Supporting Information
Figure S1. Phylogeny of Bsister genes.A RAxML tree was calculated for all known monocot
and representative eudicot Bsister genes as well as the Amborellatrichocarpahomologswith
bootstrap values given on the nodes. GGM13 from Gnetumgnemonserved as representative of
theoutgroup. OsMADS29-, OsMADS30- and OsMADS31-subclades are indicated by purple,
blue and green branches, respectively.Gene phylogeny within subclades follows species
phylogeny with two exceptions: The ortholog of OsMADS30 from Phyllostachys is basal
within the OsMADS30 subclade in our phylogeny but would be expected to be closely related
to the OsMADS30-like genes from Oryza species (Kellogg 2001, 1998). Also, the OsMADS30
ortholog of Hordeum vulgare would be expected to be the sister gene to the subclade of
OsMADS30-like genes of Triticum and Aegilops species, but instead it appears to be closely
related to one OsMADS30-like gene from Triticumurartu. The branch leading to the
OsMADS29 subclade and the branches within this subclade are short, indicating only few
substitutions in the OsMADS29-like genes, whereas the branch leading to the OsMADS31
subclade is much longer, which implies considerable sequence changes before species
divergence.
Figure S2. Alignment of Bsister proteins from grasses (Poaceae), Amborella and Gnetum.
Protein sequences of OsMADS29-, OsMADS30- and OsMADS31-like proteins as well as
Bsister protein sequences from Gnetumgenom and Amborellatrichopodawere aligned. MADS,
I, K and C-terminal domain are designated by bars above the alignment. The heterologous Cterminal domain of OsMADS30 encoded by the OsME is highlighted in red. PI-derived and
Paleo-AP3 motif are indicated by boxes. The alignment is colored according to the ClustalX
coloring scheme. The mean pairwise identity within the MADS domain of OsMADS29-like
proteins and OsMADS31-like proteins is 97 % and 93 %, respectively, while for OsMADS30-
like proteins it is only 73 %. Furthermore, almost all OsMADS30-like proteins lack the PI
derived motif and have a distorted Paleo AP3 motif.
Figure S3. Transmembrane prediction for OsMADS30.“DAS”(Dense Alignment
Surface)profile score of OsMADS30 predicted on the basis of its amino acid sequence.
Protein domain structure is depicted on top. Calculated at http://www.sbc.su.se/~miklos/DAS/
Figure S4. Alignment of the OsMADS30 genomic locus.The nucleotide sequence of the
genomic locus of OsMADS30(chr_OsM30) was aligned to the OsMADS30 mRNA
(mRNA_OsM30), OsME1, OsME2, OsME3, the putative ancestral C-terminal region
(ancstrC) and published mRNA transcribed from OsME2 (J090038P19) and OsME3 (001033-F05; J013110N09).
Figure S5. Sequence features of OsME. (a) Results from a RepBase search using OsME1 as
a query. Searching RepBase (Jurka, et al. 2005), we found that a region of 298 bp of the
OsME shows a similarity of about 70 % to a non-coding region of an En/Spm transposable
element and a 40 bp region with similarity to a MuDR transposable element. (b, c) A dot plot
comparison of OsME1 to itself was conducted with seaview (Gouy et al. 2010). For LTRs and
TIRs diagonal lines would be expected in the corners of the diagram. Window size and
identical sites per window were 4/4 (b) and 11/20 (c).
Figure S6. OsMADS30asRNA and in situ hybridization probes. OsMADS30 mRNA is
schematically drawn on top. The encoded domains are given above. Coding regions are filled
black and blue, UTRs filled white. Regions encoded by OsME are bordered orange. The
exons are indicated by broken grey lines. Red, brown and orange boxes below the
OsMADS30mRNA indicate cloned or PCR amplified fragments of complementary asRNA
and probes used for in situ hybridization, respectively.
Figure S7. Expression of Bsister genes in grasses. Data obtained from genevestigator, qTeller
and ESTs. White, grey and black boxes indicate no, weak and medium/strong expression,
respectively. See Supplementary Table 1 for details and references. nd, no data.
Figure S8. OsMADS30 cDNA of T-DNA lines.(a)cDNA from rice plants with and without TDNA insertion was used to amplify PCR products. These were used as a template for nested
PCRs with two different primer combinations (A, B) located within OsMADS30 (forward
primer, upstream T-DNA insertion) and within the GFP gene (reverse primer, part of T-DNA).
The PCR product was verified through sequencing. (b) RNA extracted from 7 day old
seedlings with and without T-DNA insertion was used for cDNA synthesis. Primer pairs were
located both upstream (A), and up and downstream (B) of the T-DNA insertion of osmads302. Actin1 was used as control.+ , plasmid DNA with the respective cloned cDNA.
Figure S9. Mature carpels of Oryza sativa. Toluidine stained mature carpels of theOryza
sativa japonicawild type (a) and the T-DNA insertion lines osmads30-1 (b) and osmads30-2
(c). Scale bars = 200 µm. a, antipods; cw, carpel wall; es, embryo sac; n, nucellus.
Figure S10. Seeds of OsMADS30 T-DNA insertion lines.Seed set (a), length (b), width (c)
and weight (d) of T-DNA insertion lines and wildtype control plants. * and ** indicate sibling
plants obtained from a selfed heterozygous plant.Differences between nontransformedDonjing cv. plants and mutant lines did occur. However, they were not correlated
with the mutant allele (osmads30-1 and osmads30-2) but rather with the T-DNA insertion line
itself, as they were found in wildtype siblings of the respective mutant line as well. Hence we
do not consider the mutation ofOsMADS30 to be the cause of the variation of respective traits.
Figure S11. Grain germination of Oryza sativa OsMADS30T-DNA insertion lines and
control plants. The fraction of germinated grains was determined for osmads30-1 (a) and
osmads30-2 (b) under different concentrations of NaCl (0 mM (duplicated), 171 mM, 207
mM) and PEG 6000 (3%) daily over the course of ten days. Significant differences between
the germination rate of the Donjing cv. and both osmads30-1 and osmads30-2grains could not
be detected. * and ** indicate that seeds were taken from sibling plants.
The differences in germination rate of osmads30-1* and Dongjin* under high salt conditions
(a) is likely not correlated with OsMADS30 since the effect cannot be seen in the second TDNA line.
Figure S12. Growth rate (a), compactness (b) and plant height (c) of OsMADS30 T-DNA
insertion lines and wild type Oryza sativa.Boxplots for each day of imaging (117-147 days)
of wild type control Oryza sativa plants of the cultivar Dongjin (white background) and
osmads30-1 (light grey background) and osmads30-2 (dark grey background). (a) Growth
rates did not significantly differ between wild type and mutant plants.
(b, c) osmads30-1 plants were found to have a significantly lower plant height (although
being comparable in terms of projected leaf area) while being significantly more compact
than osmads30-2 and wild type plants which could be due to different plant morphological
features such as differences in tiller or leaf number (see also Table S3, S4). Probably due to
the lower plant height (with shorter leaves/tillers) the hull area of osmads30-1 is furthermore
significantly smaller than that of osmads30-2 at almost all observed days (Supplementary
Table 3). Since theprojected shoot area (side) is only weakly different between the two
mutants (significant only at days 145 and 147), the calculated compactness value (the fraction
of the convex hull area filled with plant pixels) of osmads30-1 is significantly higher than that
of osmads30-2. This feature distinguishes osmads30-1 also clearly (and significantly) from
the wild type.
Table S1. Expression data of Bsistergenes in grasses.
Table S2. Genes used for phylogeny
Table S3. Extracted values of selected phenotypic traits
Table S4: Imaging derived phenotypic traits of 3 rice genotypes for day 117 and day 147
after sowing.
Table S5. Primer and Probes for quantitative RT-PCR.
Methods S1.Supplementary Experimental Procedures.