gDNA resequencing of Williams 82: leaf tissue
DNA Sequencing and Candidate Gene SearchDNA was isolated from ~40 mg of lyophilized seedling leaf tissue from a single plant each for Essex and Williams 82 using the DNeasy Plant Mini Kit (Qiagen). Isolated DNA was randomly sheared using a Covaris S2 Adaptive Focused Acoustic Disruptor instrument (Covaris, Inc., Woburn, MA, USA) to an average fragmentation size of 200 basepairs. Sheared DNA was then used to prepare indexed Illumina libraries by Global Biologics, LLC (Columbia, MO) using standard Illumina adapters.  Libraries were then paired-end sequenced (2x 100 bps) using a HiSeq 2000 instrument at the DNAcore facility at the University of Missouri.All resequencing analyses used the Wm82.a2.v1 genome sequence and annotation build, which was downloaded from (http://phytozome.jgi.doe.gov/). Read mapping and variant calling were done using CLC Genomics Workbench software Version 8.0 (Qiagen/CLC Biotech, Cambridge, MA). Read mapping used the following settings: insertion cost of 3, deletion cost of 3, a similarity fraction setting of 0.8, automatic detection of paired-end distances, and non-specific matching handling was set to ignore. For variant calling, the fixed ploidy program was used with settings: minimum coverage of 9, variant probability cutoff of 90.0, maximum variant count of 2, and non-specific read matches were ignored. Total variant calls were filtered to call genes with amino acid changes.
EL5757_Essex_gDNA-reseq_1