Illumina Genome Analyzer II paired end sequencing; Compatible and incompatible infection of potato by nematode Globodera rostochiensis
Compatible and incompatible infection of potato by nematode Globodera rostochiensis
Desiree and SW-1015 potato plants were grown in vitro. Sterile nutrient-containing agar was prepared and poured into 9 cm diameter sterile plastic petri dishes. The tips of growing shoots (0.5-1 cm long) were removed from older in vitro plants and placed into the agar with the growing point extending up out of the agar. Two tips of the same cultivar were planted in each dish, in a line approximately 2/3 of the way across the dish. The dishes were placed on end with the plantlets in the upper 1/3 of the dish and kept in a growth chamber for 2-3 weeks, until the root systems reached nearly to the bottom of the dishes. Because of the concentrated agar, most of the root system was found on the surface of the agar.  G. rostochiensis Ro1/4 cysts were elutriated from soil and dried at room temperature. Excess soil or debris was brushed off with a fine paintbrush. Approximately 30 cysts were placed in into a 20 mL plastic syringe (Sarstedt, Nümbrecht. Germany) modified by having the tip and end cut off, replaced with a 30 µm mesh heat sealed across the tube. The plunger of the syringe was replaced by pushing it half way down the tube. Cysts were washed with 90% EtOH for 15s, 2% bleach for 3-5 min and 3 times with water for 45s. The plunger was then removed from the syringes and they were placed in 50 mL centrifuge tubes (Falcon Sarstedt, Nümbrecht. Germany) containing autoclaved tap water. The centrifuge tubes and syringes were covered with paraffin film (BEMIS, Wisconsin.USA) and allowed to incubate in the dark at room temperature for 2-3 days. Potato root exudate was obtained by overwatering a Désirée potato plant grown in a pot filled with sand and collecting the water that flowed out of the bottom. The exudate was passed through a filter of size 0.2 μm and stored in the dark at 4°C for a maximum of 7 days. After 2-3 days of soaking in water, cyst-containing syringes were moved to centrifuge tubes containing filtered potato root exudate, resealed and left in the dark for 5-15 days. Every few days, the exudate was changed and the infective juvenile nematodes that had hatched and swum through the mesh into the centrifuge tube were collected. Infective juveniles were allowed to collect at the bottom of the container. Solution of infective juvenile G. rostochiensis Ro1/4 nematodes collected three days earlier were diluted to a concentration of 1000 nematodes / mL in autoclaved tap water. Five to 10 µl aliquots of the nematode solution or a sterile tapwater control were pipetted onto the root system in each plate and the plates were returned to the growth chamber in a completely randomized design. In order to investigate the potato response to G. rostochiensis Ro1/4 infection we took samples at 8 and 48 h after inoculation. The roots were removed from the other plates by grabbing them with blunt forceps and peeling them off the agar and the roots were immediately frozen in liquid N and stored at -80°C. RNA was isolated by RNeasy Plant Mini Kit (Qiagen) Beads with oligo(dT) were used to isolate poly(A) mRNA. Fragmentation buffer was added for interrupting mRNA to short fragments. Taking these short fragments as templates, Random hexamerprimer were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I, respectively. Short fragments were purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments were connected with sequencing adaptors. And, for amplification with PCR, we selected suitable fragments, as templates, with respect to the result of agarose gel electrophoresis.
Experimental Factor: cultivar
SW93-1015
Experimental Factor: infect
none
Experimental Factor: sampling time point
48