Figure S1. Alignment of the amino acid sequences of LgAP1 and other plant APs. Sequences of LgAP1, two
Arabidopsis APs (At1g31450; CDR1, At5g33340) and a poplar AP (PtAP1, XP_002304395) were aligned with
Clustal X. Sites that are conserved or show only conservative replacements among APs are marked underneath
with asterisks and colons, respectively. The amino acid groups were defined in Dyahoff et al. (1978): C, STAPG,
MILV, HRK, NDEQ, FYW. Signal peptides are boxed. Two aspartyl residues in catalytic sites are marked with
arrowheads.
Figure S2. Full phylogenetic tree for R2-R3-type MYB transcription factors. LgMYB21, subgroup 19 members
of other species and all Arabidopsis MYBs were used for construction of the phylogenetic tree. Amino acid
sequence information is listed in Table S4.
Figure S3. Alignment of the amino acid sequences of Linum GLXs. Signal peptides are boxed. Sites that are
conserved or show only conservative replacements among these GLXs are marked underneath with asterisks and
colons, respectively. Amino acid residues shown in red and black boxes are identical to those of spot C predicted
by de novo sequencing. Red boxes are peptides matched to the GLXs of other species (Table 3). The peptide
mass of the grey-boxed fragments is matched to that of the spot C trypsin fragments. Sequence coverage and
Mascot score to LgGLX5 were 36% and 55, respectively. Nineteen trypsin fragments of spot C matched
LgGLX5. Of these, 15 fragments were unique. No unique fragment matched the other GLXs. These facts
show that LgGLX5 encodes spot C and no contamination of other GLXs is involved in spot C.
Figure S4. Alignment of the amino acid sequences of LgSKS1 and other members of Group 3. Amino acid
sequences of LgSKS1 (AB617828), AtSKS12 (NM_104433), NTP303 (X61146) and SKU5 (AT4G12420)
were aligned by Clustal X. Sites that are conserved or show only conservative replacements among these SKSs
are marked underneath with asterisks and colons, respectively. Signal peptides deduced by SignalP 3.0 are boxed.
Amino acid residues shown in red and black boxes are identical to those of spot A2 predicted by de novo
sequencing. Red boxes are peptides matched to SKS of other species (Table 3). The peptide mass of the greyboxed fragments is matched to that of spot A1 and A2 trypsin fragments. Sequence coverage and Mascot score
to LgSKS1 were 18% and 54, respectively.
Figure S5. Amino acid sequence of LgMAT1. A sequence of LgMAT1 will appear in the
DDBJ/EMBL/GenBank databases under accession number AB617830. Amino acid residues shown in the red
box are identical to those of spot B predicted by de novo sequencing and matched to MAT of other species (Table
3). The peptide mass of the grey-boxed fragments is matched to that of the spot B trypsin fragments. Sequence
coverage and Mascot score to LgMAT1 were 40% and 38, respectively.
Figure S6. Amino acid sequence of LgFBA1. A sequence of LgMAT1 will appear in the
DDBJ/EMBL/GenBank databases under accession number AB649213. Amino acid residues shown in the
red and black boxes are identical to those of spot A2 predicted by de novo sequencing. Red boxes are
peptides matched to SKS of other species (Table 3). Sequence coverage and Mascot score to LgFBA1
were 44% and 853, respectively.
Figure S7. Full phylogenetic tree of the plant SKS family. A total of 96 plant SKSs, including LgSKS1 and eight
Laccases, were aligned by Clustal X. A phylogenetic tree was constructed by the neighbour-joining method
using Laccases as an outgroup. Plant SKSs formed four distinct groups, Groups 1–4, in the phylogenetic tree.
The SKSs of Group 3 were further divided into three subgroups, the SKSs of asterids, rosids and monocots.
Sequence information is listed in Table S6.
Figure S8. SNP genotyping of the floral morph-related genes. After PCR amplification, the products were
digested with an appropriate restriction enzyme and electrophoresed on a 1.5 or 3.5% agarose gel.
Figure S9. Transactivation activity of LgMYB21. (a) Alignment of the C-terminal region of MYB305 and
LgMYB21. Three acidic amino acid residues (D or E) of MYB305 marked with arrowheads are necessary for
transcriptional activity (Liu et al. 2010). An α-helix in the activation domain predicted by the PAPIA system
(http://mbs.cbrc.jp/papia/papia.html) is shown in the grey box. (b) Constructs used for transactivation activity
assay. The LacZ gene in MaV203 with the Gal4 DNA binding site on its promoter served as a reporter (c). (d)
Assays for β-galactosidase activity in yeast. Sequence analysis using the PAPIA system predicted an α-helix
structure at the C-terminal region of LgMYB21 (Fig. S3a). However, the last acidic residue was not conserved in
LgMYB21. To assay transactivation activity, we performed yeast-one hybrid analysis. We conducted PCR to
amplify the fulllength ORF of LgMYB21 and truncated one that lacked a C-terminal activation domain. These
fragments were cloned into pENTR/D-TOPO, an entry vector, using a pENTR Directional TOPO Cloning Kit
(Invitrogen). The resultant entry vectors (b) were recombined with pDEST32, a destination vector possessing a
GAL4 DNA binding domain, using Gateway LR Clonase II. The constructs were transformed into a yeast strain,
MaV203, whose genome possessed the LacZ reporter gene (c). The transformed yeast cells were subjected to a
β-galactosidase activity assay according to the manufacturer’s instructions. Intact LgMYB21 could activate
expression of the reporter gene, but the lack of a C-terminal caused loss of activity (d). These results indicated
that replacement of the last residue has no affect on transactivation activity and that LgMYB21 is expected to
function in the same manner as the other subgroup 19 MYBs.
Figure S10. Melting curve evaluation of single pollen genotyping. No fragment (a) or primer dimmer (b) was
amplified from the blank tube. Only actin fragments were amplified from the PCR tubes in which pollen with no
TSS1 gene was contained (c). In contrast, tubes containing a pollen grain with TSS1 gene should show TSS1
peak (d).
Table S1. Sequence information for SSH clones.
Table S2. Sequence information for TSS1 phylogenetic analysis.
Table S3. Sequence information for AP phylogenetic analysis.
Table S4. Sequence information for MYB phylogenetic analysis.
Table S5. qPCR primer information for LgGLXs, LgSKS1 and LgMAT1.
Table S6. Sequence information for SKS phylogenetic tree.
Table S7. Primer information for PCR genotyping.
Table S8. Primers and restriction enzymes for CAPS and dCAPS.