Kod plus

TetO-FUW-cMYC was a gift from Rudolf Jaenisch (Addgene plasmid # 20324). The pLL3.7 vector (provided by Luk Van Parijs) was modified by replacing the EGFP gene with the zeocin resistance gene. All shRNA targeting sequences (Supplementary Table 1) were designed and BLASTed to ensure specificity. The oligonucleotides encoding target shRNA were cloned as described before [59 (link)]. The pPyCAGIP expression vector was a gift from Ian Chambers. The full-length ORF of Nac1 was PCR-amplified from mouse ESC cDNA using KOD-Plus- (TOYOBO). The amplified ORF was subsequently cloned into pGEM-T Easy (Promega) for sequence verification, and then subcloned into pPyCAGIP. The 3×FLAG fragments were generated from the p3×FLAG-CMV plasmid via PCR, and subcloned in-frame into the expression vectors. The c-Myc promoter (positions −2520 to +525) was amplified by PCR from mouse genomic DNA and inserted into pGL3-basic vector (Promega). The primers for construction are listed in Supplementary Table 2.

Genomic DNA was extracted from fresh leaves of each plant using the cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson, 1980). The polymorphic simple sequence repeat (SSR) markers, randomly distributed across the 12 rice chromosomes, are listed in Table S2. PCRs for amplification of the SSRs were conducted according to the methods in Jin et al. (2010) and the products were run on an 8% denaturing polyacrylamide gel at 120 V for 100 min and the gel was visualized using silver staining. Hd1, including the 1825‐bp coding region, was amplified from genomic DNA using KOD plus (TOYOBO, Tokyo, Japan). PCRs were conducted using standard PCR protocols. The primers used for PCR and sequencing are listed in Table S4. The initial genomic sequence of Hd1 was assembled using DNA star software (DNAStar Inc., Madison, WI).

Total RNAs were extracted using either an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) or a FastPure® RNA Kit (Takara Bio, Shiga, Japan). First-strand cDNA synthesis was performed using PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara Bio) with oligo-dT primers provided by the manufacturer. Procedures for cDNA cloning are described in the Supplementary information. Briefly, partial fragments of targeted genes for SrPK, SrPEPtase, SrPEPC, and SrPEPCK were first amplified using PCR with Takara Ex Taq® (Takara Bio) and the primers are listed in Supplementary Table 2. Gene-specific primers were then designed to perform 5′- and 3′-rapid amplification of cDNA ends using SMARTer™RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA). To isolate full-length cDNAs, final PCR amplifications were performed with the KOD -Plus- (Toyobo, Osaka, Japan) with the primers shown in Supplementary Table 2. The obtained fragments were cloned into the T-Vector (pMD19, Takara Bio) and sequenced in both directions. Full-length sequences were determined by isolating at least 2 independent clones with identical sequences. DNA sequences were analyzed using GENETYX software (Genetyx, Tokyo, Japan). Complete cDNAs encoding SrPEPC, SrPK, SrPEPCK, and SrPEPtase were deposited in the DNA Data Bank of Japan with Accession numbers LC155943, LC155944, LC155945, and LC155946, respectively.

A 5000 F₂ mapping population was generated from the cross between fc17 and MH63, an indica cultivar in China. All plants were cultivated in the experimental fields at the Shenyang Agricultural University during the natural growing season. The segregation ratio in F₂ population showed that the normal plants and Brittle Culm plants segregated as 3:1. The fc17 gene was localized to 91-kb genomic region that contains the CESA4 gene. The CESA4 gene of the mutant and its corresponding WT were PCR amplified with KOD-PLUS (TOYOBO) and sequenced with a 3730 sequencer (ABI). For complementation analysis, a 8.53-kb genomic DNA fragment containing the entire CESA4 coding region, a 1.8-kb upstream sequence, and a 1.5-kb downstream sequence were cloned into the binary vector pCAMBIA 1305 to generate the transformation plasmid. The binary plasmids were introduced into Agrobacterium tumefaciens strain EHA105 and transformed into the fc17 mutant plants.