18S MiSeq sequencing of AM fungal community
Molecular diversity of AM fungi from plant DNA was assessed by 18S rRNA gene amplification with the primers NS31 and AML2 (Simon et al. 1992; Lee et al. 2008) according to Davison et al. (2012). After PCR, the amplification products (pools of 2 PCRs) were purified by using illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Sciences, Velizy-Villacoublay, France) following manufacturers guidelines. Then, DNA concentration of PCR products were quantified by using a Qubit fluorometer (Qubit Fluorometric quantitation, Invitrogen) and the Qubit dsDNA HS Assay Kit. PCR product concentration was adjusted to 10ng/l and subjected to paired-end Illumina MiSeq sequencing (2300 bp) by Molecular Research LP (MR DNA, TX, USA). MiSeq Illumina sequencing data were analysed by using Mothur software according the standard operating procedure (http://www.mothur.org/wiki/MiSeq_SOP) proposed in Kozich et al (2013), except that only forward reads were analysed because of the length of PCR products not suitable for paired reads and of a higher quality of forwards reads compared to reverse reads.
Ziziphus2013