应用ITS2序列片段对木瓜及其近缘属植物的分子鉴定研究

Molecular Identification of Chaenomeles and Its Closely Related Genera Using ITS2 Sequence Fragments

运用ITS2序列对木瓜及其近缘属植物进行DNA条形码分子鉴定,探讨利用ITS2条形码序列对木瓜及近缘物属植物鉴定的可行性。对试验样品进行DNA提取纯化、PCR扩增并双向测序得到22条ITS2序列,将所得22条序列和Gen Bank数据库下载的8条木瓜及其近缘植物的ITS2序列用Clustal X软件进行比对,Bio Edit软件人工校正;利用MEGA5.05软件计算种内、种间遗传距离,并采用K2P距离法构建NJ和ML树,评价序列的鉴定效果。木瓜及其近缘属植物种内遗传距离变异区间为0~0.0274,平均0.0029,木瓜及近缘属植物种间遗传距离变异区间为0.011~0.112,平均0.075,种间距离明显大于种内距离;构建木瓜及其近缘属各物种NJ和ML系统进化树,二者结果一致,各物种均聚为一支,形成单系类群,支持率均在50%以上。ITS2条形码序列能够快速准确地鉴定木瓜属及其近缘属植物。

Conduct molecular identification of Chaenomeles from its closely related genera by ITS2(nuclear internal transcribed spacer 2)sequence fragments,aiming at investigating the feasibility of application of methodology of ITS2. The DNA samples were extracted and purified,the PCR amplification and bidirectional sequencing of the materials were carried out to get 22 ITS2 sequences. Then the 22 ITS2 sequences obtained together with 8 ITS2 sequences from Gen Bank database were aligned by Clustal X(version 1.81),with manual adjustments assisted by Bio Edit. MEGA program(version 5.05)was employed to estimate the intraspecific and interspecific genetic distances. NJ(Neighbor-Joining)and ML(Maximum Likelihood) trees were constructed based an K2P(Kimura-2-parameter)distance of the ITS2 sequences in order to evaluate the former results identified. The range of intraspecific differences of Chaenomeles and its closely related genera was 0 to 0.0274,with a mean value of 0.0029;While the interspecific difference value was from 0.011 to 0.112,with a mean value of 0.075. These indicates a significant difference between interspecific and intraspecific values. Further on,NJ and ML phylogenetic tree of different species of Chaenomeles and its closely related genera were constructed. These two methods gave rise to a similar result. Various species were clustered together,formed a monophyletic group. The supporting rates of the above results were all above 50%. The results showed that the ITS2 barcoding was a fast and accurate method for the identification of Chaenomeles and its closely related genera.