小麦流式分选染色体的鉴定

Identification of Wheat Chromosomes Sorted by Flow Cytometry

构建染色体BAC文库对于简化拥有庞大基因组植物的测序、物理作图和基因克隆具有重要意义。分选染色体的鉴定是整个文库构建过程的关键环节之一。本文在前人研究的基础上,分别采用荧光原位杂交(FISH)、原位PCR(C-PRINS)和PCR方法,对分选的6VS、3B和7BL染色体(臂)进行了鉴定。结果表明,这3种方法都能对流式分选染色体进行有效鉴定,其中PCR法速度最快、重复性好,但结果不具可视性、也不能鉴定分选纯度;而FISH法重复性好、结果具有可视性、能够鉴定分选纯度,但操作程序复杂耗时,受探针特异性的限制;C-PRINS法则综合了上述两种方法的优点,是最具潜力的鉴定方法,如果与液体原位杂交相结合,有可能为解决染色体分选问题开辟新的途径,但也存在重复性差、杂交信号不稳定的缺点。

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 chromo-somes （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 re- sults 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 visi- ble 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.