关键词:

小麦染色体, 流式分选, PCR, 原位PCR, 荧光原位杂交

Abstract:

Construction of chromosome specific BAC library plays an important role for simplifying sequencing, physical mapping and gene cloning of plant with complexity genome such as common wheat, identification of sorted chromosome is a vital step of library construction. Although there were some reports about identification of sorted chromosomes using different methods, the sorted chromosomes were different normal chromosomes and the applicability and feature of various identification methods had also not been commented by the numbers yet. Based on previous research, the identifications of 6VS, 3B and 7BL chromosomes (arms) sorted from ditelosomic and common wheat were performed through fluorescence in situ hybridization (FISH), primed in situ DNA labeling (C-PRINS) and PCR amplification methods respectively. The results showed that all of these three methods could efficiently identify the flow sorted chromosomes. The chromosome staining before flow sorting and chromosome damage from physical shear force during chromosome suspension preparation and flow sorting did not impact obviously the results of identification. After comparing to these three protocols, the PCR approach was the fastest with better repetition which adapted to determine rapidly constitute of chromosomal peaks on the univariate flow karyotype histogram, but there were no visible signals and the purity of sorted chromosomes could not be determined were the disadvantages of this approach. The FISH approach could provide a visible and repetitive result and was suit for identifying purity of the sorted chromosomes, but it was time-consuming, complex and necessary for special probes. C-PRINS combined the advantages of FISH and PCR, had potential for chromosomes identification, although the hybridization signals was instable and repetition was not so good at present, if combined this method with in situ hybridization in suspension, a new way for chromosome flow sorting might be set up. The features of these three methods, some key points during the identification process and their applicability also were discussed.

Key words:

Wheat chromosome, Flow sorting, PCR, C-PRINS, FISH