盔孢伞属真菌DNA条形码及微条形码技术

Research on DNA Barcoding and Mini-Barcoding of Galerina species

收集盔孢伞属Galerina 16个物种40份标本,提取样品基因组DNA,使用通用引物扩增内转录间隔区(ITS)、基因内间隔区(IGS)、核糖体大亚基(nrLSU)、线粒体小亚基(mtSSU)、翻译延长因子(EF1-α)、RNA聚合酶第一大亚基(RPB1)、RNA聚合酶第二大亚基(RPB2)和β-微管蛋白(β-tubulin)8个基因序列进行双向测序,将得到的序列进行拼接校对后计算物种的种内、种间遗传距离并构建发育树。结果表明:ITS序列最适合作为盔孢伞属的DNA条形码,基于ITS序列开发出盔孢伞属的DNA微条形码、设计出微条形码引物。该引物具有良好的通用性和较强的适用性,对材料品质要求低,能够扩增由于蒸煮或消化等原因导致DNA发生降解的材料,具有一定的实际应用意义。

Forty specimens from 16 species of Galerina were collected, the genomic DNA of the samples was extracted, and 8 gene segments including internal transcribed spacer(ITS), inter-rDNA region(IGS), ribosomal large subunit(nrLSU), mitochondrial small subunit using universal primers Base(mtSSU), translation elongation factor(EF1-α), RNA polymerase first subunit(RPB1), RNA polymerase second subunit(RPB2) and β-tubulin were amplified using general primers and sequenced on both directions. The obtained sequences were spliced and proofread. The inter-species and intraspecies genetic distances were calculated,and the phylogenetic trees were constructed. The results showed that the ITS gene sequence was most suitable for DNA barcode of Galerina. The DNA minibarcode of Galerina was developed based on ITS gene sequence and mini-barcode primers. The primers have good versatility and strong applicability, and low requirements for material quality such as cooked or digested materials which DNA could be degraded, indicating its certain practical application potential.