基于液氮研磨法的流式细胞术检测马铃薯倍性的研究

染色体组倍性鉴定是马铃薯种质资源评价的重要内容,流式细胞仪能够快速、准确地对细胞核DNA含量进行测定,从而广泛用于检测植物染色体组倍性。建立适于马铃薯倍性鉴定的高通量流式细胞术体系,对马铃薯育种工作提供依据。以20份马铃薯合作88孤雌诱导后代为材料,用液氮研磨法制备叶片细胞核悬液,并将其与传统刀片切碎法制备的细胞核悬液进行比较,对已知四倍体马铃薯合作88和二倍体马铃薯IVP101进行染色体倍性测定,结果发现这两种方法在倍性测定结果之间无明显差异,但是液氮研磨法操作简单、耗时少。基于液氮研磨法的流式细胞术可快速、准确检测其倍性。另外,在液氮研磨法中,对细胞核悬液染色时间的长短(从15 min到12 h)并不会影响倍性测定结果,从而方便研究人员在实际操作中灵活选择染色时间。

Study on the Identification of Potato Ploidy Using Flow Cytometry Based on Liquid Nitrogen Grinding Method

The ploidy identification of potato chromosomes is an important part in the evaluation of potato germplasm resources.Flow cytometry can quickly and accurately determine the nuclear DNA content of cells,which is widely used to identify plant’s chromosome ploidy.A high-throughput flow cytometry system that is suitable for potato ploidy identification is established for providing evidences for potato breeding.Twenty pieces of potato cooperative 88,progeny of an induced parthenogenesis,were used as materials,a liquid nitrogen grinding method was used to prepare leaf cell nuclear suspension,which was then compared with the leaf cell nuclear suspension by traditional blade shredding method,and chromosome ploidy of the known tetraploid potato cooperative 88 and diploid potato IVP101 was determined.It was found that there was no significant difference between the ploidy identification results by the two methods,but the liquid nitrogen grinding method was simple and less time-consuming.The flow cytometry based on the liquid nitrogen grinding method quickly and accurately identified potato ploidy.In addition,fluorescent staining time(from 15 min to 12 h)of the nucleus suspension prepared by the liquid nitrogen grinding method did not affect the ploidy determination results,which is convenient for researchers to flexibly choose the staining time in actual operation.