分子标记鉴定常山胡柚优良基因型的初步研究

本研究利用RAPD和ISSR分子标记对常山胡柚的优良基因型进行鉴定,并探讨常山胡柚的起源。从100个RAPD引物中筛选出12个多态性引物用于正式扩增,共得到117条DNA带,其中多态性DNA带64条,占扩增片段的54．7%；从105个ISSR引物中筛选出11个多态性引物用于正式扩增,共得到94条DNA带,其中多态性DNA带58条,占扩增片段的61．7%。RAPD和ISSR分析揭示了常山胡柚及其近缘种的一些特异性条带。ISSR共产生了15条特异条带,RAPD共产生12特异性条带。实验数据用AMOVA软件计算遗传距离,用NTSYS-pc软件构建UPGMA聚类树状图。结果显示,所有的基因型及不同种之间均能够彼此区分,分析得到的指纹图谱对常山胡柚种和基因型的鉴定具有潜在的应用价值,可用于优良基因型的鉴定。聚类分析结果显示常山胡柚和甜柚聚为一枝,确定了甜柚是杂交亲本之一,但是常山胡柚和柚的遗传距离较远,说明常山胡柚可能是甜橙、柚和柑桔属其他种的多重自然杂交的结果。

PRELIMINARY IDENTIFICATION OF CITRUS CHANGSHAN-HUYOU ELITE GENOTYPES BY MOLECULAR MARKERS

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (IS- SR) 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 repro- ducible 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.