# Rice Sesquierpene Plays Important Roles in Antixenosis against Brown Planthopper in Rice
## 3 Materials and Methods
### Plant Volatile Collection and Identification  
The rice varieties RH, KD, and IL283 were used to assess BPH-feeding-induced volatile emissions. Volatile compounds were extracted via solid-phase microextraction (SPME) from 4-week-old rice plants infested by BPH and kept under control conditions for 10 days. Five grams of rice leaves was placed in a 250 mL Duran bottle. Extraction was performed at 75 \({}^{\circ}\)C with equilibrium and adsorption times of 20 and 30 min, respectively. The SPME sampling apparatus included an SPME fiber assembly holding a 1.0 cm fused-silica fiber (Supelco, Bellefonte, PA) with a 50/30 \(\upmu\)m divinylbenzene/carboux/polym dimethylsiloxane (DVB/CAR/PDMS) fiber used for sesquiterpene extraction. The fibers were thermally conditioned prior to adsorption at 230 \({}^{\circ}\)C in an injection port of the gas chromatography (GC) apparatus for 30 min to reduce bleeding and to prevent sample contamination. Gas chromatography-mass spectrometry (GC-MS) analysis was performed using an HP model 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to an HP model 5973 mass-selective detector. The capillary column was an AT-5 MS (5% phenylmethylpolysiloxane) column (30 m \(\times\) 0.25 mm i.d., 0.25 \(\upmu\)m film thickness (Agilent Technologies, Palo Alto, CA, USA)). The oven temperature was initially held at 45 \({}^{\circ}\)C, and then increased at a rate of 2 \({}^{\circ}\)C/min to a final temperature of 230 \({}^{\circ}\)C, which was maintained for 13 min. The injector temperature was 230 \({}^{\circ}\)C. Purified helium was used as the carrier gas at a flow rate of 1 mL/min. EI mass spectra were collected at 70 eV ionization voltages over the range of 29-550 m/z. The electron multiplier voltage was 1150 V. The ion source and quadrupole temperatures were set at 230 \({}^{\circ}\)C and 150 \({}^{\circ}\)C, respectively. Identification of volatile components was performed by the comparison of their Kovat retention indices relative to C8 to C22 n-paraffin hydrocarbon mixtures and by the comparison of the mass spectra of individual components with the reference mass spectra in the Wiley 275 and NIST 98 database. The following are available online at [[https://www.mdpi.com/article/10.3390/plants10061049/s1](https://www.mdpi.com/article/10.3390/plants10061049/s1)]([https://www.mdpi.com/article/10.3390/plants10061049/s1](https://www.mdpi.com/article/10.3390/plants10061049/s1)), Figure S1: Association between four SNPs at 5'upstream and expression of _OsSTPS2_ in 18 rice varieties. Figure S2: Sequence alignment analysis. Table S1: BPH damage score. Table S2: Antixenosis BPH feeding preference test. Table S3: Sesquiterpene products emitted by RH infested by BPH. Table S4: Sesquiterpene products emitted by IL283 infested by BPH. Table S5: Sesquiterpene products emitted by KD infested by BPH. [PERSON], [PERSON], [PERSON], and [PERSON] designed and supervised the research. [PERSON], [PERSON], [PERSON], and [PERSON] performed the experiments. [PERSON], [PERSON], and [PERSON] analyzed the data. [PERSON] and [PERSON] wrote the manuscript. All authors have read and agreed to the published version of the manuscript. This work was supported by the National Science and Technology Development Agency (NSTDA) (grant number P-18-52711) and the Office of National Higher Education Science Research and Innovation Policy Council, Program Management Unit for Human Resources & Institutional Development, Research and Innovation (grant number B16F630088). Not applicable. Data Availability Statement: Data is contained within the article or supplementary material. The authors would like to thank Ubon Ratchathani Rice Research Center, Thailand Rice Department, Ministry of Agriculture and Cooperatives, Rice Science Center and the Center of Excellence on Rice Precision Breeding for Food Security, Quality and Nutrition, Kasetsart University for rice material and BPH insect support.