基于流式细胞仪鉴定马铃薯倍性的高通量样品制备方法

doi:10.13560/j.cnki.biotech.bull.1985.2024-0611

摘要/Abstract

摘要：

【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。

关键词: 流式细胞术, 马铃薯, 倍性鉴定, 低温钢珠打样法

Abstract:

【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.

Key words: flow cytometry, potato, ploidy identification, low-temperature steel-bullet beating method

袁兰, 黄娅楠, 张贝妮, 熊雨萌, 王洪洋. 基于流式细胞仪鉴定马铃薯倍性的高通量样品制备方法[J]. 生物技术通报, 2024, 40(9): 141-147.

YUAN Lan, HUANG Ya-nan, ZHANG Bei-ni, XIONG Yu-meng, WANG Hong-yang. High-throughput Sample Preparation Method for the Identification of Potato Ploidy Using Flow Cytometry[J]. Biotechnology Bulletin, 2024, 40(9): 141-147.

图/表 5

表1 500 mL裂解液的配制

Table 1 Preparation of 500 mL lysis buffer

样品Sample	浓度Concentration	分子量 Molecular weight	添加量 Additive amount
MgCl₂·6H₂O	0.045 mol/L	203.3	4.574 g
Na₃C₆H₅O₇·2H₂O	0.030 mol/L	294.1	4.411 g
C₇H₁₅NO₄S	0.020 mol/L	209.27	2.093 g
Triton X-100	0.1%（体积比）		0.5 mL

图1 不同制样方法得到的细胞核悬液中细胞核所占的比例 A：低温钢珠打样的密度图；B：液氮研磨法的密度图；C：刀片切碎法的密度图；D：细胞核占比图；采用t检验进行差异显著性分析；****表示P<0.001；n=30

Fig. 1 Proportion of nuclei in nuclear suspension obtained by different sample preparation methods A: Density map of low temperature steel-bullet beating. B: Density map of liquid nitrogen grinding method. C: Density map of blade cutting method. D: Proportion map of nucleus. The significance of the difference was analyzed by t test. **** indicates P<0.001; n =30

图2 不同制样方法得到的细胞核悬液中细胞核DNA含量的直方图 A：低温钢珠打样法；B：液氮研磨法；C：刀片切碎法；D：3种制样法M4峰值；E：3种制样法M6峰值；F：3种制样法M4M6峰值之间比值；采用t检验进行差异显著性分析；ns表示统计学上无显著性差异；n=30

Fig. 2 Histograms of nuclear DNA content in nuclear suspension obtained by different sample preparation methods A: Low temperature steel-bullet beating method. B: Liquid nitrogen grinding method. C: Blade chopping method. D: M4 peak values of three sample preparation methods. E: M6 peak values of three sample preparation methods. F: M4M6 peak value between three sample preparation methods. The significance of the difference was analyzed by t test. ns indicates no statistically significant difference. n=30

表2 三种制样法制备样品时长的比较

Table 2 Comparison of sample preparation time of three sample preparation methods

制样方法 Sample preparation method	1个样品耗时 Time taken for 1 sample/min	30个样品耗时Time taken for 30 samples/min
低温钢珠打样法Low-temperature steel-bullet beating method	0.5-1	1-2
液氮研磨法 Liquid nitrogen grinding method	2-3	60-90
刀片切碎法 Blade shredding method	6-7	180-210

图3 四倍体马铃薯HTJ349-3和二倍体马铃薯IVP101密度图和直方图 A：二倍体IVP101的密度图；B：四倍体HTJ349-3的密度图；C：二倍体IVP101的直方图；D：四倍体HTJ349-3的直方图

Fig. 3 Density and histogram of tetraploid potato HTJ349-3 and diploid potato IVP101 A: Density map of diploid IVP101. B: Density map of tetraploid HTJ349-3. C: Histogram of diploid IVP101. D: Histogram of tetraploid HTJ349-3

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