基于ITS 2序列的肉苁蓉种子DNA条形码鉴定研究

Study on DNA Barcode Identification of Cistanche Seeds Based on ITS 2 Sequence

本研究基于ITS 2序列,对肉苁蓉和管花肉苁蓉的种子进行鉴定研究,旨在建立肉苁蓉种子的DNA条形码鉴定方法。通过提取种子的基因组DNA,经PCR扩增和双向测序,获得ITS 2序列。应用MEGA-X软件进行序列比对,计算K 2 P遗传距离,构建系统发育树。结果表明,所有种子样本均可以成功提取DNA和扩增ITS 2序列,种子的DNA分子量为10000 bp左右,扩增产物为500 bp左右。肉苁蓉种子的ITS 2序列长度为414~472 bp,GC含量为54.41%~55.07%;管花肉苁蓉种子的ITS 2序列长度为399~489 bp,GC含量为54.18%~55.14%。肉苁蓉种子和管花肉苁蓉种子的ITS 2序列共有61个差异位点,种内最大遗传距离小于种间最小遗传距离,二者在系统发育树中分别聚为一支。ITS 2序列可以作为肉苁蓉种子的DNA条形码鉴定序列。

The seeds of C.deserticola and C.tubulosa were identified based on the ITS 2 sequence in this study,aiming to establish the DNA barcode identification method for Cistanche seeds.The ITS 2 sequence was obtained by extracting the genomic DNA of seeds,PCR amplification and bidirectional sequencing.The MEGA-X software was used for sequence alignment,K 2 P genetic distance was calculated,and a phylogenetic tree was constructed.The results showed that all the seeds could extract DNA and amplify the ITS 2 suquence successfully,the DNA molecules of C.deserticola seeds and C.tubulosa seeds were about 10000 bp,and the amplified product of the ITS 2 sequence was about 500 bp.The ITS 2 sequence length of C.deserticola seeds was 414-472 bp,and the GC content was 54.41%-55.07%;the ITS 2 sequence length of C.tubulosa seeds was 399-489 bp,and the GC content was 54.18%-55.14%.There were 61 different loci in the ITS 2 sequence of C.deserticola and C.tubulosa seeds,the maximum genetic distance within the species was smaller than the minimum genetic distance between species of seeds and the two were clustered into one branch in the phylogenetic tree,respectively.The ITS 2 sequence could be used as the DNA barcode of Cistanche seeds.