作物学报2017，Vol.43Issue(7)：947-953,7.DOI:10.3724/SP.J.1006.2017.00947
基于FLUOstar平台的小麦dsDNA荧光定量与基因型分析
dsDNA Fluorescent Quantification and Genotyping in Common Wheat by FLUOstar System
摘要
Abstract
Quantitative analysis on double-stranded DNA (dsDNA) lays a foundation in molecular biology research in plants, particularly important for genotyping in molecular breeding. The objective of this study was to establish standard curve for fluo-rescence quantitative analysis by lambda DNA, to compare the difference between dsDNA value in fluorescence system and ul-traviolet spectrophotometry, and to identify the allelic variations of rust resistance genes in wheat. The fluorescent dye could be efficiently performed in the quantitative analysis with micro dsDNA concentration (< 1.1 ng μL-1). However, the fluorescent dye could lead to uncertainty of original concentrations of wheat leaf and grain genome DNA, due to more fold serial dilutions for higher DNA concentration. A downward tendency was happened in fluorescent intensity when fluorescent reaction volume was tapered, which influenced the accuracy of DNA concentration. The volume of reaction system mixed nucleic acid and fluorescent dye should be more than 200 μL for accurate determination of micro dsDNA. For genotyping on PCR products, the volume of fluorescent reaction system should be more than 40 μL. FLUOstar could be used for identifying the dominant marker, for instance csSr32#1 (Sr32) and IB-267 (Sr50), its accuracy was 100% in correspondence with that from agarose gel electrophoresis. Co-dominant marker with the characteristic of peculiarity and major difference in amplified fragment length ( ≥100 bp), such as We173 (Yr26), could also be identified by fluorescent analysis. Compared with agarose gel electrophoresis method, fluorescent method have a simple, convenient, and rapid oparetion with high repeatability, and can be used for segregating generations in marker-assisted breeding.关键词
普通小麦/dsDNA/荧光定量/显性标记/分子育种Key words
Common wheat/dsDNA/Fluorescent quantitative analysis/Dominant marker/Molecular breeding引用本文复制引用
肖永贵,Susanne DREISIGACKER,Claudia NUÑEZ-RÍOS,胡卫国,夏先春,何中虎..基于FLUOstar平台的小麦dsDNA荧光定量与基因型分析[J].作物学报,2017,43(7):947-953,7.基金项目
本研究由国家重点研发计划项目(2016YFD0101804-6), 国家自然科学基金项目(31671691), 国家科技支撑计划项目(2014BAD01B05)和科技部国际合作项目(2013DFG30530)资助.This study was supported by the Key Project of the National Research and Development Program (2016YFD0101804-6), the National Natural Science Foundation of China (31671691), the National Key Technology R&D Program of China (2014BAD01B05), and the International Science & Technology Cooperation Program of Ministry of Science and Technology (2013DFG30530). （2016YFD0101804-6）
