Taketa et al. 10.1073/pnas.0711034105. |
Fig. 5. Nucleotide and deduced amino acid sequence (227 aa) of the Nud candidate gene of the standard cultivar Haruna Nijo. TATA box, 5'-UTR (178 bp), exon 1 (83 bp), intron (200 bp), exon 2 (598 bp), and 3'-UTR (374 bp) are underlined. The putative polyadenylation signal (AATAAA) is boxed. The 84-bp tandem duplication in the 3' noncoding region is colored in yellow and green to portray each repeat unit. A 1-bp change in the mutant allele nud1.b is shown in pink. A 1-bp deletion site in the mutant allele nud1.c is marked in blue. Microsatellite regions with different motifs, (GTAT)n, (ACTC)n, and (CT)n, are shaded in gray.
Fig. 6. Similarity of the deduced amino acid sequence of Nud candidate to ERF family proteins of the Arabidopsis WIN/SHN clade members and rice homologs. A rice homologous protein BAD35470 on chromosome 6 (locus ID Os06g0604000; see http://rapdb.dna.affrc.go.jp) and another homologous rice protein BAD15859 on chromosome 2 (locus ID Os02g0202000) were abbreviated as Os06ERF and Os02ERF, respectively. (A) Alignment of deduced amino acid sequences. Amino acid identity and similarity are indicated with an asterisk and dot(s). In this figure, "mm" and "cm" respectively denote conserved middle motif and conserved C-terminal motif. The single nucleotide change in Mut.4129 (nud1.b) causes V134 ® D amino acid substitution (marked in red) within the "mm" motif. The 1-bp deletion in Mut.3041/6a (nud1.c) causes frame shift starting at position 164 (T164 ® S, marked in blue) and an early stop codon appears at 200 within the "cm" motif. (B) Neighbor-joining tree based on entire region of deduced amino acid sequences of the ERF family transcription factor genes. The tree was drawn using MEGA3.1 software. Confidence estimates for junction points are from bootstrap tests with 1,000 replicates.
Fig. 7. Comparison of pericarp permeability of 2-week-old dehulled caryopses in the Bowman isogenic lines. (A) Chlorophyll leaching assay by immersion in 80% ethanol. (B) Water loss assay in an incubator at 30°C. The values are means of two replications (±SD).
Fig. 8. TLC of surface lipids. Surface lipids were extracted from dehulled caryopses or detached hulls of each of Bowman (lanes marked with N) and nud-Bowman (lanes marked with n). Materials analyzed were 3-week-old grains. After TLC separation, lipids were detected using 50% sulfuric acid spraying (A), iodine vapor exposure (B), 0.7% ninhydrin spraying (C), or 0.05% anthrone reagent spraying (D). Sprayed plates required subsequent heating on a hot plate. The amount of lipid extract loaded per lane was adjusted by grain number so that each lane contained the amount equivalent to 10 grains. The first lane marked with M is glyceryl trilinoleate (standard). Bars on the upper right indicate the front of the solvent; the bottom bar indicates the origin.
SI Methods
Plant Materials.
Two populations for genetic mapping were Kobinkatagi (nud, Okayama University accession no. OUJ369) ´ Triumph (Nud, a gift from D. A. Laurie, John Innes Centre, Norwich Research Park, Norwich, United Kingdom) and Karafuto Zairai (Nud, OUJ301) ´ Aizu Hadaka 3 (nud, OUJ323). In the former cross, we used F2 and F3 plants segregating for nud, whereas, in the latter cross, F2 plants were used for mapping. Compared with our study in ref. 1, 94 and 814 additional F2 plants were studied for the former and the latter cross, respectively.Bowman is a two-rowed covered cultivar. nud-Bowman is an isogenic line of Bowman carrying the nud gene, which was raised by eight backcrossings (a kind gift from J. Franckowiack, Hermitage Research Station, Warwick, Australia). The source of the nud gene for nud-Bowman cannot be traced back to a single naked cultivar because of its complicated pedigrees. Bowman and its isogenic naked line (nud-Bowman) are collectively called "the Bowman isogenic lines". Plants were grown in a room with a controlled environment at a constant temperature of 15°C under natural light. The Bowman isogenic lines are almost photoperiod-insensitive and started flowering approximately 2 months after sowing under this growing condition. When the anthers in the middle part of the spike opened, the spikes were tagged. Grain samples were harvested from plants flowered during summer to autumn seasons, during which the daylength varied from 14.5 to 10.5 h. Under this growing condition, grains were dehulled without difficulty until 3 weeks after anthesis, and reached physiological maturity after an additional 3-4 weeks.
Chromosome Walking.
BAC clones near the nud locus were selected either by PCR screening of DNA pools or hybridizing the arrayed BAC colony filters with digoxigenin (DIG)-labeled probes. BAC DNA was extracted by using a standard alkaline-SDS procedure and the size of the insert was analyzed by using pulse field gel electrophoresis after digestion with NotI, AscI or PmeI. All BAC clones were end-sequenced by using M13 primers. Selected BAC clones were subjected to subcloning after fingerprinting with 6-bp restriction enzyme digestion; unique bands were extracted from gels and cloned into plasmid vectors for sequencing. Overlapping BACs were identified either by Southern hybridization or by PCR marker analysis. Primers designed from the end sequences were used to perform PCRs to construct a BAC minimum tiling path.BAC Sequencing and Annotation.
BAC clone HNB106O20 was shotgun-sequenced by the TaKaRa Dragon Genomics Center (www.takara-bio.co.jp) with an approximate 7-fold coverage; gaps were filled by PCR primer walking. Three other BACs (HNB 233N01, 035P04, and 589B20) were shotgun-sequenced (10-fold coverage) according to the standard dideoxy terminator chemistry protocol of the Rice Genome Research Program (2), but HNB 589B20 was only partially sequenced to fill a small gap between clones 035P04 and 106O20.Annotation analyses were carried out as follows: First, the entire sequence was processed by using Repeat Masker (www.repeatmasker.org/cgi-bin/RepeatProteinMaskRequest). Then, the masked sequence was analyzed by using gene prediction program, RiceGAAS (http://ricegaas.dna.affrc.go.jp) and GeneMark.hmm ver.2.2a (http://opal.biology.gatech.edu/GeneMark/eukhmm.cgi). For identification of repetitive elements, the unmasked sequence data were analyzed by using a BLASTN search against the Triticeae Repeat database (TREP) (http://wheat.pw.usda.gov/ITMI/Repeats). The resultant data were sorted manually with a threshold E value of e-20. Predicted genes were also analyzed by BLASTN using the DDBJ EST database of plant species including Arabidopsis, rice, maize, wheat, and barley. A database of approximately 170,000 barley cDNA end sequences derived from cultivar Haruna Nijo (Matsumoto et al. manuscript in preparation) was also referred for BLASTN analysis. Genes were predicted based on the combined results of the analyses described above.
Long PCR and Rapid Amplification of cDNA Ends (RACE).
For determination of the extent of deletion in naked cultivars, long PCR amplification was attempted by using Phusion High-Fidelity DNA Polymerase (Finnzymes) and primer pair HNB32C2 F13-R8 (for the primer sequence, see SI Table 4). A 3.6-kb fragment was cloned by using a PCR cloning Kit (Zero Blunt TOPO; Invitrogen). Three independent clones were sequenced to avoid PCR-induced errors. Using three primers, wF2-kR1-tR2, PCR can detect the presence or absence of the 17-kb deletion as an amplicon length polymorphism (853 bp for covered barley and 785 bp for naked barley).Total RNA was isolated from 1-week-old caryopses of Bowman. For 3' RACE, cDNA was synthesized from total RNA using SuperScript II RNase H-reverse transcriptase (Invitrogen) according to the manufacturer's protocol. For 5' RACE, the 5' RACE System for Rapid Amplification of cDNA End kit (ver.2; Invitrogen) was used according to the manufacturer's protocol. The RACE primers are presented in SI Table 4.
Sequence Analysis of Nud Alleles.
Allelic variation of approximately 1.7-kb Nud region including the coding region, intron and 5' and 3' noncoding regions, was studied by direct sequencing, using a sequencing kit (BigDye Terminator v3.1 Cycle; Applied Biosystems) and a DNA sequencer (Model 3100; Applied Biosystems). The sequenced region was amplified as four separate overlapping fragments using primer sets described in SI Table 4.Histochemical Analysis.
Caryopses with the hull were fixed with FAA solution (3.7% p-formaldehyde, 5% acetic acid) under low pressure and then embedded (Technovit-7100; Heraeus Kulzer). Using a microtome, 10-mm-thick grain sections were prepared, and staining was done with 1% Toluidine blue O or 0.1% Sudan black B (Wako). For Sudan black B staining of caryopses, hulls and dehulled caryopses were stained in a 0.1% solution of Sudan black B dissolved in 70% ethanol for 10 min, followed by a rinse in 50% ethanol for 2 min.Chlorophyll Leaching and Water Loss Analyses.
For chlorophyll leaching and water loss analyses, dehulled caryopses at 2 weeks after anthesis from the Bowman isogenic lines were used. All experiments were carried out in duplicate. For the chlorophyll leaching assay, 30 dehulled caryopses were placed in a plastic vial containing 7.5 ml of 80% ethanol at room temperature with gentle agitation in the dark. From each sample, 500 ml of ethanol was removed at fixed times for absorbance measurements. Chlorophyll concentration was calculated according to Ahanori et al. (3). For water loss experiments, 15 dehulled caryopses were placed in a Petri dish in an incubator maintained at 30°C for 48 h, then further dried at 60°C for 48 h to measure the dry matter weight. The samples were weighed periodically. Water loss was calculated according to Ahanori et al. (3).Surface Lipid Extraction.
Three-week-old grains were collected from six spikes of each Bowman isogenic line. Surface lipids of hulls and dehulled caryopses were extracted separately, using 2:1 (vol/vol) chloroform/methanol by dipping with gentle agitation for 5 min at room temperature.RT-PCR and RNA in Situ Hybridization.
Total RNA was extracted by using a kit (Isogen; Nippon Gene) from hulls, caryopses and flag leaves of the Bowman isogenic lines. Samples were collected on the day of anthesis (0 week) and at 1-week intervals until 3 weeks after anthesis. For caryopses, the embryo and endosperm were removed by pinching with fingers and blotting onto paper to minimize mRNA contamination from other parts. Subsequently, DNA was removed by digestion with DNaseI before reverse transcription. Using beads (Ready-To-Go You-Prime First-Strand Beads; GE Healthcare) according to the manufacturer's instructions, cDNA was synthesized. Then RT-PCR was performed by using ExTaq DNA polymerase (TaKaRa) and actin primers as a control. For Nud and the actin control, 30 and 25 amplification cycles were used, respectively. The primer sets that were used are shown in SI Table 4.Young grains were fixed in FAA as described above, dehydrated and embedded in Paraplast (Oxford Labware). Then RNA in situ hybridization was performed as described by Kouchi and Hata (4) with modifications used by Mai et al. (5). The 262-bp sequence containing the entire 5'-UTR plus an initial 84 bp of the coding region of the Nud gene was used as a probe, and DIG-labeled sense and antisense RNA probes were prepared (TOPO TA Cloning Kit Dual Promoter with TOP; Invitrogen).
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