Genetic Diverisity Of Arbuscular Mycorrhizal Fungi (AMF)in Canadian Priaire Through Different Soil Types

Arbuscular mycorrhizal fungi (AMF) are kind of the popular mycorrhizal fungi, which could colonize most of the terrestrial vascular plant on the earth, and play an important role in the natural ecosystem, that is the reason of people interesting in AMF research. In order to rationally take advantage of AMF, we need to understand how these fungi could improve growth of plant. However, AMF can not grow in vitro, which limited their study of metabolizable physiology, biochemistry and genetic knowledge, especially on the aspect of their diveristy of natural resource. Recently developed several effective and short time consume methods with high-throughput base sequences and high accuracy produced which represented by the next generation sequencing (named454pyrosequencing hereafter). While nested-PCR works well for amplifying little amount of mycorrhizal fungi from environmental samples but might not good procedure for downstream pyrosequencing owing to accumulated PCR cycles is one of the probable causes of sequence errors and chimeras which strongly impact the estimates of diversity and richness.This study tested different methods for amplifying target gene, and purifying PCR amplicons, thus modify the optimum protocol of yield AMF target DNA fragment for pyrosequencing. Every experiments of this study were stated below in details:1. Effect of different ways to process raw DNA in soil for pyrosequencing. Here, we made test to compare the difference of using nest-PCR and PCR protocol to amplify mycorrhizal fungi DNA from the same soil samples respectively, and effect of PCR-induced sequence bias and assessing fungal community structure and richness.10commercial wheat fields were selected for soil sampling and18S rDNA was considered as target DNA region (Fig.1). Fig.1Map of ribosome repeat unit gene.(Source:Vilgalys lab)We designed three different DNA processing methods:Method â…  using nested-PCR within agarose gel for purifying PCR products, Method â…¡ using simple PCR with multiple reactions also within agarose gel for purification and Method â…¢ adopt simple PCR with parallel replicated reactions follow by magnetic beads using charge switch principle for PCR purification. Results show that at97%similarity level Method â…¢ detected valid sequences (80875),14736of which are mycorrhizal fungal sequences and generated410AMF operational taxonomic units (OTUs); the other two methods (for method â… , there are totally1319sequences and209of which belong to AM fungi with25AM fungal OTUs. For Method â…¡, totally7222sequences and931of which were identified AM fungi with89OTUs) were much less than Method â…¢. Almost OTUs detected by Method â…  and â…¡ were shared by Method â…¢. In order to explore the optimal PCR amplicons for pyrosequencing with time saving method, we also using PCR purification kit instead of running agarose gel electrophoresis to clear PCR products as another part of test work. We hypothesized that â…°) run parallel PCR replica and then pool into large volume for subsequent concentration will obtain sufficient mycorrhizal fungal DNA yield. â…±) nest-PCR will generate more based-sequence artifacts than simply PCR protocol. â…²) PCR purification kit is a good choice of processing PCR amplicon for enough DNA quantities and time saving.2. Study of AMF diveristy and phylogenetic analysis by pyrosequencing. Insufficient knowledge of the AM fungal resources naturally present in the landscape is a major impediment to the management of AM symbioses in crop production in the Canadian Prairie provinces where an important agricultural industry has developed over the last century. The composition of the AM fungal communities in76wheat fields distributed over28million ha in the Canadian Prairie was described using a tag-encoded massively parallel pyrosequencing protocol. Of the33dominant AMF operational taxonomic units (AMF OTUS) found in the76wheat fields surveyed at anthesis in2009,14clustered as Funneliformis/Rhizophagus,16as Claroideoglomus, and3as Diversisporales. Multiresponse permutation procedure (MRPP) analysis showed that AMF OTUS community composition was better explained by the Chernozem great groups (P=0.0044) than by measured soil properties. Black Chernozem is the most conducive to AMF proliferation. AMF are generally distributed according to Chernozem great groups in the Canadian Prairie, although some taxa are evenly distributed in all soil groups.3. Study of AMF distribution and associated environmental factors. Lots of research indicated that environmental factors signifcantly effected AMF distribution, such as soil pH can affect AM functional diveristy by inducing changes in root colonization and species diveristy. The third part of this study is on the perspective that distribtuion and richness of AMF community could be affected by soil nutrients and properties. The content of research work included soil description for the four soil types (Black Chernozem, Brown Chernozem, Dark Brown Chernozem, Gray Chernozem), soil nutrients (N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, B, S, Al, Pb, Cd), soil pH, bulk density, Olsen P, hyphal trap, AM root colonization, wheat dry biomass, wheat tissue N, P and pyrosequencing for AMF18S rDNA. Redundancy analysis (RDA) found that AMF distribution was significantly affect by soil type (P=0.036), Black and Dark Brown Chernozem have higher levels of AMF diversity and richness,while Brown Chernozem was the lowest. AMF community was strongly affected by soil nutrients (P=0.005). AMF contributed plant nutrients uptake (e.g., Cu, Zn)(P=0.047). This study also found that soil P was negatively correlated with AMF root colonization (P=0.0002).