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

双链DNA(dsDNA)定量分析是植物分子生物学研究的基础,对基因型分析尤为重要。本研究以λ噬菌体dsDNA为标准样品,建立了荧光定量标准曲线,探讨荧光核酸定量通量性及其与紫外法定量的差异,并分析荧光染料在基因分型中的应用。结果表明,荧光染料能够对dsDNA进行高效微量定量分析(<1.1ngmL-1),但因鉴定核酸的浓度较低,对小麦籽粒和叶片全基因组DNA定量时稀释倍数较大,易增大浓度误差。降低反应体系量导致标准曲线决定系数降低,影响测量准确性。精确定量dsDNA浓度时,总反应体系应大于200mL;对PCR产物进行基因分型时,总反应体系应不低于40mL。相同DNA模板浓度下,FLUOstar平台可以对抗秆锈病基因显性标记csSr32#1(Sr32)和IB-267(Sr50)的PCR产物进行基因型分型,判断准确率为100%。对特异性强且等位基因片段差异大(≥100bp)的共显性标记,如抗叶锈病基因标记We173(Yr26)等,用荧光染料同样可以进行基因分型。与琼脂糖凝胶电泳相比,荧光染料鉴定等位基因价格略高,但方法简单、准确快速、重现性好,可用于分子育种中世代材料快速筛选。

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 fluorescencequantitative analysis by lambda DNA, to compare the difference between dsDNA value in fluorescence system and ultravioletspectrophotometry, and to identify the allelic variations of rust resistance genes in wheat. The fluorescent dye could beefficiently performed in the quantitative analysis with micro dsDNA concentration （〈 1.1 ng ·L^-1）. However, the fluorescent dyecould lead to uncertainty of original concentrations of wheat leaf and grain genome DNA, due to more fold serial dilutions forhigher DNA concentration. A downward tendency was happened in fluorescent intensity when fluorescent reaction volume wastapered, which influenced the accuracy of DNA concentration. The volume of reaction system mixed nucleic acid and fluorescentdye should be more than 200 μL for accurate determination of micro dsDNA. For genotyping on PCR products, the volume offluorescent 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 asWe173 （Yr26）, could also be identified by fluorescent analysis. Compared with agarose gel electrophoresis method, fluorescentmethod have a simple, convenient, and rapid oparetion with high repeatability, and can be used for segregating generations inmarker-assisted breeding.