不同PCR引物在根系丛枝菌根真菌群落研究中的应用比较(英文)

Comparison of different PCR primers on detecting arbuscular mycorrhizal communities inside plant roots

【目的】基础PCR的各种分子技术已广泛地应用于丛枝菌根真菌(AMF)的群落研究。为探讨不同PCR引物对丛枝菌根真菌群落的特异性差异扩增。【方法】本研究选取4对AMF特异性引物(NS31-AM1,AML1-AML2,NS31-AML2和SSUm Cf-LSUm Br),通过PCR、克隆及测序技术对AMF的多样性及群落结构进行了分析比较。【结果】不同引物对AMF的扩增特异性及覆盖度均有显著性差异,且不同引物得到的AMF群落结构也存在显著性差异。SSUm Cf-LSUm Br的扩增特异性及覆盖度最高,NS31-AML2和NS31-AM1次之,而AML1-AML2则相对较差。【结论】NS31-AML2的扩增区段能很好地与越来越被认可的AMF VT分类数据库(http://maarjam.botany.ut.ee)相匹配,且扩增片段长度也适合于目前的高通量测序技术。基于此,本文推荐NS31-AML2为AMF群落研究中的首选引物。

[Objective] Communities of arbuscular mycorrhizal fungi（ AMF） colonizing roots have been increasingly investigated by molecular approaches with AMF-specific PCR primers. However,it is difficult to compare the species diversity and species compositions of AMF communities across various studies due to the PCR primers used differently,and also little is known if significant difference of community compositions is characterized by different primers. We aim to compare the difference of efficiency of four primers for AMF. [Methods] We chose four commonly used AMF-specific primer combinations（ NS31-AM1,AML1-AML2,NS31-AML2 and SSUm Cf-LSUm Br）,and used 18 S r DNA clone libraries to describe the AMF diversity and community. [Results] Our results showed that the specificity and coverage varied among the tested primers,different primer combinations would yield distinct patterns of species diversity and composition of AMF community. SSUm Cf-LSUm Br had the best specificity and coverage in amplifying AMF sequences,followed by NS31-AML2 and NS31-AM1,and AML1-AML2 showed the lowest specificity towards AMF sequences.[Conclusion]SSUm Cf-LSUm Br is not the optimal primer pair for AMF community study in current stage due to limited reference sequences and large DNA size. As an alternative,NS31-AML2 is more suitable in AMF community study,because its target r DNA region could well match the increasingly used virtual taxonomy database（ http： / / maarjam.botany. ut. ee） and also its suitable DNA size could be efficiently used in high-throughput sequencing.