Supplementary Experimental Procedures
Phylogenetic Analyses – Identification of Bsister genes
The Bsister genes from Musa accuminata were taken from Gramzow and Theißen, 2013
(Gramzow and Theissen 2013) and named MUSA ACUMINATA BSISTER1 and 2, (MaBS1,
and MaBS2). The Bsister genes from Elais oleifera and Phoenix dactylifera were identified via
BLAST searches (Altschul et al. 1990) of the genomes using OsMADS29 as query (AlMssallem et al. 2013; Singh et al. 2013). They were termed ELAIS OLEIFERA BSISTER
(EoBS) and PHOENIX DACTYLIFERA BSISTER (PdBS), respectively. The Bsister gene of
Spirodela polyrhiza was taken from Wang et al., 2014 (Wang et al. 2014). Furthermore, we
searched the transcriptome data of the 1kp project (http://onekp.com/) to identify monocot
Bsister genes. In frame of another project we identified all MADS-box genes in the
transcriptome data using Hidden Markov Model searches (Gramzow and Theissen 2013) and
identified Bsister genes using phylogenies. Complete or almost complete Bsister gene sequences
were identified from Posidonia australis, Maianthemum spec., Agave tequilana, Traubia
modesta, Narcissus viridiflorus and Triodia aff and termed PaBS, MsBS, AteBS, TmBS, NvBS
and TafBS, respectively. The Bsister genes from Oryza spec. were identified by BLAST
(Altschul et al. 1990) searching the genomes of O. glabberima, O. punctata, O. brachyantha,
O. barthii, O. rufipogon, O. meridionalis and O. nivara using OsMADS29, OsMADS30 and
OsMADS31 as queries. Genes were named according to the O. sativa orthologs. The
OsMADS30 orthologs of O. glaberrima and O. glumaepatula did not contain a complete
MADS box due to incomplete sequencing and possibly incorrect gene prediction, respectively
and were hence not included in the phylogenetic analyses. The Bsister genes from Aegilops
tauschii, Triticum urartu and Triticum aestivum were identified by BLAST search (Altschul et
al. 1990) of the respective genome and named according to species name and their position in
the phylogeny, BS1, BS2 or BS3 if they were orthologous to OsMADS29, OsMADS30 or
OsMADS31, respectively.
Selection analysis
Selection analysis was carried out using all grass Bsister genes that were used for the phylogeny.
Due to high sequence similarity to other genes in the dataset all Oryza sequences except the
ones from O. sativa, and all sequences from Aegilops tauschii and Triticum urartu were
excluded from the analysis. Remaining protein sequences were aligned using ClustalW
(Thompson et al. 1994). The resulting alignment was reverse translated using RevTrans
(Wernersson and Pedersen 2003). This nucleotide alignment and the previously obtained
phylogeny were used as input for PAML (Yang 2007). We used different subsets of the
alignment, corresponding to the full alignment except the part encoding for the OsMADS30
C-terminal domain (MIKC), the full alignment without the part encoding for the C-terminal
domain of all genes in the analysis (MIK), only the MADS-box and only the K-box. We also
used different branch models, the one-ratio model allowing only one ratio for all branches in
the phylogeny, a two-ratio model allowing the OsMADS30 subclade to have a different ωvalue to all the other branches and a four ratio model which has different ω-values for each of
the three subclades, OsMADS29, OsMADS30 and OsMADS31 and the branch leading to the
OsMADS31 subclade. To find out which model fits the data best, Likelihood ratio tests were
carried out, comparing the two ratio model with the one ratio model and the four ratio model
with the one ratio model.
Imaging-based plant phenotyping
Seeds of the osmads30-1 and osmads30-2 mutant lines as well as Oryza sativa ssp. japonica
cv. Dongjin wildtype were sown in trays filled with a 2:1 mixture of clay substrate
(Klasmann-Deilmann GmbH, Germany) and vermiculite (1/2mm, Gärtnereibedarf Kammlott,
Germany) . Seedling were cultured in a climate controlled glass house at 24/17°C day/night,
60% relative air humidity, and 205–245 μmol m−2s−1 PAR with the light period set to 16 h
(06:00–22:00 h). Due to the use of shading (when outside sun light exceeded 65 klux), total
light intensity (natural sunlight + supplemental illumination) only rarely exceeded 380 μmol
m−2s−1 PAR. For the analysis of growth dynamics and plant architectural as well as
physiological features expressed upon cultivation in an acclimatized greenhouse. After an
initial cultivation period of four months and before reaching the booting stage, ten plants per
mutant and the Oryza sativa wild type were transferred to an automated phenotyping platform.
After eleven weeks of pre-culture seedlings were transferred to 5.5l pots with clay/vermiculite
mixture, stabilized with blue cages and the substrate mixture was covered with a blue rubber
mat. Watering from the bottom was performed every second day and fertilizer was applied
once per week (40ml/10l Wuxal (Manna, Germany)).
The plants were grown for further six weeks with daily imaging, weighing and watering. The
relative growth rate was calculated as RGR = (log(LA[tn+4]) - log(LA[tn])) / ((tn+4) - tn) in a
sliding window manner to represent the increase in projected leaf area (side view) every 4
days as described elsewhere (Poorter and Lewis 1986). Plant compactness was calculated as
Compactness = 4∏ (projected shoot area/border pixels²) and the hull area represents the area
(in pixels) of the convex hull, which is the shortest convex line drawing
around the plant. Extracted values of selected traits were subjected to statistical analysis using
GenStat (ANOVA and subsequent post-hoc analysis by Tukey's range test).
Additional References
Al-Mssallem, I.S., Hu, S., Zhang, X. et al. (2013) Genome sequence of the 11 date palm
Phoenix dactylifera L. Nat. Commun. 4, 2274.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local
alignment search tool. J. Mol. Biol. 215, 403–12 410.
Gramzow, L. and Theissen, G. (2013) Phylogenomics of MADS-box genes in
plants – two opposing life styles in one gene family. Biology (Basel), 2,
1150–14 1164.
Poorter, H. and Lewis, C. (1986) Testing differences in relative growth-rate –
a method avoiding curve fitting and pairing. Physiol. Plant. 67, 223–16 226.