1
Full Legends for Supporting Information
2
3
Figure S1
4
Phylogenetic relationships of Lepidium and Arabidopsis fruit developmental genes
5
based on Bayesian analysis: Lepidium genes (highlighted in grey) are sister to those of
6
other Brassicaceae and thus seem to represent orthologs of Arabidopsis genes. (a)
7
MADS-box genes including SHATTERPROOF1,2 and FRUITFULL, (b) BELL
8
homeobox genes including REPLUMLESS, and (c) bHLH genes including
9
INDEHISCENT and ALCATRAZ. Thick branches indicate posterior probability (PP)
10
values > 0.95, PP values < 0.80 are indicated by dashed lines.
11
12
Nucleotide sequences were conceptionally translated into amino acids sequences,
13
aligned using the ClustalW software (Larkin et al., 2007) and edited manually.
14
Subsequently, the alignment was translated back to nucleotide sequences. Nucleotide
15
sequence similarities (p-distances) and amino acid similarities and identities were
16
calculated in PAUP* 4.0 b 10 (Swofford 2003) and MatGAT 2.01 (Campanella et al.,
17
2003), respectively. Best substitution models were established using Mrmodeltest2
18
(Nyylander 2004). Datasets were implemented in MrBayes 3.1.2 (Huelsenbeck and
19
Ronquist 2001; Ronquist and Huelsenbeck 2003). Bayesian analyses were run for
20
1.000.000 generations at a sample frequency of 100. The 50% majority rule consensus
21
trees were established after a burn in of 250.000 generations. Ratios of non-synonymous
22
to synonymous substitution rates (Ka/Ks, ω) were determined using the KaKs-Calculator
23
(Zhang et al., 2006).
24
1
1
Figure S2
2
Southern blot hybridisation to analyse gene copy number of Lepidium fruit
3
developmental gene orthologs: DNA gel blot analysis was generally conducted as
4
described by Southern (1975) but employing the non-radioactive DIG-labelling system
5
and using Biodyne B membranes (Pall, USA). A few analyses were performed using 32P
6
labelled probes and Zeta Probe nylon membranes (BioRad, Germany). Probes were
7
labelled using the PCR-DIG-labelling mix (Roche, Germany) and the NEBlot-Kit
8
(NEB, England) using Klenow fragments for radiolabelling of probes. Probe templates
9
were produced using primers given in the supplementary data (Table S2). Hybridisation
10
signals of gene-specific probes on digested genomic DNA of L. appelianum and L.
11
campestre are indicated by black dots. Fragment sizes of the molecular ladder (DNA
12
Molecular Weight Marker VII, Roche) are indicated. Abbreviations: La, Lepidium
13
appelianum; Lc, Lepidium campestre. All genes seem to be single copy genes.
14
15
Figure S3
16
Amino acid alignments of genes under study including sequences of further
17
Brassicaceae species with dehiscent fruits: In total, 20 amino acids changes were
18
detected solely in genes of indehiscent L. appelianum and they are marked by arrows.
19
20
Figure S4
21
Gene expression analysis of Lepidium fruit developmental genes in different tissues
22
via Northern blot hybridisation: RNA was isolated using RNAiso-G+ (Segenetic,
23
Germany). 15 µg of total RNA were separated on a 0.8% (w/v) agarose gel and
24
transferred under denaturing conditions to Zeta Probe nylon membranes (BioRad,
2
1
Germany) via a vacuum blotter. Probes were radiolabelled with a NEBlot-Kit (NEB,
2
England) using Klenow fragments. Primer information is given in the supplementary
3
data (Table S2). Hybridisation was performed following the manufacturer’s instruction
4
manual of the Zeta Probe nylon membrane (BioRad, Germany). An 18S rRNA specific
5
probe has been used as a positive control. Northern hybridisation on distinct plant
6
tissues of L. campestre and L. appelianum using radiolabelled (α32P-(d)CTP) DNA-
7
probes. (a) positive control using an 18S gene specific probe. (b-g) gene expression of
8
putative fruit developmental genes in total RNA of distinct tissues. No hybridisation
9
results could be obtained for LcALC, LcIND, and LcSHP2. Abbreviations: R, Root; S,
10
Stem; L, Leaf; B, buds; 13-14, Flower stage 13-14; 15, Flower stage 15.
11
12
Figure S5
13
Overexpression phenotypes of RPL orthologs. Constitutive expression of AtRPL (b),
14
LaRPL (c), or LcRPL (d) in A. thaliana results in alterations in phyllotaxy and irregular
15
elongation of internodes in some transformants compared to the wild-type (a).
16
17
Figure S6
18
Shl sequences of dehiscent Brassicaceae species (green) and indehiscent Lepidium
19
appelianum (blue): The boxed nucleotide, responsible for replum differentiation in
20
Brassica and Sinapis, seems not to have any effect on the evolution of indehiscence in
21
Lepidium appelianum. All sequences except that of L. appelianum are from (Arnaud et
22
al., 2011).
23
Amplification of the Shl region was performed using PhusionTM DNA polymerase.
24
Specific primers with an optimised annealing temperature of 52°C were designed basing
3
1
on sequence information from Arabidopsis lyrata, A. thaliana and Capsella rubella
2
(accessions given in Table S1). PCR products of about 600 bp were gel eluted using
3
NucleoSpin® Gel and PCR Clean-up (Macherey & Nagel) and directly sequenced.
4
5
Table S1
6
GenBank accession numbers of all genes used for this manuscript.
7
8
Table S2
9
Details of primers used for cloning and preparation of probes for Northern and Southern
10
blot hybridisation.
11
12
Table S3
13
Details of qRT-PCR primers and amplicons for each of the 20 analyzed genes
14
15
16
Supplementary References
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Arnaud, N., Lawrenson, T., Ostergaard, L. and Sablowski, R. (2011) The Same
Regulatory Point Mutation Changed Seed-Dispersal Structures in Evolution and
Domestication. Current Biology, 21, 1-5.
Campanella, J.J., Bitincka, L. and Smalley, J. (2003) MatGAT: An application that
generates similarity/identity matrices using protein or DNA sequences. BMC
Bioinformatics, 4.
Huelsenbeck, J.P. and Ronquist, F. (2001) MRBAYES: Bayesian inference of
phylogenetic trees. Bioinformatics, 17, 754-755.
Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A.,
McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson,
J.D., Gibson, T.J. and Higgins, D.G. (2007) Clustal W and clustal X version
2.0. Bioinformatics, 23, 2947-2948.
Nyylander, J.A.A. (2004) MrModeltest v2. Program distributed by the author.
Evolutionary Biology Centre, Uppsala University.
4
1
2
3
4
5
6
7
8
9
10
11
Ronquist, F. and Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic
inference under mixed models. Bioinformatics, 19, 1572-1574.
Southern, E.M. (1975) Detection of Specific Sequences Among DNA Fragments
Separated by Gel Electrophoresis. J Mol Biol, 98, 503-517.
Swofford, D.L. (2003) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other
Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
Zhang, Z., Li, J., Zhao, X.-Q., Wang, J., Wong, G.K.-S. and Yu, J. (2006)
KaKs_calculator: Calculating Ka and Ks through model selection and model
averaging. Genomics Proteomics & Bioinformatics, 4, 259-263.
5