Genemapper v4

We used 26 microsatellite loci specific to the morpho‐species M. phoenicea (Postaire et al., 2015), using the same PCR conditions. PCR products were genotyped using an ABI 3730 genetic analyzer (Applied Biosystems), and allelic sizes were determined on GeneMapper v.4.0 (Applied Biosystems) using an internal size standard (GeneScan LIZ‐500; Applied Biosystems). Considering the whole dataset, over the 26 available loci for M. phoenicea sp., 15 amplified correctly M. phoenicea α individuals, that is, presented less than 10% of missing data, and were considered for all analyses. For each sampling site, identical multilocus genotypes (MLGs) were identified with GenClone v.2.0 and clonal richness R [(N_MLG − 1)/(N − 1)] was assessed (Arnaud‐Haond & Belkhir, 2007; Table 1).

Each of the four rings for each atmospheric treatment at the SoyFACE experiment can be viewed as a true replicate. In order to utilize this level of replication, a higher throughput method for community analysis was required. The AM fungal communities of three of the plant root systems from each of the 16 treatment rings from 2006 and 2008 were therefore characterized using TRFLPs. Dual-labelled partial SSU rRNA gene sequences of AM fungi were produced as described previously for clone library production, but with the AM1 and NS31 primers labelled with FAM and HEX dyes, respectively (Eurofins MWG Operon, Ebersberg, Germany) and with an annealing temperature of 63°C. Labelled PCR products of c. 550 bp were cleaned by gel extraction using a 1.2% agarose gel and QIAquick Gel Extraction Kit (Qiagen) following the manufacturer's instructions.
Eight microlitres of each cleaned PCR product were separately digested using five units of HinfI and AluI (Promega) in a reaction volume of 15 μl containing the manufacturer's buffer and 2 μg BSA for 15 h, and restriction products were cleaned using a QIAquick PCR Purification Kit (Qiagen). The sizes and quantities of FAM-labelled AluI digests and HEX-labelled HinfI digests were determined against the GS600 LIZ size standard employing an ABI 3130 genetic analyser (and its supplied software: GeneMapper v4.0; Applied Biosystems).

Genomic DNA was isolated from peripheral leukocytes according to a standard salting out procedure (Miller et al. 1988 (link)). Microsatellite markers were fluorescently labeled and amplified by PCR, run on ABI 3130xl or 3730 (Applied Biosystems) and analyzed with Gene Mapper v4.0. Pedigrees were drawn with Progeny (Progeny Genetics LLC) and haplotypes phased manually with the least number of recombination events. To test for linkage in the NL family, five affected members (PIDs II-2, II-3, II-6, II-9, III-2; otosclerosis confirmed by surgery) and two of unknown clinical status (PIDs III-5, III-6) (Fig. 2a) were genotyped for markers spanning each OTSC disease interval and bracketing three otosclerosis susceptibility genes (Supplementary Table 1). As well, a total of 17 relatives were genotyped with nine extra markers (D16S518, D16S3049, D16S3098, D16S422, D16S2625, D16S520, D16S413, D16S3023, D16S3026) mapping qter of the OTSC4 disease interval. Two-point parametric linkage analyses (MLINK ver 5.1) were run for three markers per locus (assuming autosomal dominant inheritance, 99% penetrance and a gene frequency of 0.00). LOD scores were calculated at recombination fractions 0.000 to 0.5000. The proband was also sequenced for rare otosclerosis-associated variants in SERPINF1 (Ziff et al. 2016 (link)).

DNA was extracted from individual mosquitoes using a cetyltrimethylammonium bromide (CTAB) protocol [39 ]. A total of 564 individuals were genotyped (24–30 individuals per site). In each site, half of the analyzed individuals were males and the other half females. A set of 16 microsatellite loci previously developed for Ae. albopictus was used [32 (link), 34 (link)] (Additional file 2: Table S2). Microsatellite locus primers were pooled in three PCR-mixes based on non-overlapping size ranges and amplified by multiplex PCRs using fluorescent primers (Additional file 2: Table S2). All PCR reactions (10 µl) contained 0.5 U of Taq DNA polymerase, 6 µM of dNTPs, 37.5 µM of MgCl₂, 1.2–7 µM of each primer (Additional file 2: Table S2) and 10 ng of mosquito DNA. The following program was used to amplify all 16 loci: 10 min at 95 °C followed by 35 cycles at 95 °C for 30 s, 57 °C for 30 s and 72 °C for 1 min and a final elongation step of 72 °C for 10 min. PCR products were run on a 3730XL DNA Genetic Analyzer (Applied Biosystems, California, USA) with a GeneScan 500LIZ size standard. Microsatellite alleles were read using the software GeneMapper v. 4.0 (Applied Biosystems).