Massarray typer version 4

As described previously (Khosla et al., 1999 (link)), total genomic DNA was extracted from young fresh leaves (3 g) taken from the parental cultivars, RILs, first F₁, first F₂, second F₁, and second F₂ plants using guanidine hydrochloride (Sigma-Aldrich) and proteinase K (QIAGEN). Multiplex assays for 513 SNPs, distributed throughout the genome, were designed (Supplementary Table 2) using Sequenom Assay Design 3.1 (Sequenom). Genotyping was conducted using the Sequenom MassARRAY system (Oeth et al., 2009 (link)). Multiplex PCR, followed by template-directed single-base extension at each SNP, was conducted using the MassARRAY iPLEX Gold kit (Sequenom), following the instructions of the manufacturer. Genotypes were determined using MassARRAY Typer version 4.0 (Sequenom).

Genotyping was performed at the Research Unit of Molecular Epidemiology at Helmholtz Zentrum Munich, Germany, as previously described [25 (link)]. DNA was extracted from the buffy coat of umbilical artery blood by the Puregene DNA isolation kit (Gentra Systems, Hilden, Germany). Genotyping was performed using the iPLEX Gold Chemistry (Sequenom, Hamburg, Germany) and matrix-assisted laser desorption ionization-time of flight mass spectrometry, with methods to detect allelic differences. In brief, locations containing certain SNPs were amplified by polymerase chain reaction, using specific primers. After deactivation by alkaline phosphatase, single-base elongation was performed in accordance to the print order. After salt ion removal by ion switch and elongation reaction, the specimen was transferred to a silicone chip and covered with 3-hydroxypicolinic acid. The differences from specific alleles were measured by the matrix-assisted laser desorption ionization-time of flight mass spectrometry. Allele recognition from SNPs was performed by Mass ARRAY Typer version 4.0.5 (Sequenom, Hamburg, Germany). SNPs for FADS genes were selected based on 3 criteria: (1) the SNP has been studied in previous publications; (2) the SNP candidates being considered are SNPs that have already been demonstrated to be associated with LC-PUFA status; and (3) minor allele frequency (MAF) >10%.