Full legends for Supporting Information
Figure S1. Alignment of casein kinase I isoforms from various species. Boxed residues indicate
the catalytic loop (purple) and the L-0D loop (yellow). The blue box indicates the human family
advanced sleep phase syndrome (FASPS) short-period mutation site. The red boxes indicate the
Drosophila double-time short-period (dbtS) and long-period (dbtL) mutation sites, and the golden
hamster tau mutation site. The aqua box indicates the Koshihikari Hd16 mutation site. Gene
names and accession numbers are as follows: Homo sapiens CKI (NP_001885.1), Mesocricetus
auratus CKI (AAF65549.1), Mus musculus CKI (NP_038795.3), Drosophila melanogaster
double-time (AAC39134), Arabidopsis thaliana dual-specificity kinase 1 (AAB47968.1), Oryza
sativa similar to dual-specificity kinase 1 (AK111554, Os02g0810400), and O. sativa Hd16
(AB753041, Os03g0793500).
Figure S2. Phylogenetic relationships among the casein kinase I genes composed of 240 amino
acids from different animal and plant species. The evolutionary history was inferred using the
neighbor-joining method. Bootstrap values based on 1000 replicates are shown next to the
branches. The tree is drawn to scale, with branch lengths in the same units as the evolutionary
distances used to infer the phylogenetic tree. Evolutionary distances were calculated using the
Poisson correlation method and are expressed in units representing the number of amino acid
substitutions per site. Gene ID and locus numbers were obtained from data registered in the
following public databases: NCBI in human, golden hamster, mouse, and Drosophila
(http://www.ncbi.nlm.nih.gov/), TAIR in Arabidopsis (http://www.arabidopsis.org/), RAP-DB in
rice (http://rapdb.dna.affrc.go.jp/), and SbGDB in sorghum (http://www.plantgdb.org/SbGDB/).
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Figure S3. RNA interference (RNAi) with Hd16, showing mRNA production in T2 lines under
long-day (LD) conditions. Four genes sharing sequences highly homologous with Hd16
(Os07g0171300, Os01g0772600, Os01g0198200, and Os05g0200500) were also investigated in
the RNAi T2 lines. Primer sequences are listed in Table S1.
Figure S4. Days to flowering of RNAi plants in N-NIL(Hd16) genetic background under
different photoperiod conditions. Values are means±standard deviation (n=10). SD, short-day
conditions (10 h light/14 h dark); LD, long-day conditions (14.5 h light/9.5 h dark).
Figure S5. Levels of Hd16 mRNA in different tissues during development under long-day (LD)
conditions in Nipponbare and a Nipponbare NIL, N-NIL(Hd16). UBQ, ubiquitin (used to
normalize the values).
Figure S6. Localization of GUS expression at the seedling stage. T1 transgenic rice plants
containing an Hd16 promoter::GUS fusion construct in the Koshihikari genetic background were
stained to detect GUS activity.
Figure S7. (a) Distributions of the Nipponbare-type allele and the Koshihikari-type allele of the
Hd16 gene in cultivated rice (Oryza sativa) and wild rices (O. rufipogon). Numbers inside the
graphs indicate the number of accessions investigated in the present study. "Old" japonica
cultivars are those that were released before 1954, whereas "modern" japonica cultivars are those
that were released from 1954 to the present. (b) Genealogical identification of the origin of the
Koshihikari-type SNP in the Hd16 gene. Details of the accessions and additional data are
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provided in Table S2.
Figure S8. Diurnal changes in transcript levels of OsLHY and OsPRR1 under constant light (LL)
conditions in Nipponbare and N-NIL(Hd16). Transcript levels were investigated at every 3 h
from plants grown under LL conditions for 3 days. Seedlings were grown under long-day (LD)
conditions for 14 days before transfer to LL conditions. Values are means±standard deviation of
three biological replicates. UBQ, ubiquitin (used to normalize the other values). ZT, Zeitgeber
time.
Figure S9. Bioluminescence analysis of Cab1R:LUC expression in plants exposed to (a) a 12 h
dark/12 h light cycle (DL), (b) constant light (LL), or (c) constant dark (DD). Seedlings were
grown under dark/light cycles for 4 days before transfer to DD or LL conditions. White
background, the light period; dark background, the dark period; light gray, the nominal 12-h
interval if light conditions changed during the LL treatment. ZT, Zeitgeber time.
Figure S10. Autoradiograph from the SDS-PAGE analyses of Hd1 and Ghd7 after in vitro Hd16
phosphorylation assays. rHd16(Ni), recombinant protein derived from the Nipponbare cDNA
sequence; rHd16(Ko), recombinant protein derived from the Koshihikari cDNA sequence.
Figure S11. Autoradiograph from the SDS-PAGE analyses of SLR1 after in vitro Hd16
phosphorylation assays. rHd16(Ni), recombinant protein derived from the Nipponbare Hd16
cDNA sequence; rSLR1, recombinant protein derived from the SLR1 cDNA sequence.
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Figure S12. (a) Comparison of gibberellin (GA3) and paclobutrazol (PBZ) responses in
Nipponbare, N-NIL(Hd16), and the RNAi transformant T2-1 under natural day-length (12 h
light/12 h dark) conditions. Relative length (%) and actual measured length (mm) are presented
for the second leaf sheaths of 7-day-old seedlings in the presence of exogenous GA3 or PBZ. (b)
Culm length under long-day (LD) conditions in Nipponbare, N-NIL(Hd16), and the RNAi
transformant T2-1. Values are means±standard deviation (n = 10). Values followed by different
letters differ significantly (P < 0.05, heteroscedastic t-test).
Figure S13. Comparison of agronomic traits between Koshihikari and K-NIL(Hd16) under shortday (SD), long-day (LD), and natural day-length (ND) conditions. Values are means±standard
deviation (n = 10). Asterisks indicate a significant difference between Koshihikari and KNIL(Hd16) (Student’s t-test: * P < 0.05, ** P < 0.01, *** P < 0.001).
Table S1. Primer and probe sequences used in this study.
Table S2. Rice accessions used in this study. Remarks indicate the accession number in the NIAS
Core Collection (Kojima et al., 2005; Ebana et al., 2008).
Table S3. Compositions of the culture media used in this study.
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