目的采用18 S rRNA基因、ITS2序列、SCAR标记等3种DNA分子标记法鉴别草珊瑚,为其分子鉴定提供依据。方法通过PCR扩增、克隆测序后获得草珊瑚18 S rRNA基因序列,并进行Blast比对;通过PCR扩增、测序并注释后获得草珊瑚ITS2序列,从GenBank...目的采用18 S rRNA基因、ITS2序列、SCAR标记等3种DNA分子标记法鉴别草珊瑚,为其分子鉴定提供依据。方法通过PCR扩增、克隆测序后获得草珊瑚18 S rRNA基因序列,并进行Blast比对;通过PCR扩增、测序并注释后获得草珊瑚ITS2序列,从GenBank上收集混伪品和其他植物的ITS2序列,使用MEGA5.5软件,计算种内种间遗传距离,构建系统聚类树;通过RAPD法获得草珊瑚SCAR分子标记,克隆测序后,设计特异性引物扩增草珊瑚及其混伪品。结果获得的草珊瑚18 S rRNA基因长度为1820 bp,Blast比对显示草珊瑚与金粟兰科同源性最高,同源性为99%,证明其为草珊瑚的18 S rRNA基因。获得的草珊瑚ITS2序列长度为500 bp,草珊瑚与其混伪品之间的遗传距离为0.190~0.219,混伪品之间的遗传距离为0~0.074,聚类分析显示草珊瑚聚为一支,混伪品聚为一支,与其他植物距离较远。获得草珊瑚的SCAR分子标记,用特异性引物扩增出现草珊瑚特异性产物,混伪品未出现特异性产物。结论3种分子标记法联合可更有效地鉴别草珊瑚及其混伪品,从而建立一套新的鉴别草珊瑚与混伪品的方法,为其鉴别提供新的思路。展开更多
The ciliate genus Protocruzia belongs to one of the most ambiguous taxa considering its systematic position,possibly as a member of the classes Heterotrichea,Spirotrichea or Karyorelictea,which is tentatively placed i...The ciliate genus Protocruzia belongs to one of the most ambiguous taxa considering its systematic position,possibly as a member of the classes Heterotrichea,Spirotrichea or Karyorelictea,which is tentatively placed into Spirotrichea in Lynn’s 2008 system.To test these hypotheses,multigene trees(Bayesian inference,evolutionary distance,maximum parsimony,and maximum likelihood) were constructed using the small subunit rRNA(SSU rRNA) gene,internal transcribed spacer 2(ITS2) and a protein coding gene(histone H4).All analyses agree that:(1) four morphotypes of Protocruzia from different geographical origins group together and form a monophyletic clade,which cannot be assigned to any of the eleven described ciliate classes;(2) it is invariably positioned on an isolated branch separated from the class Spirotrichea suggesting that this clade should be clearly removed from Spirotrichea;(3) this leads us to hypothesize that this taxon may indeed represent a lineage on a class rank.Based on the fact that it is,both morphologically and in molecular features,closely related to the heterotrichs,Colpodea and Oligohymenophorea,Protocruziida might be an ancestral form for the subphylum Intramacronucleata in the evolutionary line from the class Heterotrichea(subphylum Postciliodesmatophora) to higher taxa.展开更多
文摘目的采用18 S rRNA基因、ITS2序列、SCAR标记等3种DNA分子标记法鉴别草珊瑚,为其分子鉴定提供依据。方法通过PCR扩增、克隆测序后获得草珊瑚18 S rRNA基因序列,并进行Blast比对;通过PCR扩增、测序并注释后获得草珊瑚ITS2序列,从GenBank上收集混伪品和其他植物的ITS2序列,使用MEGA5.5软件,计算种内种间遗传距离,构建系统聚类树;通过RAPD法获得草珊瑚SCAR分子标记,克隆测序后,设计特异性引物扩增草珊瑚及其混伪品。结果获得的草珊瑚18 S rRNA基因长度为1820 bp,Blast比对显示草珊瑚与金粟兰科同源性最高,同源性为99%,证明其为草珊瑚的18 S rRNA基因。获得的草珊瑚ITS2序列长度为500 bp,草珊瑚与其混伪品之间的遗传距离为0.190~0.219,混伪品之间的遗传距离为0~0.074,聚类分析显示草珊瑚聚为一支,混伪品聚为一支,与其他植物距离较远。获得草珊瑚的SCAR分子标记,用特异性引物扩增出现草珊瑚特异性产物,混伪品未出现特异性产物。结论3种分子标记法联合可更有效地鉴别草珊瑚及其混伪品,从而建立一套新的鉴别草珊瑚与混伪品的方法,为其鉴别提供新的思路。
基金supported by the “Natural Science Foundation of China” (Grant No 30870264)the Deutsche Forschungsgemeinschaft (DFG) STO414/2-4supported by the Center of Excellence in Biodiversity Research and the DNA Research Chair, King Saud University
文摘The ciliate genus Protocruzia belongs to one of the most ambiguous taxa considering its systematic position,possibly as a member of the classes Heterotrichea,Spirotrichea or Karyorelictea,which is tentatively placed into Spirotrichea in Lynn’s 2008 system.To test these hypotheses,multigene trees(Bayesian inference,evolutionary distance,maximum parsimony,and maximum likelihood) were constructed using the small subunit rRNA(SSU rRNA) gene,internal transcribed spacer 2(ITS2) and a protein coding gene(histone H4).All analyses agree that:(1) four morphotypes of Protocruzia from different geographical origins group together and form a monophyletic clade,which cannot be assigned to any of the eleven described ciliate classes;(2) it is invariably positioned on an isolated branch separated from the class Spirotrichea suggesting that this clade should be clearly removed from Spirotrichea;(3) this leads us to hypothesize that this taxon may indeed represent a lineage on a class rank.Based on the fact that it is,both morphologically and in molecular features,closely related to the heterotrichs,Colpodea and Oligohymenophorea,Protocruziida might be an ancestral form for the subphylum Intramacronucleata in the evolutionary line from the class Heterotrichea(subphylum Postciliodesmatophora) to higher taxa.
