Snplex

Venous blood samples were collected in tubes containing EDTA in order to obtain genomic DNA using the Chemagic system (Chemagen, Baesweiler, Germany). DNA was quantified and diluted to a final concentration of 100 ng ⁄µL. The IL18RAP single nuclear polymorphisms (SNPs) were selected for genotyping based on the conjunction of several parameters selected in the SYSNPs web tool25 (link): heterozygosity in a Caucasian population (>10% for the minor allele frequency), position and spacing along the gene and a possible functional effect (http://www.ensembl.org/index.html). Five SNPs in the IL18RAP gene were selected (table 2) and were genotyped using an oligonucleotide ligation assay (SNPlex; Applied Biosystems, Foster City, California, USA), according to the manufacturer’s guidelines. The genomic information about the selected SNPs was obtained from Ensembl release 89 and the single nucleotide polymorphism database (dbSNP) build 149.

We isolated DNA from peripheral blood cells using Chemagic System (Chemagen), and quantified it with PicoGreen dsDNA Quantification Reagent (Invitrogen, Carlsbad, CA, USA). DNA was diluted to a final concentration of 100 ng/µl. We used bibliography searches and the SYSNPS program [24] (link), based on public data sources including Ensembl and HapMap (GRCh36), to identify 597 single nucleotide polymorphisms (SNPs) from 155 candidate genes implicated in diabetes metabolic pathways such as inflammation, redox homeostasis and response to insulin or gluconeogenesis. We included SNPs previously reported to be related to health outcomes in humans or to have functional implications.
The SNPs were genotyped using an oligo-ligation-assay (SNPlex, Applied Biosystems, Foster City) following the manufacturer's guidelines. The polymorphisms nomenclature was based in Den Dunnen JT et al. recommendations. The mean genotyping coverage across all genotyped SNPs was 95%. We excluded 42 SNPs because of a genotyping coverage <90%, 59 SNPs because they had less than 3 genotypes and 41 SNPs because they did not meet a Hardy-Weinberg equilibrium p-value <0.01. 101 SNPs were further excluded because they had a MAF<0.05 or a minor genotype frequency <20 in the study population. The final number of SNPs included in gene-environment interaction analyses was 354.

Genomic DNA was extracted from young leaves from the parents and the 599 seedlings of the ‘Royal Gala’ × ‘Braeburn’ segregating population using the Qiagen Plant DNeasy Plant Mini kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Ten nanograms of genomic DNA from each sample were then amplified by whole-genome amplification^{23 (link)} using the GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK). A subset of the 951 SNP markers used for anchoring the ‘Golden Delicious’ genome sequence to a genetic map was selected for map construction, on the basis of even distribution along the ‘Golden Delicious’ pseudo-chromosomes. SNPlex (Applied Biosystems Inc., Foster City, CA, USA) genotyping assays were carried out using 1 µL (from 45 to 225 ng) of WGA-DNA according to the manufacturer's protocol. Samples were run on a 3730xl DNA Analyzer (Applied Biosystems Inc.) and data were analysed using the Gene Mapper v.4.0 software (Applied Biosystems Inc.). Genotype analysis was performed according to the SNPlex_Rules_3730 method, in accordance with the manufacturer's default settings.