Climate-driven hotspots and spatial patterns of arbuscular mycorrhizal fungi in Ugandan coffee agroforestry systems

The study sites were localized in the main Robusta coffee-growing regions of Uganda, distributed in 10 districts (Masaka, Lwengo, Rakai, Nakaseke, Nakasongola, Buikwe, Bushenyi, Mitooma, Mityana, and Mubende). Twelve smallholder coffee farms were selected in each district. Five samples were collected along a transect at an interval of 20 m from 5–30 cm depth and mixed to form a composite sample as representative of the farm, making a total of 120 samples. The samples were collected in insulated containers, transported to the laboratory, and stored at 4°C for further analysis. Total DNA was extracted from 1 g of soil using the Fast DNA SPIN kit for soil (MP Biomedicals Europe, Illkirch, France) according to the manufacturer's instructions. DNA purity was improved by adding 40 mg of polyvinylpolypyrrolidone (PVPP) during the first step of DNA extraction. An additional washing step was performed with 5.5 M guanidine thiocyanate before using the washing buffer SEWS-M. DNA extraction was performed in duplicate and stored at -20°C for further analysis. Amplification of 18S rRNA gene specific to AM fungi was performed using AMV4.5NF/AMDGR primers [35]. A first batch of amplifications were carried out in duplicates in 25 µl reaction volumes containing 12.5 µl AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), forward and reverse primers in equimolar concentration (0.2 µM each), and 2 µL of template DNA. The PCR duplicates were then pooled and diluted to 1:10 (v/v). A volume of 2 µl of the pool PCR products was added to Taq 2× Master Mix (New England Biolabs, Ipswich, MA, USA) for a second batch of amplifications, which incorporated dual barcode indices and sequencing adapters. All final PCR products, including negative controls were quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Thermo Fisher Scientific, USA) and a Tecan Multimode Microplate Reader Spark (Tecan Group, Männedorf, Switzerland). Equimolar pooling of PCR samples was purified using NucleoMag NGS Clean-up and Size Select (Macherey-Nagel, Düren, Germany), followed by a TapeStation (Agilent Technologies, Santa Clara, CA, USA) analysis and quantification by qPCR using SYBR Green (Thermo Fisher Scientific, Waltham, MA, USA) on a LightCycler 480 (Roche, Basel, Switzerland). The sequencing-ready library was sequenced on an Illumina MiSeq PE250 platform (MGX genomics platform, Cirad-AGAP, Montpellier, France).