title: epiGBS of Rhizophora mangle: WB6
design: For genetic and epigenetic analyses, we isolated total genomic DNA from a total of 247 samples, including 60 maternal trees from the field and 187 offspring grown in the greenhouse. The 187 individuals represented 46 maternal families across the 6 populations (5-10 families per population). We increased replication of some families for genetic (not epigenetic) diversity analyses with 29 plants that had received either high salt or high nitrogen treatments. By population, in the final group of samples that made it through the filtering process these 29 samples included AC (3 of 10 individuals), FD (5/47), HI (3/19), UTB (8/49), WB (4/24), WI (6/38) (Table S1).To prepare the epigenotyping-by-sequencing (epiGBS) libraries, we disrupted approximately 80 mg of leaf tissue using stainless steel beads in a Qiagen TissueLyser II. Then, we extracted the DNA using the Qiagen DNeasy Plant Mini Kit following the manufacturer instructions with slight modifications that included an extended lysis step, a post-extraction clean-up with Buffer AW2, and elution in molecular grade water. The final concentration of DNA was quantified using the Qubit 3.0 Fluorometric dsDNA BR assay kit (Life Technologies).We prepared libraries for epiGBS following the methods outlined in van Gurp et al., (2016). In brief, we digested 400 ng of genomic DNA from each sample with the methylation-sensitive restriction enzyme PstI, and ligated methylated, non-phosphorylated barcoded adapters to the resulting fragments. The barcoded adaptors were designed so that we can identify forward (Watson) and reverse (Crick) strands for each fragment within each individual. Having the strand information allows for differentiating between C/T polymorphisms and methylation polymorphisms because we can recreate when unmethylated cytosines were present in either strand before bisulfite treatment (for details see (van Gurp et al., 2016)).We concentrated the libraries (NucleoSpin Gel and PCR Clean-up Kit), and size selected the fragments using 0.8x SPRI beads (Agencourt AMPure XP, Beckman coulter). We performed nick translation, bisulfite converted the fragments (EZ Lightning methylation kit, Zymo Research), and performed PCR amplification with the KAPA HIFI Uracil+ Hotstart Ready Mix (Roche). Finally, we quantified the libraries using the Qubit dsDNA assay kit, pooled them with equimolar concentrations (each sequenced library consisted of 96 multiplexed samples), and assessed their quality by analyzing 1 l on a High Sensitivity DNA chip using an Agilent 2100 Bioanalyzer. We prepared libraries and sequenced paired-end reads of the 60 maternal plant samples and 36 randomly chosen offspring at the University of Florida Interdisciplinary Center for Biotechnology Research on one lane of the Illumina HiSeq 3000 (2 x 150bp) in February 2017. In August 2017, we prepared separate libraries for an additional 151 offspring and sequenced them at Novogene (HK) Company Limited in Hong Kong on two lanes of the Illumina HiSeq X-Ten System (2 x 150 bp): one lane contained 96 offspring samples, a second lane held 55 offspring samples along with 40 samples of another species prepared with the same protocol for another study (Ceratodon purpureus; Boquete, et al., unpublished).