Hiscan system

All microarrays were scanned with an Illumina HiScan system. For the EPIC array data, we used the most current manifest file, “Infinium MethylationEPIC v1.0 B4 Manifest File,” released by Illumina on May 26, 2017 and consisting of 865,918 probes, whereas for the 450K we used the “HumanMethylation450 v1.2 Manifest File” with 485,577 probes. Both manifest files are available at https://support.illumina.com/downloads.html. In addition to unprocessed (raw) data, we used data preprocessed in three different ways (1) color corrected/background subtracted in Genome Studio (GS), (2) quantile-normalized using “preprocessQuantile” [29 (link)], (3) normal-expontential out-of-band (noob)-normalized with “preprocessNoob” [30 (link)]. Raw data and data that were to be quantile or noob normalized were uploaded directly into R from IDAT files using the ‘minfi’ package function “read.metharray” [8 ]. For the color correction/background subtracted preprocessing, data were background subtracted/color corrected with GenomeStudio, and then uploaded into R with the package ‘methylumi,’ function ‘lumiMethyR’ [5 ].

Genomic DNA was extracted using standard methods (Qiagen, CA, USA). Genotyping was performed using the HiScan^® system with the whole-genome Infinium^® Multi-Ethnic Hispanic AMR/AFR MEGA BeadChip array according to manufacturer instructions (Illumina, San Diego, CA, USA). Standard quality control (QC) procedures were implemented, which included: removal of single nucleotide polymorphisms (SNPs) with excessive missingness (> 5%) and individuals with high missing genotype (> 5%) or high heterozygosity rates (three standard deviations [SD] away from the mean rate). Hardy-Weinberg equilibrium (HWE) was calculated for all genotyped SNPs. Any SNPs that deviated from HWE were flagged but not removed. Gender misspecification was checked using X chromosome zygosity. Individuals who did not match known sample data were excluded. Identity-by-descent (IBD) check was performed to identify sample duplicates, contaminated samples, and cryptic relationships. For each pair of samples with estimated IBD coefficients greater than 0.185, only the sample with the highest call rate was retained. Only SNPs with a minor allele frequency (MAF) greater than 5% were included in our analyses. We then imputed the QCed genotype data using the TOPMed Imputation server (https://imputation.biodatacatalyst.nhlbi.nih.gov/#!), which were performed following standard procedures described elsewhere.^{18 (link)}

LV apical myocardial tissue samples were washed in ice-cold saline (0.9% NaCl) and stored in liquid nitrogen until DNA and RNA were extracted. DNA was extracted using a DNA Kit (Qiagen). Purity and concentration were determined using a Bioanalyzer (Agilent Technologies). For DNA methylation profiling, DNA was bisulfite converted using a commercially available kit (Zymo Research). Illumina Infinium Human Methylation 450K Bead Chip and EPIC Bead Chip were used for genome-wide profiling of DNA methylation (58 (link)). The EPIC chip measures over 850,000 methylation sites, with high reproducibility compared with the previous 450k chip (59 (link)). After whole genome DNA amplification, the samples were applied following the Illumina Infinium Human Methylation DNA chip manufacturer protocols (Illumina). Chips were analyzed using the Illumina Hi-Scan system at the McDonnell Genome Institute (MGI) at Washington University.

Ten-day old seedlings of all lines were collected and their genomic DNA was extracted using plant DNA extraction kits (Qiagen, Hilden, Germany). At least 1 μg genomic DNA was used for each cultivar according to the manufacturer’s instructions (Illumina, San Diego). Then all 99 cultivars were genotyped by Rice50K chip25 (link) on Illumina HiScan system, with all experimental procedure following the standard protocol provided by Illumina. All raw genotyping data were adjusted by golden standard genotypes designed in our chip platform with GenomeStudio software (Version 2.0, http://www.illumina.com/techniques/microarrays/array-data-analysis-experimental-design/genomestudio.html). And finally a total of 51,479 SNP markers were obtained for the subsequent analysis. Among them, 22,334 SNPs are high quality chip data with genotyping missing rates <80% and minor allele frequency >5% among all lines.

DNA was extracted from 200 μL of blood, using the Quick-DNA™ Miniprep kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s protocol. The DNA obtained was purified using the DNA Clean & Concentrator™ -5 kit (Zymo Research). The DNA concentration was measured with a Nanodrop Lite (Thermo Scientific®, Wilmington, DE, USA), and integrity was visualized on a 1.5% agarose gel. The genotyping of the two experimental groups was performed with a medium-density array Illumina OvineSNP50 Beadchip (54, 241 SNPs) in the Illumina HiScan System according to the manufacturer’s instructions, at GeneSeek (Lincoln, NE, USA).