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目的探讨破布叶Microcos paniculata不同地理居群间的DNA序列变异与地理分布的关系,为其种质资源评价和基原植物的分子鉴定提供参考。方法对破布叶14个居群14份个体样品以改良的3×CTAB法提取基因组DNA,选择特异的引物扩增ITS2和psb A-trnH序列;PCR扩增产物直接双向测序分析。DNAMAN 8.0软件处理测序数据,MEGA6.0软件计算K2P遗传距离,运用NJ法构建系统聚类树。结果破布叶14个居群的ITS2序列均为462 bp,共检测到14个变异位点,居群间遗传距离0.000 0~0.019 8。除云南景洪居群样本的psb A-trnH序列存在8 bp缺失外,其他13个居群样本的psb A-trnH序列均长387 bp,无变异,居群间遗传距离为0.000 0。结论不同地理居群破布叶的psb A-trnH较ITS2更为保守,二者PCR扩增引物特异性好,扩增效率和测序成功率高,对其药材及基原植物的分子鉴定可采用psb A-trnH为主,ITS2序列为辅的DNA条形码技术。
Objective To investigate the relationship between the DNA sequence variation and geographic distribution of Microcos paniculata populations in different geographical populations and to provide references for the evaluation of germplasm resources and the molecular identification of primordial plants. Methods Genomic DNA was extracted from 14 individuals of 14 populations of ragweed leaves by modified 3 × CTAB method. Specific primers were used to amplify ITS2 and psb A-trnH sequences. The PCR products were directly bi-directionally sequenced. DNAMAN 8.0 software to process sequencing data, MEGA6.0 software to calculate K2P genetic distance, the use of NJ method to build a system clustering tree. Results The ITS2 sequences of 14 populations of Sabina vulgaris were all 462 bp, 14 loci were detected, and the genetic distance between populations was 0.000 0 ~ 0.019 8. Except for the 8 bp deletion in the psb A-trnH sequence in the population of Jinghong population in Yunnan province, the psb A-trnH sequences of the other 13 populations were both 387 bp in length with no variation and the genetic distance between populations was 0.000 0. Conclusion psb A-trnH of ragweed leaves is more conservative than ITS2 in different geographic populations. Both PCR primers have good specificity, high amplification efficiency and high success rate of sequencing. The molecular identification of their medicinal materials and plants can be used psb A-trnH-based, ITS2 sequences supplemented DNA barcoding technology.