基于ITS2序列的球兰属植物种间鉴定研究

Study on Interspecies Identification of Hoya Plants Based on ITS2 Sequence

目的:利用ITS2序列对球兰属植物进行DNA分子鉴定,为其物种鉴定和遗传多样性研究寻求新路径。方法:提取80份球兰样品的DNA,通过PCR扩增ITS2序列并测序,预测其ITS2二级结构。运用MEGA6.0等软件构建邻接(NJ)系统发育树,计算各类别遗传距离。通过叶片形态学特征对其聚类结果进行验证。结果:80份球兰属植物ITS2序列长度约为250 bp,样品CG含量在60.44%~72.88%之间,平均CG含量为68.12%。种内恒定位点数为32,变异位点数为217。NJ系统发育树中80份样品可聚类为11类。序列总体平均遗传距离为0.171,对80份球兰样品的鉴别成功率约为81.25%,结合ITS2序列二级结构可直观地将大部分球兰属植物区分。基于ITS2序列的NJ进化树可阐述球兰属植物的种间关系,聚为一类的球兰属植物叶片颜色和叶形较为相似,也在一定程度上验证了聚类树构建的合理性。结论:ITS2序列可作为解决球兰属植物物种鉴定难题的有效方法。

Objective:To use ITS2 sequence to identify the DNA of Hoya plants,so as to find a new way for species identification and genetic diversity study.Methods:The DNAs of 80 samples of Hoya plants were extracted,ITS2 sequence was amplified by PCR and sequenced and the secondary structure information of ITS2 was predicted.MEGA6.0 software was used to construct adjacent(NJ)trees.The genetic distance of each species was calculated.The clustering results were verified by morphological characteristics of leaves.Results:The length of ITS2 sequence of 80 samples of Hoya plants was about 250 bp,and the CG content of the samples ranged from 60.44%to 72.88%,with an average CG content of 68.12%.The number of constant bit points and the number of mutant bit points were 32 and 217,respectively.The 80 samples in NJ phylogenetic tree could be clustered into 11 classes.The average genetic distance of the sequence was 0.171,and the success rate of identification was 81.25%for 80 samples.The ITS2 secondary structure of the sequence could be used to distinguish most of the plants.NJ evolutionary tree based on ITS2 sequence could basically explain the relationships between species of Hoya plants.Most of the Hoya plants clustered into a group had very similar leaf color and leaf shape,which also verified the rationality of the construction of evolutionary tree to a certain extent.Conclusion:ITS2 sequence can be used as an effective method to solve the difficult problem of species identification of Hoya plants.