利用流式细胞仪鉴定鸭茅倍性

doi:10.11686/cyxb2018316

摘要/Abstract

摘要： 鸭茅为世界重要禾本科牧草之一,多为四倍体或二倍体。快速鉴定其倍性可为鸭茅遗传背景研究及多倍体材料的创制提供技术支撑,加快育种进度。采用流式细胞术,筛选4种细胞核DNA解离液,对比2种流式细胞仪检测方法,最终建立鸭茅倍性鉴定高效技术体系。结果表明,不同解离液处理后的细胞核DNA含量存在一定差异。利用Otto制备鸭茅细胞核悬浮液效果较理想,峰型完整,碎片峰较少且样本峰信号清晰,其荧光变异系数为5%左右。内外标法检测各有优势,外标法检测更快速更简便,而内标法准确性更高。在以外标检测为主的基础上,共在46份鸭茅材料中鉴定出二倍体鸭茅22份,四倍体鸭茅24份,为鸭茅遗传育种提供材料。

关键词: 流式细胞仪, 鸭茅, 倍性, 外标法, 内标法

Abstract: Orchardgrass (Dactylis glomerata) is one of world’s most important forage grasses. Various ploidy levels exist, though most varieties are tetraploid or diploid. Rapid ploidy determination is needed to ascertain genetic background of collected germplasm and facilitate progress in breeding programmes. In this study, four kinds of nuclear DNA lysis buffer screen and two flow cytometry methods were compared in order to establish a rapid orchardgrass ploidy determination method. It was found that DNA extract quality varied with different lysis buffers. A superior nuclear suspension with complete peaks, small percentage of fragments, and clear fluorescent signal was obtained using Otto lysis buffer. In flow cytometry, both internal and external standard methods were tested. The external standard method was faster and simpler, while the internal standard method had higher accuracy in plant ploidy detection, with coefficient of variation being about 5%. There were 22 diploids and 24 tetraploids among 46 accessions tested, mainly using the external standard method. This work provided information on ploidy status of accessions, to support an orchardgrass breeding program.

Key words: flow cytometry, Dactylis glomerata, ploidy level, external standard method, internal standard method

许蕾, 陈佩琳, 冯光燕, 钟旻依, 景婷婷, 黄琳凯, 张新全. 利用流式细胞仪鉴定鸭茅倍性[J]. 草业学报, 2019, 28(3): 74-84.

XU Lei, CHEN Pei-lin, FENG Guang-yan, ZHONG Min-yi, JING Ting-ting, HUANG Lin-kai, ZHANG Xin-Quan. Ploidy determination of orchardgrass (Dactylis glomerata) using flow cytometry[J]. Acta Prataculturae Sinica, 2019, 28(3): 74-84.

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