Impact of scoring error and reproducibility RAPD data on RAPD based estimates of genetic distance
Impact of scoring error and reproducibility RAPD data on RAPD based estimates of genetic distance
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评分误差和再现性 RAPD 数据对基于 RAPD 的遗传距离估计的影响
DOI：
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发表时间：
1995
影响因子：
5.4
通讯作者：
J. Nienhuis
中科院分区：
文献类型：
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作者：
P. Skroch;J. Nienhuis
以10个四季豆(Phaseolus ual garis L.)为材料，对50个随机扩增条带(RAPD)的重复性和评分误差进行了评价。基因分型。比较了基于不同DNA条带的遗传距离，以评价评分误差、重复性和相对扩增强度的差异对基于RAPD的遗传距离估计的重复性的影响。测定的随机扩增多态性DNA数据评分误差为2%。重复性为76%，表示的是在重复数据中获得的随机扩增条带的百分比。结果表明，在重复数据中获得一条被评分的RAPD条带的概率强烈地依赖于实验之间扩增条件的一致性，以及该条带的相对扩增强度。通过减少重复之间反应条件的差异，显著改善了条带的重复性，并减少了一些评分误差。观察到的用于标记的RAPD重复性的引物的可变性也可能有助于引物的选择，从而显著改善种质研究中使用的RAPD数据的重复性。在192条随机扩增带中，由于抽样误差引起的重复间遗传距离的变异比由于评分误差引起的遗传距离变异大6倍以上。根据重复数据中的随机扩增带和未被重复数据中得分的随机扩增带计算的遗传距离矩阵没有显著差异。在所得到的遗传距离矩阵中，溴化乙锭染色强度的差异与产生的遗传距离矩阵的差异不相关。假设抽样误差是误差的唯一来源，足以解释观察到的跨独立的几组随机扩增带的遗传距离估计的差异。
RAPD band reproducibility and scoring error were evaluated for RAPDs generated by 50 RAPD primers among ten snap bean (Phaseolus vulgaris L.) genotypes. Genetic distances based on different sets of RAPD bands were compared to evaluate the impact of scoring error, reproducibility, and differences in relative amplification strength on the reproducibility of RAPD based genetic distance estimates. The measured RAPD data scoring error was 2%. Reproducibility, expressed as the percentage of RAPD bands scored that are also scored in replicate data, was 76%. The results indicate that the probability of a scored RAPD band being scored in replicate data is strongly dependent on the uniformity of amplification conditions between experiments, as well as the relative amplification strength of the RAPD band. Significant improvement in the reproducibility of scored bands and some reduction in scoring error was achieved by reducing differences in reaction conditions between replicates. Observed primer variability for the reproducibility of scored RAPDs may also facilitate the selection of primers, resulting in dramatic improvements in the reproducibility of RAPD data used in germplasm studies. Variance of genetic distances across replicates due to sampling error was found to be more than six times greater than that due to scoring error for a set of 192 RAPD bands. Genetic distance matrices computed from the RAPD bands scored in replicated data and RAPD bands that failed to be scored in replicated data were not significantly different. Differences in the ethidium bromide staining intensity of RAPD bands were not associated with significant differences in resulting genetic distance matrices. The assumption of sampling error as the only source of error was sufficient to account for the observed variation in genetic distance estimates across independent sets of RAPD bands.