Complex dominance hierarchy controlled by polymorphism of small RNAs and their targets
Supplementary Methods Genome sequencing
The nucleotide sequence of an 86.4 kb partial S-locus region spanning the SP11, SRK and SLG genes
was previously reported in the S60-haplotype (AB097116)16. We determined the entire S60-genomic
sequence by obtaining the lacking SP11 downstream sequence by PCR amplification using primers
designed from S60-SP11 and the flanking S-locus region of the class-I S46-haplotype (AB257128). The
14–18 kb S44-, S40- and S29-genomic sequences between SP11 and a partial SRK sequence were
previously reported17. The sequence from SP11 downstream to the flanking S-locus region of the S40-
haplotypes were also PCR-amplified using the primer designed from the S46-flanking region and
specific S40-SP11 primer. For the S44-haplotype, a partial SP11 downstream sequence was obtained
using Universal GenomeWalkerTM 2.0 (Clontech). Then, the sequence from SP11 downstream to the
flanking S-locus region were also PCR-amplified using the primers designed from the S46-flanking
region and obtained partial S44-SP11 downstream sequence. For the S29-haplotype, a partial SP11
downstream sequence was PCR-amplified using the primers designed from S29-SMI and S29-SP11.
Then, about 1 kb of downstream of SP11 was sequenced. Full-length SRK genomic fragments of S44-,
S40- and S29-haplotypes were amplified using specific primers designed from SRK cDNA sequences
(AB211198, AB211197 and AB008191, respectively). In the S44-, S40- and S29-haplotypes, each SRK–
SMI2 region was amplified using an SRK primer and SMI2 primers specific for each S-haplotype. For
the S29-haplotype, the full-length SMI2 genomic fragment was amplified using primers designed based
on the S60-SMI2 sequence. The S60-SMI2 genomic region was independently sequenced. The SMI2–
SLG regions of S44- and S40-haplotypes were amplified using specific SMI2 primers and the primers
designed based on each SLG cDNA sequence (AB054059 and AB054058). The regions from SLG to
the flanking region of the S-locus were amplified using a specific SLG primer and a primer designed
based on the sequence of the S60 S-locus flanking region. Each PCR-amplified product was fragmented
using the DNA Fragmentation Kit (Takara), cloned into pGEM-T Easy Vectors (Promega) and
sequenced.
Prediction of sRNA precursor regions
Inverted repeats in each class-II S-locus were predicted using three programs. First, we used the
einverted program in the EMBOSS package30 with the default scoring matrix and maximum extent of
repeats of 350 bp. From these results, we selected inverted repeats with a maximal terminal loop size
of 50 bp. Second, we performed a hairpin search using the GENETYX-WIN software with the
following score matrix: match % of stem parts = 75; max size of stem parts = 200; min size of stem
parts = 30; max size of loop parts = 50; min size of loop parts = 4. Third, we identified inverted repeats
structures of the predicted inverted repeats using the RNAfold program32 and identified hairpin
structure with low free energy (< ˗25 kcal mol˗1). Predicted stem-loops carrying homologous
sequences to the four S-haplotypes of SP11 sequences ± 1 kb were further selected by a BLAST search.
Phylogenetic analyses
The deduced amino acid sequences of the SRK ectodomain were multiply aligned using Clustal
Omega33. Conserved selection blocks from the alignment were selected using Gblocks34 with default
parameters, yielding a 330 amino acid alignment without gaps. The phylogenetic tree was constructed
based on Bayesian inference using the MrBayes 3.2.2 program35 by setting B. rapa SLR1-2
(AB016534) and SLR1-4 (AB016535) as the outgroup sequences. Four chains of Metropolis-coupled
Markov Chain Monte Carlo processes were run for 2,000,000 generations, with trees sampled for
every 1,000 generations. The first 25% of trees were discarded, and the remainder were used to support
the majority rule consensus tree topology with posterior probabilities.
Supplementary Figure 1 | Sequence alignment of Smi2 precursors. Identical sequences are
indicated by asterisks. Mature Smi2 sequences are underlined in magenta. Arrows indicate the inverted
repeat region.
Supplementary Figure 2 | Phylogenetic tree of SRK alleles from B. rapa. The phylogenetic tree is
based on the SRK ectodomain. GenBank accession numbers are provided in parentheses.
Supplementary Figure 3 | Small RNA processing pattern from Smi2 precursors. sRNA
sequencing analysis of anther sRNA from each class-II homozygote. The arrows indicate sRNAs
mapped on the stem regions of S44-Smi2 (a), S60-Smi2 (b), S40-Smi2 (c) and S29-Smi2 (d) precursor
sequences. Numbers on the head side of arrows show total sRNA reads, and the numbers on the tail
side describe nucleotide length. Smi2 sequences are indicated in magenta.
Supplementary Figure 4 | Smi2 and Smi sequences homologous to the promoter region of class-
II SP11 alleles. (a) Class-II SP11 promoter region targeted by Smi and Smi2. Nucleotide substitutions
among sRNAs and promoter regions are indicated in blue and magenta, respectively. The translation
start site of SP11 is assigned position +1. (b) Sequence complementarity of the S9-Smi2 (class-I) and
S60-Smi2 (class-II) against the antisense strand of the SP11 promoter regions. ‘Matched bases’ indicates
the number of matched bases between the 21 nt region 5’ of Smi2 and class-II SP11 promoters. The
box indicates the core segment. The mispair score was calculated as described in the Methods.
Mismatched bases relative to Smi2 are indicated in magenta. G:U pairs are indicated in blue. (c)
Sequence alignment of Smi2 sequences and the class-II SP11 promoter regions.
Supplementary Figure 5 | Self-incompatibility phenotype of stigma of homozygotes with S60-
SMI2 transgene. (a) Stigmas from S40S40 homozygotes with S60-SMI2 transgene were pollinated with
pollen from S40S40 and S60S60 homozygotes, respectively. (b) Stigmas from S29S29 homozygotes with
S60-SMI2 transgene were pollinated with pollen from S29S29 and S60S60 homozygotes, respectively.
Bundle of pollen tubes (PT) indicates compatible pollination (arrow).
Supplementary Table 1 | Summary of inverted repeat regions from the S44 S-locus predicted by
three programs. Predicted stem-loop sequences were used as the query in a homology search against
the four alleles of SP11 sequences ± 1 kb. ‘Seq. Pos.’ indicates the location of each stem-loop sequence
in the S44 S-locus. * We did not detect sRNAs homologous to the target. †No sRNAs are produced
from this stem-loop sequence. E; einverted, G; Genetyx, M; miRPara. N. H., no hits found.
E-value
Seq. Pos. Strand Software S44-SP11 S60-SP11 S40-SP11 S29-SP11 7,809-7,931 + E, G 0.011 9.00E-04* 0.011 0.011 7,803-7,936† - E, G 0.012 0.001 0.012 0.012 8,692-8,799 + G 0.032 0.110 8.00E-04* 0.032 8,692-8,799† - G 0.032 0.110 8.00E-04 0.032 8,772-8,842 + G 0.840 0.240 2.900 0.840 14,041-14,132 (SMI) + E, G, M 0.002 0.002 0.002 0.002 14,041-14,132 - E, G, M 0.002* 0.002* 0.002* 0.002*
26,529-26,752 - E N. H. N. H. N. H. N. H.
28,873-28,935 + E, G, M 2.500 0.210 0.059 2.500 28,873-28,935 - E, G, M 2.500 0.210 0.059 2.500 46,183-46,291 + M 0.390 4.800 0.032 0.110 46,410-46,491 + E, G N. H. N. H. N. H. 3.500
46,415-46,482 - E, G, M 9.700 N. H. 9.700 2.800 46,493-46,548 + G 2.200 7.600 7.600 7.600 46,495-46,546 - G 2.000 6.900 6.900 6.900 51,286-51,457 (SMI2) + E, M 0.053 8.00E-06 0.001 6.00E-08 51,295-51,448 - E 0.048 3.00E-05* 0.001* 5.00E-08*
Supplementary Table 2 | Summary of inverted repeat regions from the S60 S-locus predicted by
three programs. Predicted stem-loop sequences were used as the query in a homology search against
the four alleles of SP11 sequences ± 1 kb. ‘Seq. Pos.’ indicates the location of each stem-loop sequence
in the S60 S-locus. †No sRNAs are produced from this stem-loop sequence. E; einverted, G; Genetyx,
M; miRPara. N. H., no hits found.
E-value
Seq. Pos. Strand Software S44-SP11 S60-SP11 S40-SP11 S29-SP11 4,092-4,182 - M 0.320 0.320 1.100 0.320 11,417-11,508 (SMI) + E, G, M 0.002 0.002 0.002 0.002 11,417-11,508† - E, G, M 0.002 0.002 0.002 0.002 37,098-37,225 (SMI2) + E, G, M 0.140 0.011 2.00E-05 3.00E-10 37,098-37,225† - E, G 0.140 0.011 2.00E-05 3.00E-10 42,424-42,498 - G 0.900 N. H. N. H. N. H.
42,450-42,552 - E, G N. H. N. H. N. H. N. H.
42,464-42,538 + E, G N. H. N. H. N. H. N. H.
43,043-43,124 + G 1.000 0.290 3.500 1.000 43,044-43,123 - G, M 0.970 0.280 3.400 0.970 43,091-43,208 + G 0.120 0.120 0.430 0.120 43,184-43,231 - G 0.510 N. H. N. H. 1.800
43,204-43,299 + G 1.200 4.100 1.200 1.200 43,204-43,299 - G 1.200 4.100 1.200 1.200 45,514-45,625 - M 0.120 0.010 0.010 0.033 58,869-58,955 + M 1.100 0.300 1.100 1.100 71,289-71,390 - M 0.360 4.400 4.400 1.300
Supplementary Table 3 | Summary of inverted repeat regions from the S40 S-locus predicted by
three programs. Predicted stem-loop sequences were used as the query in a homology search against
the four alleles of SP11 sequences ± 1 kb. ‘Seq. Pos.’ indicates the location of each stem-loop sequence
in the S40 S-locus. * We did not detect sRNAs homologous to target. †No sRNAs are produced from
this stem-loop sequence. E; einverted, G; Genetyx, M; miRPara. N. H., no hits found.
E-value
Seq. Pos. Strand Software S44-SP11 S60-SP11 S40-SP11 S29-SP11 4,536-4,616 + E, M N. H. N. H. 0.081 3.400 5,769-5,876 + M 1.400 1.400 1.400 1.400 7,404-7,556 + E 0.580 2.000 7.000 2.000 7,404-7,556 - E, M 0.580 2.000 7.000 2.000 10,713-10,804 (SMI) + E, G, M 0.008 0.002 0.008 0.008 26,768-26,870† + G 0.370 0.009 0.370 6.00E-24 26,773-26,865† - G 0.330 0.027 0.330 6.00E-23 36,180-36,333† + G 0.048 3.00E-04 0.014 0.004
36,180-36,333† - G 0.048 3.00E-04 0.014 0.004 46,261-46,434 (SMI2) + E, G, M 0.540 1.00E-04 0.001 3.00E-11 46,271-46,424† - E, G 0.048 3.00E-04 0.001 3.00E-11 47,644-47,805 + E 0.001* 1.00E-04* 0.014 0.014
47,644-47,805† - E 0.001 1.00E-04 0.014 0.014
Supplementary Table 4 | Summary of small RNA sequences obtained from each class-II S-
homozygote. ‘Unique’ indicates non-redundant sequence reads of a particular type.
Sample Total reads 18-45 nt reads Unique (18-45 nt ) Smi2
S44S44 17,257,506 8,142,282 2,520,432 302 S60S60 12,417,899 4,535,608 1,821,768 2 S40S40 16,628,424 7,443,950 2,766,551 268 S29S29 11,384,786 3,225,969 1,294,497 0
Supplementary Table 5 | Primer sequences.
Primers for genome sequencing
S46 S-flanking F 5’-CGGTACCAAGATCAAGCACATTCCAG-3’
S60 SP11 downstream R 5’-CTGAGGTAACTCAAGCAGATGTGATCTG-3’
S40 SP11 downstream R 5’-CACCAAATCTTCCAATTTGTGATCTGAG-3’
S44 SP11 downstream GenomeWalk R
5’-GGGAGGATTAATTGCTACTGTTGCAAAG-3’
S44 SP11 downstream GenomeWalk R nested
5’-CTATGCAATATACGGCGGCAGTGGATC-3’
S44 SP11 downstream R 5’-TGGGTTCATGCATGTACCTGAGAGAAC-3’
S29 SMI F 5’-CCTCGATTTGGTACATACAAGTACAACTG-3’
S29 SP11 downstream R 5’-CACTAGATGTGGGAGCTAGGAAC-3’
S44 full SRK 5’ F 5’-TACACCTTCTCGTTCTTGCTAGTC-3’
S40 full SRK 5’ F 5’-AAAGGGTACATAACATTTACCAC-3’
S29 full SRK 5’ F 5’-TTGTCGGGGAGCGATGAAAAG-3’
Class-II full SRK 3’ R 5’-TGGTGATTTGGTTCACTGTCC-3’
Class-II SRK 3’ F 5’-GAACCAAATCACCATGTCGATCATTGACG-3’
S44 SMI2 R 5’-ACTACATGCGAGTCTATCAGTCACGAAG-3’
S40 SMI2 R 5’-ACACGTTATAGACTACATGTGAGTCTATC-3’
S29 SMI2 R 5’-TATCAACACGTTATAGACTACTTGTGAGTCTATCAG-3’
S29 full SMI2 F 5’-GAAGCTTCCTCTCTTTTCTTTATTCTCC-3’
S29 full SMI2 R 5’-TTGGTAAAATATATATTTATGYTC-3’
S44 SMI2 F 5’-TTCGTGACTGATAGACTCGCATGTAGTC-3’
S44 SLG R 5’-TTATTACTAGAGTTAACCGGTGCATGTGC-3’
S40 SMI2 F 5’-TCTTTGTGACCGATAGACTCACATGTAGTC-3’
S40 SLG R 5’-CCTTATTAGAGTTAACCGGTGCATGTGC-3’
S44 SLG F 5’-GCACATGCACCGGTTAACTCTAGTAAT-3’
S40 SLG F 5’-GCACATGCACCGGTTAACTCTAATAAGG-3’
S-flanking region R 5’-ATCTTTTGCTGGAACTTGGGTTCAC-3’
Supplementary Table 5 | Primer sequences. Continued.
Primers for stem-loop RT-PCR
S604029 Smi2-RT 5’-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACAAGATA-3’
S44 Smi2-RT 5’-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACAAGACA-3’
miR166-RT 5’-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACGGGGAA-3’
miR166 F 5’-CAGCATCGGACCAGGCTTCA-3’
S60 Smi2 F 5’-CGGCGGACACACCTTATTTGTGTA-3’
S40 Smi2 F 5’-CGCCGTACACACTTTATTCGTGTA-3’
S29 Smi2 F 5’-CGCGACACACGTTATTCGTGTA-3’
universal RT 5’-GTGCAGGGTCCGAGGT-3’
Primers for S60-Smi2 expression constructs
S60 SMI2 Hind F 5’-CCATGTCGATCATTGAAGCTTGGTAA-3’
S60 SMI2 BamH R 5’-CTAGGCCCGTCAGTATCACCGCTATTTTG-3’
Primers for quantitative real-time PCR
S44 SP11-RT-F 5’-TTGACATATGTTCAAGCTCTAGATGTGG-3’
S44 SP11-RT-R 5’-TCGTGGAGTTTAAGCATGATCCTCTG-3’
S60 SP11-RT-F 5’-TGACATCTGTTCAAGCACTAGATGTGG-3’
S60 SP11-RT-R 5’-TTACACTCTGTGCTCCTGGAATTAATGC-3’
S40 SP11-RT-F 5’-TTGACATATGTTCAAGCACTAGATGTGG-3’
S40 SP11-RT-R 5’-TAGACAGTCTTCGCTCACTGAATTTACG-3’
S29 SP11-RT-F 5’-TGACATCTGTTCAAGCACTAGATGTG-3’
S29 SP11-RT-R 5’-TGACAGTCTCTGCTCTTGGTATTTAAG-3’
GAPDH-F 5’-GACCTTACTGTCAGACTCGAG-3’
GAPDH-R 5’-CGGTGTATCCAAGGATTCCCT-3’
Primers for bisulphite sequencing
S40 SP11 F 5’-TTTATTAATTAAAATTTAAAGTGTATTT-3’
S40 SP11 R 5’-AATCCTAAATCCTCAACAAAAAAAA-3’
S40 SP11 F nested 5’-GTATTTTGAAGAAATATGAGAGGAG-3’
S40 SP11 R nested 5’-CTATATATATATTTTTCCTTCACATATC-3’
S29 SP11 F 5’-TGTGAAATTATTTTTAAAATGTTATTTTGT-3’
S29 SP11 R 5’-AAACAATTCCTAACTCCCACATCTA-3’
S29 SP11 F nested 5’-ATGTTATTTTGTTATTATGTAAGG-3’
S29 SP11 R nested 5’-CTCTAAATATATATATATTTTTTTCTTCAC-3’
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