Identification of arbuscular mycorrhizal fungi in soils of the North Caucasus based on Illumina MiSeq data for ITS1 and ITS2 regions

Abstract

The objective of our research was to analyze the efficiency of identification of arbuscular mycorrhizal fungi (AMF) for 2 regions: ITS1 and ITS2 regions of AMF DNA isolated from the soils of the North Caucasus (Karachay-Cherkessia). For the first time the necessity of different AMF species identification using both ITS regions was revealed, but not one region. The research demonstrated: 1) the set of taxa is different using ITS1- and ITS2-based identification; 2) analysis of the ITS1 region reveals a greater number of operational taxonomic units; 3) ITS2 allows identification of AMF at the species level more often. Sample preparation for Illumina MiSeq analysis was optimized. Obligatory stages in the sample preparation were the purification of DNA in the agarose gel in Silica after isolation, as well as separate amplification of ITS1 and ITS2 followed by combining and joint sequencing for each sample. The results showed the highest AMF biodiversity for the 176Te sample from the ecosystem of the subalpine meadow of the southeastern slope of Malaya Hatipara mountain (43°25′48.0″N 41°42′31.0″E; 2401 m above sea level), in which 8 species of AMF were identified (Archaeospora spainiae, Claroideoglomus claroideum, Diversispora versiformis, Entrophpora infrequens, Funneliformis mosseae, Glomus indicum, Paraglomus laccatum, Rhizophagus irregularis).

Illumina-based identification of arbuscular mycorrhizal fungi from Karachay-Cherkessia soils for development of bio-fertilizers

“…A comparison of the different STPs suggests that we have discovered a new region with high AM fungal biodiversity, STP #3 (the number of AM fungal species is 23). The previously studied fungal diversity for three other STPs in Karachay-Cherkessia showed significantly lower AM fungal diversity with the highest number of species equal up to 8 per STP [13]. It should be noted that such a high diversity of AM fungi species is also characteristic of other mountain region, such as the Andes [18].…”

“…ITS1 and ITS2 regions were amplified separately. For amplification of ITS1 region we used the primers ITS-5 [12] and ITS-6RK [13]. For amplification of ITS2 region we used the primers ITS-3 and ITS-4 [12].…”