identification of Wheat Chromosomes Sorted by Flow Cytometry

Abstract Construction of chromosome-specific BAC library plays an important role for simplifying sequencing, physical mapping, and gene cloning of plant with complex genome such as common wheat ( Triticum aestivum L.), and identification of sorted chromosomes is a vital step of library construction. Based on previous studies, the identification of chromosomes (arms) 6VS, 3B, and 7BL sorted from ditelosomic and normal wheat were performed through fluorescence in situ hybridization (FISH), primed in situ DNA labeling (C-PRINS), and PCR amplification methods, respectively. The results indicated that all these methods could efficiently identify the flow sorted chromosomes. Chromosome staining before flow sorting and chromosome damage from physical shear force during suspension of chromosome preparation and flow sorting did not impact obviously the results of identification. Amongst the 3 methods, PCR is the fastest one with good repetition, and better for rapid determination of the constitution of chromosomal peaks on the univariate flow karyotype histogram, but there are no visible signals hybridized on the sorted chromosomes and the purity of sorted chromosomes cannot be determined in this method. FISH can provide a visible and repetitive result and is suitable for identifying the purity of the sorted chromosomes, but it is time-consuming, complex, and obligatory for special probes. C-PRINS, combining the advantages of FISH and PCR, has the potential for chromosomes identification, although the hybridization signal was not stable enough and its repetition was not satisfactory at present. If combined with in situ hybridization in suspension, C-PRINS is probably a new way for chromosome flow sorting.