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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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Sample Information

Title Oryza sativa L.
Organism - Scientific Name Oryza sativa L.
Organism - ID NCBI taxonomy:4530
Compound - ID
Compound - Source
Preparation Genotype

The rice diversity research set (RDRS) consists of 68 accessions and Nipponbare and Kasalath as reference cultivars. We furthermore chose to include the salinity resistant cultivar Pokkari. Four additional varieties outside the RDRS (Yumetoiro, Hoshiyutaka, Kinmaze and Soft158) and two amylose hyper accumulating Starch synthase IIIa (SSIIIa) knock-out lines (Tos17 retrotransposon insert): e1, a single knock-out (Nipponbare background) [9] and 4019, double knockout, with Nipponbare/Kinmaze backgroundwere used for the validation experiment.


Organ specification
Brown rice

Sample Preparation Details ID SS1

Sample Preparation Details Information

Title Sample Preparation
Description Growth condition

Twenty-five rice seeds for each of RDRS were sown at a rice field in NIAS, Tsukuba (Lat., 36.030753; Long. 140.099858), Japan in Spring. For the external set of samples, seeds were grown at a rice field in Akita (Lat. 39.803897; Long. 140.046451), Japan.

Sampling and sampling date
For RDRS, seeds were harvested independently for each variety after 40 days, starting from the day on which the first panicle of rice was observed in 2005 and 2006. For others, seeds were also harvested independently for each variety in 2005 (for Yumetoiro, Hoshiyutaka, Kinmaze, Soft158, el and Nipponbare) and 2008 (for 4019 and Nipponbare).


Analytical Method Information

Method Details ID MS3
Sample Amount 11.25 µl of extracts, ca. 0.6 µg of each sample

Analytical Method Details Information

Title Extraction for CE-MS
Instrument CE:Agilent CE capillary electrophoresis system / MS: Agilent G3250AA LC/MSD TOF system
Instrument Type
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 CE-MS
50 mg of each sample was extracted in 20 volumes of methanol containing 8µM of two reference compounds (methionine sulfone for cation and camphor 10- sulfonic acid for anion analyses) using a Retsch mixer mill MM310 at a frequency of 27 Hz for 1 min. The extracts were then centrifuged at 20,400 x g for 3 min at 4 °C. Five hundred-µl aliquot of the supernatant was transferred into a tube. Five hundred µl of chloroform and 200 µl of water was added into the tube to perform liquid-liquid distribution. The upper layer was evaporated for 30 min at 45°C by a centrifugal concentrator to obtain two layers. For removing high-molecular-weight compounds such as oligo-sugars, the upper layer was centrifugally filtered through a Millipore 5-kDa cutoff filter at 9,100 g for 120 min at 4°C. The filtrate was dried for 120 min by a centrifugal concentrator. The residue (ca. 25 mg of each sample) was dissolved into 20 µl of water containing 200 µM of internal standards (3-aminopyrrolidine for cation and trimesic acid for anion analyses) that were used for compensation of migration time in the peak annotation step.

CE-TOF-MS conditions
All CE-TOFMS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany), an Agilent G3250AA LC/MSD TOF system (Agilent Technologies, Palo Alto, CA), an Agilent 1100 series binary HPLC pump, and the G1603A Agilent CE-MS adapter and G1607A Agilent CE-ESI-MS sprayer kit. The G2201AA Agilent ChemStation software for CE and the Analyst QS software for TOFMS were used.
Separation column and electrolytes:
Separations were carried out using a fused silica capillary (50 µm i.d. x 100 cm total length) filled with 1 M formic acid for cation analyses or with 20 mM ammonium formate (pH 10.0) for anion analyses as the electrolyte. The capillary temperature was maintained at 20 °C.
Sample injection:
The sample solutions (11.25 µl of extracts, ca. 0.6 µg of each sample) were injected at 50 mbar for 15 sec (15 nl). The sample tray was cooled below 4 °C.
Separation parameters:
Prior to each run the capillary was flushed with electrolyte for 5 min. The applied voltage for separation was set at 30 kV. Fifty percent (v/v) methanol/water containing 0.5 µM reserpine was delivered as the sheath liquid at 10 µL/min.
ESI-TOFMS was conducted in the positive ion mode for cation analyses or in the negative ion mode for anion analyses, and the capillary voltage was set at 4 kV. Dry gas condition: A flow rate of heated dry nitrogen gas (heater temperature 300 °C) was maintained at 10 psig.
Voltage settings in TOF-MS:
The fragmentor, skimmer, and Oct RFV voltage were set at 110V, 50V, and 160V for cation analyses or at 120V, 60V, and 220V for anion analyses, respectively.
Mass calibration:
Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards. The methanol dimer ion ([2M+H]+, m/z = 65.0597) and reserpine ([M+H]+, m/z = 609.2806) for cation analyses or the formic acid dimer ion ([2M-H]-, m/z = 91.0037) and reserpine ([M-H]−, m/z = 607.2661) for anion analyses provided the lock mass for exact mass measurements.
Mass data acquirement:
Exact mass data were acquired at a rate of 1.5 cycles/sec over a 50-1000 m/z range.
Quality control:
In an every single sequence analysis (maximum 36 samples) on our CE-MS system, we analyzed the standard compound mixture at the first and the end of sample analyses. The detected peak area of standard compound mixture was checked in point of reproducible sensitivity. Standard compound mixture composed of major detectable metabolites including amino acids and organic acids, and this mixture was newly prepared at least once a half year. In all analyses in this study, there were no differences in the sensitivity of standard compounds mixture.

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