咖啡树细胞核DNA含量的可靠流式细胞术估计

Reliable flow cytometric estimation of nuclear DNA content in coffee trees.

作者信息

Barre P, Noirot M, Louarn J, Duperray C, Hamon S

机构信息

Laboratoire des Ressources Génétiques et Amélioration des Plantes Tropicales, Centre ORSTOM de Montpellier, France.

出版信息

Cytometry. 1996 May 1;24(1):32-8. doi: 10.1002/(SICI)1097-0320(19960501)24:1<32::AID-CYTO4>3.0.CO;2-K.

DOI:10.1002/(SICI)1097-0320(19960501)24:1<32::AID-CYTO4>3.0.CO;2-K

PMID:8723900

Abstract

Flow cytometry gave high resolution of genome size in two coffee taxa (Coffea liberica dewevrei and C. pseudozanguebariae). Propidium iodide (PI) and Petunia hybrida were used as dye and internal standard, respectively. Proportionally between the DNA content and the digitized fluorescence signal was checked. Five main results were evident. First, optimal experimental conditions were established for peak location estimation (mean or mode), staining time (at least 2 minutes), high voltage (557 V) for the photomultiplier tube (PMT), and PI concentration (333 micrograms/ml). Second, a parameterization of the effects of high voltage and PI concentration were deduced from curve fitting. Third, two biases in DNA content estimation were recorded for high voltage and PI concentration, and were minimized. Fourth, the genome sizes of C. liberica dewevrei and C. pseudozanguebariae were estimated with accuracy 2C = 1.421 +/- 0.005 pg and 2C = 1.129 +/- 0.005 pg, respectively. Fifth, between-genotype variance was emphasized within each taxon.

摘要

流式细胞术在两种咖啡分类群（利比里亚咖啡德韦夫雷变种和拟桑给巴尔咖啡）中实现了基因组大小的高分辨率测定。碘化丙啶（PI）和矮牵牛分别用作染料和内标。检查了DNA含量与数字化荧光信号之间的比例关系。有五个主要结果很明显。第一，确定了用于峰位置估计（均值或众数）、染色时间（至少2分钟）、光电倍增管（PMT）的高电压（557 V）和PI浓度（333微克/毫升）的最佳实验条件。第二，通过曲线拟合推导出高电压和PI浓度影响的参数化。第三，记录了高电压和PI浓度在DNA含量估计中的两种偏差，并将其最小化。第四，利比里亚咖啡德韦夫雷变种和拟桑给巴尔咖啡的基因组大小分别准确估计为2C = 1.421±0.005皮克和2C = 1.129±0.005皮克。第五，强调了每个分类群内基因型间的差异。