Flow chart illustrating the physical map contig-specific fragment preparation.

The minimal tilling path BAC clones from each physical contig were selected (highlighted). The pooled BAC DNA from each physical map contig was digested with two 4-bp restriction enzymes, Mse I and Bfa I, respectively. The digestion product was then ligated with in-house designed adaptors, followed by PCR using in-house designed primers. The combination of adaptor and primer formed a specific tag representing each physical contig ID. All PCR products with a physical map contig-specific tag then were pooled together, and sequenced using Illumina HiSeq 2000 platform.