两种混合样品策略对揭示杜鹃花根部真菌多样性的影响

摘要

由于土壤微生物群落物种组成的高度空间异质性,混合样品(sample pooling)被广泛应用于微生物多样性与群落结构研究.在根部真菌的分子检测中,样品混合策略以及测序的克隆数或序列数均对揭示真菌群落结构的准确性有影响.[目的]为建立一套能快速准确地反映杜鹃花属植物根部真菌的物种组成与群落结构的分子检测技术平台,[方法]本研究采集锈红杜鹃和亮鳞杜鹃多份根系样品分别提取DNA,比较PCR扩增前和扩增后混合策略构建的克隆文库中真菌物种组成的差异.[结果]在2种宿主植物根系中,多份样品在PCR扩增后混合构建的克隆文库检测到的根部真菌物种丰富度、真菌群落的Shannon-Wiener多样性指数均高于扩增前混合的克隆文库.高频度的根部真菌在2种克隆文库中均检测到,但低频度的真菌物种组成在2种克隆文库中完全不同.更重要的是,当采用广泛应用的真菌通用引物ITS1f和ITS4扩增根部真菌ITS序列时,PCR扩增后混合的方法能有效地减轻杜鹃花属植物ITS序列被优先扩增的现象.真菌物种累积曲线显示,当测序的真菌ITS片段克隆数达到50个左右,即能较全面地反映2种杜鹃花根部真菌物种组成.[结论]独立扩增多份根系样品DNA,再将PCR产物混合构建克隆文库的方法能更全面地揭示杜鹃花属植物根部真菌物种丰富度与物种组成.%[Objective] Pooling of multiple samples is widely used in studying general patterns of microbial communities that are heterogeneously structured in space.Pooling strategies and the number of sequence reads generate biases in the description of diversity and community structure of root-associated fungi.Therefore,we developed a molecular toolbox for fast and accurate identification of the root-associated fungal community of Rododendron species.[Methods] Multiple root samples of R.lutescens and R.bureavii were collected for DNA extraction.Effects of two different pooling strategies,i.e.pooling samples prior to vs.post PCR,on fungal species composition were studied by comparing results within host species.[Results] Species richness and Shannon-Wiener index of fungal communities of clone library constructed by pooling samples after PCR were higher than that of pooling prior to PCR.High frequency fungal species were detected by both pooling strategies,whereas infrequent species detected by the two strategies differed.Notably,the prior to PCR pooling strategy effectively alleviated the unwanted amplification of host plant sequences when fungal specific primer ITS 1 f and ITS4 were used.Accumulation curves of fungal species suggested that sequencing at least 50 clones can fully reflect species composition of clone library of the two Rhododendron root-associated fungal community.[Conclusion] Clone library constructed by post PCR pooling of samples is better in providing accurate views of fungal diversity and community structure of Rhododendron species.