基于流式细胞仪鉴定马铃薯倍性的高通量样品制备方法

High-throughput Sample Preparation Method for the Identification of Potato Ploidy Using Flow Cytometry

【目的】染色体倍性鉴定是马铃薯种质资源评价的重要内容。建立马铃薯染色体倍性的高通量样品制备方法,可为后续大规模开展马铃薯倍性鉴定工作奠定基础。【方法】以30份马铃薯孤雌生殖诱导后代作为实验材料,比较使用低温钢珠打样法、液氮研磨法和刀片切碎法制备细胞核悬液的效果,并对已知二倍体马铃薯IVP101和四倍体马铃薯HTJ349-3进行倍性鉴定。【结果】低温钢珠打样法制备的细胞核悬液,荧光信号明显,细胞裂解充分,杂质少。同时,由于低温钢珠打法简单易操作,大大缩短了样品制备时间,其鉴定效率比起液氮研磨法提高45-60倍,比刀片切碎法提高105-180倍。利用该方法可以准确检测出马铃薯IVP101和HTJ349-3的染色体倍性。【结论】基于流式细胞仪的低温钢珠打样法,其检测结果同液氮研磨法、刀片切碎法制样方法一样准确,可实现高通量鉴定马铃薯染色体倍性。

【Objective】Chromosome ploidy identification is an important part of the evaluation of potato germplasm resources.The establishment of a high-throughput sample preparation method for the identification of potato chromosome ploidy based on flow cytometry would lay a foundation for the subsequent large-scale identification of potato ploidy.【Method】The 30 parthenogenetic offspring of potato were used to compare the effects of low-temperature steel-bullet beating,liquid nitrogen grinding and blade shredding to prepare nuclear suspensions.The ploidy of diploid potato IVP101 and tetraploid potato HTJ349-3 was identified.【Result】The suspension of cell nuclei prepared by the low-temperature steel-bullet beating method had obvious fluorescence signal,sufficient cell lysis and few impurities.Due to the simple and easy operation of the low-temperature steel-bullet beating method,the sample preparation time was greatly shortened,and the identification efficiency was 45-60 times higher than that of the liquid nitrogen grinding method,and 105-180 times higher than that of the blade shredding method.The chromosome ploidy of potato IVP101 and HTJ349-3 was accurately detected by this low-temperature steel-bullet beating method.【Conclusion】The results by the low-temperature steel-bullet beating method are as accurate as that by the liquid nitrogen grinding method and the blade cutting method,which can achieve high-throughput identification of potato chromosome ploidy.