Plant material and growth conditions
The panel of 196 rice accessions was used for nucleotide sequencing analysis . The varieties were grown in Wuhan (N30.52, E114.3) under natural LD conditions and in Hainan (N18.48, E110.02) under natural SD conditions for the measurements.
Two advanced backcross lines harboring the genomic regions with either functional Ghd7, Ghd8 or Hd1 were used to generate F1 hybrids . The F1 plants were then backcrossed with ZS97 twice to create the BC2F1 generation. The segregating population of BC2F2 containing approximately 200 individuals was grown in Hainan. The SSR markers RM5436, PID2, and RM121 were used to select the heterozygous segments at Ghd7, Ghd8 and Hd1 during the crossing scheme. The four homozygous genotypes of Ghd8Ghd7, Ghd8ghd7, ghd8Ghd7 and ghd8ghd7 with the common background of ZS97 were selected as the near-isogenic lines (NILs) in which Hd1 is functional.
To compare the HD and PH of the NILs under different light/dark conditions, 10 plants for each NIL were grown in LD (14 h light: 10 h dark) and SD (10 h light: 14 h dark) conditions in growth chambers. Flowering time was scored from 8 individuals per line. HD was defined as the time when the first panicle appeared from the flag leaf.
The rice core collection was genotyped by sequencing at the coding region of Ghd7, Hd1, Ehd1, Hd3a and Ghd8 covering the functional site described in recent papers [11, 12, 27]. The PCR amplified fragments were sequenced directly using BigDye Terminator Cycle Sequencing v3.1 (Applied Biosystems, Foster, USA) after digestion and purification according to the manufacturer’s specifications. The primers used are listed in Additional File 2: Table S2.
Gene expression analyses
Leaves from the main culm of NILs of 35-day-old plants were harvested to analyze the transcription levels of Ghd8, Ghd7, Hd3a, Ehd1 and Hd1. The NILs were grown in the growth chambers respectively under SD and LD conditions at 30°C in the light and 26°C in the dark. Samples were collected at Zeitgeber time (ZT) 4 under SD conditions and at ZT 8 under LD for gene expression analysis. ZT 0 indicates the beginning of day or the light phase. The samples were collected for RNA extraction using an RNA extraction kit (TRIzol Reagent, Invitrogen). The time points for sampling corresponded to the expression peak for each gene according to the reports [11, 27]. Approximately 2 μl of RNA was reverse-transcribed using M-MLV reverse transcriptase (Invitrogen) in a volume of 20 μl to obtain cDNA. Quantitative RT-PCR was performed in a total volume of 25 μl, which contained 2 μl of the reverse-transcribed product, 0.25 mM gene-speciﬁc primers and 12.5 μl of SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems 7500 Real-Time PCR System according to the manufacturer’s instructions. The expression data were obtained using the relative quantiﬁcation method . All experiments were conducted in at least three biological and three technical replicates. The primers used for the transcription analyses are listed in Additional File 2: Table S2.
Yeast two-hybrid assays
The protein-coding regions of Ghd7, Ghd8 and OsHAP5b were amplified using gene-specific primers with added restriction enzyme sites, respectively (Additional File 2: Table S2). Then, the Ghd7 or OsHAP5b product amplified by PCR was fused into the activation domain (AD) vector pGADT7 as a prey system with EcoRI and XhoI sites, and the Ghd8 product with EcoRI and BamHI sites was fused to the DNA-binding domain (BD) vector pGBKT7 as bait system. All constructs were verified by sequencing. The cotransformation of two plasmids carrying the Ghd8, OsHAP5b and Ghd7 genes into the AH109 yeast strain and cell culture were performed according to the manufacturer’s protocols (Matchmaker Gold Yeast Two-Hybrid System, Clontech). Constructs of pGBKT7-53 (pBD-53) and pGADT7-T (pAD-T) served as positive controls, and constructs of pGBKT7-Lam and pGADT7-T (pAD-T) served as negative controls.
In vitro pull-down assay
The coding region of Ghd8 was cloned into the pET-32a vector (Novagen) and pGEX-6P-1 vector (GE Healthcare) with EcoRI and XhoI sites, respectively. To obtain the OsHAP5b protein, we used the same method for GHD8. The recombinant expression vector was expressed in Escherichia coli Transetta (DE3) cells (Transgen). The pull-down experiment was performed as described previously 49]. In brief, supernatants with equal amounts of Glutathione S-transferase (GST) or OsHAP5b-GST with GHD8-His recombinant proteins were incubated for 6 h at 4 °C in a total volume of 2 ml of pull-down buffer (20 mmol Tris-HCl, pH 8.0, 200 mmol NaCl, 1 mmol EDTA, 0.5% Lgepal CA-630 and protease inhibitor), after which 200 μl of GST resin was added (GE Healthcare; 17-5132-01) and the mixture incubated for 2 h at 4 °C. The binding reaction was then washed 5 times (10 min each time at 4 °C) using the pull-down buffer. After extensive washing, the pulled-down proteins were eluted by boiling at 95 °C for 10 min, separated on 12% SDS-PAGE and detected by immunoblots using an anti-GST antibody (Abcam; ab19256) and anti-His antibody (Abcam; ab9108), respectively.
ChIP (chromatin immunoprecipitation)
For ChIP assays, wild-type and GHD8-GFP transgenic lines were used for chromatin extraction and immunoprecipitation following the method described . Briefly, young leaves from approximately 35-day-old seedlings were collected under LD conditions (15 h light: 9 h dark) and treated with formaldehyde, and the nuclei were isolated and sonicated using an Ultrasonic Crasher Noise Isolating Chamber (SCIENTZ). The soluble chromatin fragments were isolated and preabsorbed with sheared salmon sperm DNA/protein A-agarose (Sigma-Aldrich) to remove nonspecific binding. Immunoprecipitation with anti-GFP (Abcam; ab290) was performed for wild-type and GHD8-GFP transgenic lines with 3 repeats. The precipitated DNA was analyzed by quantitative RT-PCR using specific primer sets listed in Additional File 2: Table S2, designed to cover the CCAAT-box element within a 2-kb primer region upstream of ATG of Ghd7.
To construct the GHD8-GFP fusion, the GUS fragment of pCAMBIA1391Xb was replaced by the maize (Zea mays) ubiquitin promoter-GFP cassette from pU1301. The conservative domain within the Ghd8 coding region involving in the binding and interaction function was amplified and inserted into the KpnI and BamHI sites of the modified pCAMBIA1391Xb to obtain the construct Ubi::GHD8-GFP for rice transformation. The Agrobacterium mediated genetic transformation was used to generate the transgenic plants with Ubi::GHD8-GFP construct in the Nipponbare background.
Electrophoretic mobility shift assay (EMSA)
The pET32-based expression vectors for GHD8 and OsHAP5b were used to express recombinant proteins fused to a thioredoxin and a His (polyhistidine)-tag in tandem at their N-terminal ends. An E. coli BL21 strain (Transgen) was transformed with vectors and grown at 37°C. pMAL vector (New England Biolabs, E8000S) was used to obtain the recombinant HD1 proteins with the maltose-binding protein (MBP) at the down-stream. The recombinant proteins pET32-GHD8, pET32-OsHAP5b and pMAL-c2X-HD1 were purified with Ni-NTA agarose (QIAGEN; No. 30210) and amylose resin beads (New England Biolabs) respectively. The Ghd7 promoter fragment F1 (including the putative binding site CCAAT-box) were produced by annealing of 3’-biotin-labeled oligonucleotides Ghd7F/R (Sangon Biotech), respectively. DNA binding reactions were performed in the presence or absence of unlabeled F1 and labeled mutated F1 (MF1 and MF2) fragments at room temperature for 20 min in 5 mM Tris, pH7.5, 25 mM KCl, 0.5 mM DTT, 5 mM MgCl, 2.5% glycerol, 0.05% NP-40 and 50 ng/uL poly (dI-dC). We followed the protocol from the LightShift Chemiluminescent EMSA Kit (Thermo; No.20148), and the samples were run on 5% polyacrylamide gels. The primers used for EMSA are listed in Additional File 2: Table S2.
Linkage disequilibrium analysis
Linkage disequilibrium analysis of Ghd7, Ghd8, OsHAP5b, Hd1 and Hd3a was performed using the software TASSEL 3.0.147(http://www.maizegenetics.net/). The sequences including single nucleotide polymorphisms in the promoter region (1.5 kb upstream of ATG), coding region and 3’UTR (1 kb downstream of the stop codon) of these genes in 532 varieties were obtained from the website (http://ricevarmap.ncpgr.cn/django/snp_id/). All SNPs in each gene from the varieties were input into TASSEL to conduct linkage disequilibrium analysis. Out of 532 varieties, 2 groups, each including 295 indica and 156 japonica were also separated for linkage disequilibrium analysis, and a threshold of 1% was set to filter the SNPs with low frequencies.
Analysis of variance (ANOVA) of gene interaction on the assayed traits in the F2 segregating population derived from the cross of NIL (Ghd7) × NIL (Ghd8) was conducted using Statistica software . Tukey's HSD (honest significant difference) test in JMP software was employed to determine which groups were significantly different .