杨柳科植物倍性测定不同解离缓冲液效果比对分析

doi:10.7525/j.issn.1673-5102.2016.05.021

植物研究 ›› 2016, Vol. 36 ›› Issue (5): 782-789.doi: 10.7525/j.issn.1673-5102.2016.05.021

杨柳科植物倍性测定不同解离缓冲液效果比对分析

郭炜, 胡南, 李小平, 李淑娴

南京林业大学南方现代林业协同创新中心, 南京 210037

收稿日期:2016-03-16 出版日期:2016-09-15 发布日期:2016-09-27
通讯作者: 李淑娴,E-mail:shuxianli@njfu.com.cn E-mail:shuxianli@njfu.com.cn
作者简介:郭炜(1989-),男,博士,主要从事林木分子育种研究。
基金资助:
林业行业公益重大项目（201304102）；国家自然科学基金项目（31570662）；江苏省优势学科项目（PAPD）

Comparative Analysis of the Performance of Different Lysis Buffers for Measuring the Ploidy Level of Salicaceae Species

GUO Wei, HU Nan, LI Xiao-Ping, LI Shu-Xian

Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037

Received:2016-03-16 Online:2016-09-15 Published:2016-09-27
Supported by:
Supported by the Key Forestry Public Welfare Project(201304102);The National Natural Science Foundation of China(31570662);The Priority Academic Program Development of Jiangsu Higher Education Institution(PAPD)

摘要/Abstract

摘要： 多倍体育种是杨柳科植物育种的重要手段，利用流式细胞仪可以对植物的倍性进行检测，然而解离缓冲液选择是否合适对检测结果有重要影响。本研究利用不同缓冲液对杨柳科植物叶片进行了解离，然后用流式细胞仪测定了解离细胞核的前向散射光（FS）、侧向散射光（SS）、相对荧光强度（FL）、G₀/G₁期峰的变异系数（CV）以及背景碎片系数（DF）等参数。结果比对分析表明，SLB-3缓冲液解离出的细胞核DNA平均荧光强度高、变异系数小，同时对所有被测物种均有较理想的解离效果，是利用流式细胞仪测定杨柳科植物倍性的理想解离缓冲液。SLB-3解离缓冲液的配方为：0.5 mmol·L^-1四盐酸精胺，30 mmol·L^-1柠檬酸钠，200 mmol·L^-1 Tris，80 mmol·L^-1 KCl，0.5%（v/v） Triton X-100，pH7.5。通过研究，我们建立了利用FCM测定杨柳科植物倍性的实验体系，为杨柳科植物多倍体育种提供了可靠的实验技术手段。

关键词: 杨柳科植物, 流式细胞仪, 解离缓冲液, 倍性测定

Abstract: Polyploid breeding is an important approach in the breeding programs of Salicaceae species. Flow cytometer was commonly used to measure the ploidy level of plants. However, lysis buffer affects the result of ploidy measurement significantly. In this study, a variety lysis buffers were tested to isolate nuclei from leaf cells of different Salicaceae species. Subsequently, the nuclei suspensions were measured by a flow cytometer to obtain the values of parameters including forward light scatters(FS), side light scatters(SS), relative fluorescence intensity of propidium iodide-stained nuclei(FL), coefficient of variation of G₀/G₁ DNA peaks(CV), and debris background factor(DF). By analyzing these parameters, SLB-3 was the optimal lysis buffer in measuring the ploidy level of Salicaceae species, as it had a higher fluorescence intensity and a low coefficient of variance. SLB-3 buffer contains 0.5 mmol·L^-1 spermine·4HCl, 30 mmol·L^-1 sodium citrate, 200 mmol·L^-1 Tris, 80 mmol·L^-1 KCl, and 0.5%(v/v) Triton X-100 with pH of 7.5. In conclusion, there was a reliable experimental protocol for measuring the ploidy level of Salicaceae species, which provided a useful tool for the polyploidy breeding program in these species.

Key words: Salicaceae species, flow cytometer, lysis buffer, ploidy measurement

中图分类号:

Q949.733

郭炜, 胡南, 李小平, 李淑娴. 杨柳科植物倍性测定不同解离缓冲液效果比对分析[J]. 植物研究, 2016, 36(5): 782-789.

GUO Wei, HU Nan, LI Xiao-Ping, LI Shu-Xian. Comparative Analysis of the Performance of Different Lysis Buffers for Measuring the Ploidy Level of Salicaceae Species[J]. Bulletin of Botanical Research, 2016, 36(5): 782-789.

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杨柳科植物倍性测定不同解离缓冲液效果比对分析

Comparative Analysis of the Performance of Different Lysis Buffers for Measuring the Ploidy Level of Salicaceae Species

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