流式细胞术的优化及其在甘薯倍性鉴定中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2026-0247

摘要/Abstract

摘要：

目的染色体倍性鉴定是甘薯种质资源评价过程中的重要环节，为了克服传统方法在甘薯取材中的局限性，优化流式细胞术在甘薯倍性鉴定中的应用，建立一种高效的倍性鉴定方法，为后续不同倍性甘薯材料大规模鉴定提供基础。方法以‘徐紫薯8号’为试材，系统比较液氮研磨叶片法与刀片切碎根尖法在细胞核悬液制备中的效果，重点评估两种方法在细胞核得率、样品制备耗时及流式检测图谱质量方面的差异；同时，针对珍贵材料取材难的问题，通过调整裂解液中MgSO₄‧7H₂O、DTT及Triton X-100的浓度，探索利用甘薯成熟展开叶进行倍性鉴定的可行性，并利用不同倍性材料对液氮研磨叶片法进行了单独及混合检测验证。结果液氮研磨叶片法在保证检测精度的同时，显著提升制备效率，单个样品在破碎环节和上机检测环节的耗时均明显缩短。在成熟叶鉴定中，将裂解液中DTT和Triton X-100浓度加倍后，有效细胞核释放量显著增加，成功克服成熟叶难以检测的问题。该方法在二倍体、四倍体及六倍体材料的单独及混合检测中均表现出优异的区分度，荧光强度比值与理论倍性比（1∶2∶3）高度吻合，验证了其稳定性和普适性。结论基于液氮研磨法改进后的流式细胞术在甘薯倍性鉴定中，不受根尖等取材限制，可以快速、准确检测倍性和筛选材料。

关键词: 甘薯, 流式细胞术, 倍性鉴定, 液氮研磨叶片法

Abstract:

Objective Chromosome ploidy identification is an important step in evaluating sweetpotato germplasm resources. To overcome the limitations of traditional methods in sweetpotato sampling, this study optimized the application of flow cytometry for ploidy identification, established an efficient method, and provided a foundation for subsequent large-scale identification of sweetpotato materials with different ploidy levels. Method Using ‘Xuzishu 8’ as the experimental material, this study systematically compared the effects of the liquid nitrogen-ground leaf method and the blade-chopped root tip method in preparing cell nucleus suspensions. The comparison focused on nucleus yield, sample preparation time, and the quality of flow cytometry profiles. To address the difficulty of sampling precious materials, we explored the feasibility of ploidy identification using fully expanded mature sweetpotato leaves by adjusting the concentrations of MgSO₄·7H₂O, DTT, and Triton X-100 in the lysis buffer. The method was validated by individual and mixed detection of materials with different ploidy levels. Result The liquid nitrogen-ground leaf method significantly improved preparation efficiency while maintaining detection accuracy, and both the crushing step and the on-machine detection time per sample were markedly reduced. For mature leaf identification, doubling the concentrations of DTT and Triton X-100 in the lysis buffer remarkably increased the release of intact nuclei, successfully overcoming the difficulty of detecting mature leaves. This method showed excellent resolution in both individual and mixed detection of diploid, tetraploid, and hexaploid materials, and the observed fluorescence intensity ratio was highly consistent with the theoretical ploidy ratio (1:2:3), confirming its stability and universality. Conclusion For sweetpotato ploidy identification, the flow cytometry method improved by the liquid nitrogen-ground leaf approach is not restricted by sampling limitations such as root tip dependence, and can rapidly and accurately determine the ploidy of sweetpotato materials and facilitate material screening.

Key words: sweetpotato, flow cytometry, ploidy identification, liquid nitrogen-ground leaf method

李建功, 邓逸桐, 赵路宽, 王珧, 曹清河. 流式细胞术的优化及其在甘薯倍性鉴定中的应用[J]. 生物技术通报, 2026, 42(6): 77-86.

LI Jian-gong, DENG Yi-tong, ZHAO Lu-kuan, WANG Yao, CAO Qing-he. Optimization of Flow Cytometry and Its Application in Ploidy Identification of Sweetpotato[J]. Biotechnology Bulletin, 2026, 42(6): 77-86.

图/表 7

图1 两种制样方法得到的细胞核悬液中细胞核所占比例A：刀片切碎根尖法散点图；B：液氮研磨叶片法散点图；C：细胞核占比；采用t检验进行差异显著性分析；**表示P0.01；n=9

Fig. 1 Proportion of cell nuclei in the nuclear suspension obtained by the two sample preparation methodsA: Scatter plot of the blade-chopped root tip method. B: Scatter plot of the liquid nitrogen-ground leaf method. C: Proportion of cell nuclei. A t-test was used to analyze significant differences; ** indicates P0.01; n=9

图2 两种制样方法得到的细胞核悬液中细胞核DNA含量的直方图A：刀片切碎根尖法的直方图；B：液氮研磨叶片法的直方图；C：两种方法G1荧光强度对比；D：两种方法G1峰荧光强度；E：两种方法G2峰荧光强度；F：两种方法 G2/G1峰比值；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=9

Fig. 2 Histograms of nuclear DNA content in nuclear suspension obtained by the two sample preparation methodsA: Histogram of the blade-chopped root tip method. B: Histogram of the liquid nitrogen-ground leaf method. C: Comparison of G1 fluorescence intensity between the two methods. D: G1 peak fluorescence intensity of the two methods. E: G2 peak fluorescence intensity of the two methods. F: Ratio of G2/G1 peak of the two methods. A t-test was performed to analyze significant differences; ns indicates no statistically significant difference; n=9

图3 两种方法所需时间的比较A：两种方法样品破碎时间对比；B：两种方法上机时间对比；***表示P0.001；n=9

Fig. 3 Comparison of the time required by the two methodsA: Comparison of sample crushing time for the two methods. B: Comparison of on-machine detection time of the two methods; *** indicates P0.001; n=9

图4 两种裂解液处理效果的比较A：传统裂解液处理的直方图；B：改良裂解液处理的直方图

Fig. 4 Comparison of the effects of two lysis buffersA: Histogram of samples treated with traditional lysis buffer. B: Histogram of samples treated with modified lysis buffer

图5 I. cordatotriloba、 I. batatas（4x）和‘徐菜薯7号’染色体数目、散点图和直方图A： I. cordatotriloba的染色体数目；B：I. cordatotriloba的散点图；C： I. cordatotriloba的直方图；D：I. batatas（4x）的染色体数目；E：I. batatas（4x）的散点图；F：I.batatas（4x）的直方图；G：‘徐菜薯7号’的染色体数目；H：‘徐菜薯7号’的散点图；I：‘徐菜薯7号’的直方图；J：3种不同倍性材料的荧光强度统计图；K：3种不同倍性材料的细胞核聚集区散点图；L ：3种不同倍性材料的细胞核聚集区直方图；标尺为10 μm

Fig. 5 Chromosome number, scatter plots, and histogram of I. cordatotriloba, I. batatas (4x) and ‘Xucaishu 7’A: Chromosome number of I. cordatotriloba. B: Scatter plot of I. cordatotriloba. C: Histogram of I. cordatotriloba. D: Chromosome number of I. batatas (4x). E: Scatter plot of I. batatas (4x). F: Histogram of I. batatas (4x). G: Chromosome number of ‘Xucaishu7’. H: Scatter plot of ‘Xucaishu7’. I: Histogram of ‘Xucaishu7’. J: Statistical graph of fluorescence intensity of three different ploidy materials. K: Scatter plot of nuclear aggregation regions of three different ploidy materials. L: Histogram of nuclear aggregation regions for cells of three different ploidy materials. Scale bar=10 μm

图6 I. cairica、 I. ochracea和‘徐紫薯8号’染色体数目和直方图A： I. cairica的染色体数目；B：I. ochracea的染色体数目；C：‘徐紫薯8号’的染色体数目；D：I. cairica与I. ochracea混合检测直方图；E：I. cairica与‘徐紫薯8号’混合检测直方图；F：倍性比值统计图；标尺为10 μm

Fig. 6 Chromosome numbers and histogram of I. cairica, I. ochracea and ‘Xuzishu 8’A: Chromosome number of I. cairica. B: Chromosome number of I. ochracea. C: Chromosome number of ‘Xuzishu8’. D: Histogram of the mixed detection of I. cairica and I. ochracea. E: Histogram of the mixed detection of I. cairica and ‘Xuzishu8’. F: Statistical graph of ploidy ratio. Scale bar=10 μm

图7 使用裂解液A和B样品的散点图、直方图和显微镜观察A：裂解液A的散点图；B：裂解液A的直方图；C：裂解液A在显微镜下的染色细胞核数目；D：裂解液B的密度图；E：裂解液B的直方图；F：裂解液B在显微镜下的染色细胞核数目；标尺为200 μm

Fig. 7 Scatter plot, histograms, and microscopic observation of samples treated with lysis buffer A and BA: Scatter plot of lysate A. B: Histogram of lysate A. C: Number of stained nuclei of samples treated with lysis buffer A under a microscope. D: Density plot of lysate B. E: Histogram of lysate B. F: Number of stained nuclei of samples treated with lysis buffer B under a microscope. Scale bar=200 μm

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