An Invisorb Spin Plant Mini Kit (Stratec Molecular) was used to isolate genomic DNA and prepare short-read libraries for the Roche-454 and Illumina sequencing platforms. DNA concentrations were determined using a Quant-iT dsDNA Assay Kit (Life Technologies) and a Qubit Fluorometer (Invitrogen). Agarose-embedded high-molecular weight (HMW) DNA was prepared to construct Illumina TruSeq Synthetic Long Read (TSLR) libraries. DNA concentrations were quantified with the Quant-iT dsDNA Assay Kit. DNA quality was then checked using an Argus Qcard Kit (OpGen) and was estimated at 20–100 kb. 454 single-end read libraries according to the standard procedure provided by Roche, with RL adaptors (GS FLX Titanium Rapid Library Preparation Kit; Roche Diagnostic). The libraries were sequenced with titanium chemistry on a 1/2 Pico Titer Plate on a 454 GS FlX instrument (Roche Diagnostic). Longest reads were assembled together (obtained from 454 and Moleculo libraries) to maximize the separation of the two haplotypes of accession 3P and to overcome the high level of heterozygosity. An assembly of 300,113 contigs with an N50 of 9.3 kb and a cumulative size of 1.31 Gb, corresponding roughly to the size of the two haplotypes.
