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Sample Set Information

ID TSE1303
Title Exploring molecular backgrounds of quality traits in rice by predictive models based on high-coverage metabolomics
Description BACKGROUND:

Increasing awareness of limitations to natural resources has set high expectations for plant science to deliver efficient crops with increased yields, improved stress tolerance, and tailored composition. Collections of representative varieties are a valuable resource for compiling broad breeding germplasms that can satisfy these diverse needs.

Here we show that the untargeted high-coverage metabolomic characterization of such core collections is a powerful approach for studying the molecular backgrounds of quality traits and for constructing predictive metabolome-trait models. We profiled the metabolic composition of kernels from field-grown plants of the rice diversity research set using 4 complementary analytical platforms. We found that the metabolite profiles were correlated with both the overall population structure and fine-grained genetic diversity. Multivariate regression analysis showed that 10 of the 17 studied quality traits could be predicted from the metabolic composition independently of the population structure. Furthermore, the model of amylose ratio could be validated using external varieties grown in an independent experiment.

Our results demonstrate the utility of metabolomics for linking traits with quantitative molecular data. This opens up new opportunities for trait prediction and construction of tailored germplasms to support modern plant breeding.

Authors Redestig H, Kusano M, Ebana K, Kobayashi M, Oikawa A, Okazaki Y, Matsuda F, Arita M, Fujita N, Saito K
Reference BMC Syst Biol. 2011 Oct 28;5:176.

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Analytical Method Details Information

Title Extraction for LC-MS
Instrument HPLC, Waters Acquity UPLC system; MS, Waters Q-Tof Premier
Instrument Type UPLC-QTOF-MS
Ionization ESI
Ion Mode positive and negative
Description BioSource amount

For RDRS and Hoshiyutaka, 100 seeds of each variety were selected according to the average weight and length of seeds. After separating the husks from the seeds, the brown rice seeds obtained were bulked and crushed by using a Retsch mixer mill MM301 at a frequency of 20 Hz for 2 min at 4 °C. Successively, the obtained powder was divided into three to four pools. For external set of samples harvested in Akita, 100 seeds of each biological replicate were selected and crushed in the same way as RDRS.

Extraction for LC-MS
100 mg of each sample was extracted with extraction buffer [methanol/water (5:95, v/v)] at a concentration of of 100 mg/ml using a Retsch mixer mill MM310 at a frequency of 20 Hz for 10 min at 4°C. After centrifugation for 10 min at 15,000 x g, 500 µl of the supernatant was transferred into a tube and diluted in 0.1% acetic acid solution. and then it was filtered using an Oasis® HLB µ-elusion plate (30 µm, Waters Co., Massachusetts, USA). The extracts ca. 0.1 mg of each sample) were evaporated to dryness in an SPD2010 SpeedVac® concentrator.The extracts were dissolved by 200 µl of water containing five reference compounds as follows:
0.5 mg/l of lidocaine,
1.0 mg/l of ampiciline,
1.0 mg/l of torperizone,
0.5 mg/l of 10-camphor sulfonic acid and
1.0 mg/l of 2-naphthalene-4-sodium sulfate.

LC-q-TOF-MS conditions
After filtration of the extracts (Ultrafree-MC, 0.2 µm pore size;Millipore), 5 µl of extracts (ca. 0.1 mg each sample) was analyzed using an LC-MS system equipped with an electrospray ionization (ESI) interface (HPLC, Waters Acquity UPLC system; MS, Waters Q-Tof Premier). The analytical conditions were as follows. HPLC: column, Acquity bridged ethyl hybrid (BEH) C18 (pore size 1.7 µ m, length 2.0 x 100 mm, Waters); solvent system, acetonitrile (0.1% formic acid):water (0.1% formic acid); gradient program, 1:99 v/v at 0 min, 1:99 v/v at 0.1 min, 64.0 : 0.5 at 10.0 min, 99.5 : 0.5 at 11.5 min, 1:99 v/v at 11.6 min and 1:99 at 14.0 min; flow rate, 0.3 ml min−1; temperature, 38°C; MS detection: capillary voltage, +3.0 keV; cone voltage, 23 V for positive mode and 35 V for negative mode; source temperature, 120°C; desolvation temperature, 450°C; cone gas flow, 50 l h−1; desolvation gas flow, 800 l/ h; collision energy, 2 V for positive mode and 5 V for negative mode ; detection mode, scan (m/z 100–2000; dwell time 0.45 sec; interscan delay 0.05 sec, centroid). The scans were repeated for 14.0 min in a single run. The data were recorded using MassLynx version 4.1 software (Waters).


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