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Supporting Information
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Method S1
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Isolation of total RNA and genomic DNA
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Isolations of total RNA and genomic DNA were carried out by the phenol/chloroform
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method. First, 10–20 mg of seeds were ground with a TissueLyser (Qiagen, Venlo,
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Netherlands) pre-cooled with liquid nitrogen and immediately extracted with 900 l of
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TLE buffer (0.18 M Tris-HCl pH 8.2, 90 mM LiCl, 4.5 mM EDTA, 1% SDS) and 1.1
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ml of phenol/chloroform (5:1 = v/v). After vortexing at room temperature (RT) for 5
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min and subsequent centrifugation at 19,000 × g for 2 min at RT, the aqueous phase was
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mixed with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1 = v/v/v),
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and centrifuged at 19,000 × g for 2 min at RT. After extraction with
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phenol/chloroform/isoamyl alcohol twice more, the aqueous phase was further extracted
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with an equal volume of chloroform. After centrifugation at 19,000 × g for 2 min at RT,
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0.12 volume of 20% polyvinylpyrrolidone was added to the aqueous phase and the
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mixture was centrifuged at 19,000 × g for 10 min at RT. The resultant supernatants were
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mixed with 10 M LiCl to achieve a final concentration of 2 M and stored at –30°C
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overnight. After thawing, mixing, and centrifugation at 25,000 × g for 30 min at 4°C,
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the pellets containing total RNA were rinsed with 2 M LiCl and 70% (v/v) ethanol, then
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dried and dissolved in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA). Supernatants
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containing genomic DNA were mixed with 0.6 volume of isopropanol, held at RT for 5
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min, and centrifuged at 25,000 × g for 10 min at RT. After rinsing with 70% (v/v)
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ethanol, the pellets were dried and dissolved in TE buffer. Isolated total RNA and
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genomic DNA samples were stored at –80°C or –30°C, respectively, until use.
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Quantitative reverse transcription PCR analysis
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Quantitative reverse transcription PCR analysis was performed as described previously
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(Yano et al., 2009) with minor modifications. Briefly, reverse transcription was
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performed with the QuantiTect Reverse Transcription Kit (Qiagen) using 1 g of total
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RNA, according to the manufacturer’s protocol, and qPCR assay was performed in an
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Mx3000p QPCR System (Agilent, Santa Clara, CA, USA). For multiplex, singleplex,
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and SYBR green qPCR, QuantiTect multiplex qPCR (Qiagen), Quantitect qPCR
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(Qiagen), and Thunderbird qPCR kits (Toyobo, Osaka, Japan) were used, respectively.
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The PCR primers and dual-labeled probes used for qRT-PCR analyses are listed in Data
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S5. Welch’s t-test was carried out with the “t-test” function in R 2.13.0.
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Dideoxy sequencing of the HD2B genomic DNA region
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For dideoxy sequencing of the HD2B genomic DNA region, a 2.5-kb genomic DNA
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fragment of HD2B was amplified by PCR in 83 accessions using the primers HD2B-F
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(5’-CACCGCACTTCAAGGCTTCTTTTTTTGGTG-3’)
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(5’-CTCAAAATAGCTCATCCGATAACAAG-3’). After eliminating the remaining
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dNTPs and PCR primers with ExoSap-IT (GE Healthcare, Little Chalfont, UK), PCR
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mixtures were directly sequenced with an ABI PRISM 3100 Genetic Analyzer (Applied
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Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. DNA
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primers used in sequencing were listed in Data S6. The resultant sequences were joined
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with Perl scripts to obtain a 2.5-kb sequence of HD2B. Biallelic polymorphisms
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including both SNPs and indels were identified with BioEdit software (Ibis Biosciences,
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Carlsbad, CA, USA) and Microsoft Excel®. For GWA mapping, identified DNA
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polymorphisms were joined with the AtSNPtile1 database to reconstruct a genotype
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and
HD2B-R
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database, and computation of p-values was performed as described in the corresponding
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section of EXPERIMENTAL PROCEDURES.
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