Chen Shi Chao, Yang Hong, Zheng Yong Ping, Chen Yue Lei, Qiu Ying Xiong
College of Life Science and Technology, Tongji University, Shanghai.
Fen Zi Xi Bao Sheng Wu Xue Bao. 2006 Dec;39(6):502-8.
Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses were used in elite genotypes identification of Citrus changshan-huyou and analysis of its origin. 12 out of 100 RAPD primers and 11 out of 105 ISSR primers could generate reproducible polymorphic fragments. The RAPD-PCR and ISSR-PCR assays revealed that 64 bands out of 117 (the percentage of polymorphic bands, PPB=54.7%) and 58 bands out of 94 (PPB = 61.7%) were polymorphic, respectively. ISSR and RAPD produced 15 and 12 genotype-specific and species-specific molecular markers. Analysis of molecular variance (AMOVA) was used to calculate the similarity values according to these polymorphic bands, and a dendrogram was constructed using NTSYS-pc software. Each genotype and species in this study could be distinguished from others, suggesting that DNA profiles based on ISSR and RAPD markers have produced potential diagnostic fingerprints for various species, and also for genotypes. The molecular phylogenetic tree shows that C. changshan-huyou and C. sinensis formed a subcluster, so we can conclude that C. sinensis is an assured parent of C. changshan-huyou. However the largest genetic distance was found between C. grandis and C. changshan-huyou, it might be explained that C. changshan-huyou is the origin of multitude of natural hybrids from C. sinensis, C. grandis and other species of Citrus.
采用随机扩增多态性DNA（RAPD）和简单序列重复区间（ISSR）分析方法对常山胡柚优良基因型进行鉴定并分析其起源。100条RAPD引物中有12条、105条ISSR引物中有11条能产生可重复的多态性片段。RAPD-PCR和ISSR-PCR分析显示，117条带中有64条（多态性带比例，PPB = 54.7%）、94条带中有58条（PPB = 61.7%）分别具有多态性。ISSR和RAPD分别产生了15个和12个基因型特异性及物种特异性分子标记。利用分子方差分析（AMOVA）根据这些多态性带计算相似性值，并使用NTSYS-pc软件构建聚类图。本研究中的每个基因型和物种都能相互区分，这表明基于ISSR和RAPD标记的DNA图谱为不同物种及基因型产生了潜在的诊断指纹。分子系统发育树表明，常山胡柚和甜橙形成了一个亚聚类，因此可以得出结论，甜橙是常山胡柚确定的亲本之一。然而，柚与常山胡柚之间的遗传距离最大，这可能是因为常山胡柚是甜橙、柚和其他柑橘属物种众多天然杂种的起源。
