RNA sequencing for EST-based SNP discovery in Pinus thunbergii

To develop EST-based SNP markers in Pinus thunbergii, RNA sequencing was carried out using leaves and stems collected from five resistant clones and three plus-tree clones. Firstly, a 2 cm segment of stem and leaves of the main stem were collected before inoculation of nematode. Secondly, 2 cm at the tip of the main stem was cut off, the cut edge was quickly crushed with pliers, and 10,000 nematodes that had been suspended in 100 microliter sterile water were injected into the cut edge. Leaf and stem tissue of inoculated samples was collected 5 cm below the inoculated stem apex at 3 day post inoculation. Total RNA was isolated from each sample using the RNeasy plant mini kit (QIAGEN) following the protocol supplied by the manufacturer. Equal masses of total RNA from the non-inoculated and inoculated samples were poopled on each clones and used for RNA-sequencing.