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A complex dominance hierarchy is controlled bypolymorphism of small RNAs and their targetsShinsuke Yasuda1‡, Yuko Wada1‡, Tomohiro Kakizaki2‡, Yoshiaki Tarutani1†, Eiko Miura-Uno1,Kohji Murase1, Sota Fujii1, Tomoya Hioki1, Taiki Shimoda1, Yoshinobu Takada3, Hiroshi Shiba1†,Takeshi Takasaki-Yasuda4, Go Suzuki5, Masao Watanabe3* and Seiji Takayama1,6*

In diploid organisms, phenotypic traits are often biased byeffects known as Mendelian dominant–recessive interactionsbetween inherited alleles. Phenotypic expression of SP11alleles, which encodes the male determinants of self-incompat-ibility in Brassica rapa, is governed by a complex dominancehierarchy1–3. Here, we show that a single polymorphic 24nucleotide small RNA, named SP11 methylation inducer 2(Smi2), controls the linear dominance hierarchy of the fourSP11 alleles (S44 > S60 > S40 > S29). In all dominant–recessiveinteractions, small RNA variants derived from the linkedregion of dominant SP11 alleles exhibited high sequence simi-larity to the promoter regions of recessive SP11 alleles andacted in trans to epigenetically silence their expression.Together with our previous study4, we propose a new model;sequence similarity between polymorphic small RNAs andtheir target regulates mono-allelic gene expression, whichexplains the entire five-phased linear dominance hierarchy ofthe SP11 phenotypic expression in Brassica.

Most Mendelian dominance relationships are explained by loss-of-function mutations in haplosufficient genes, which make thewild-type allele dominant over the mutant allele. In a few cases,however, dominance is thought to be controlled by other geneticelements called ‘dominance modifiers’5,6. In one such case, a smallRNA (sRNA) controls the dominance of pollen self-incompatibilityphenotypes in Brassica rapa4. Self-incompatibility is a geneticmechanism, controlled by multiple S-haplotypes at the S-locus,for avoiding self-fertilization by rejecting self-pollen to maintaingenetic diversity in the species7. For the Brassicaceae self-incompat-ibility, each S-haplotype encodes a male determinant, S-locusprotein 11 (SP11, also called SCR)8–10, and a female determinant,S-locus receptor kinase (SRK)11,12; the S-haplotype-specific inter-action between these determinants triggers a self-incompatibilityresponse in the stigma epidermis13,14.

SP11 is sporophytically expressed in the anther tapetum, and theself-incompatibility phenotypes in pollen are determined by domi-nance relationships between the two S-haplotypes that the plantcarries. According to the dominance of pollen self-incompatibilityphenotypes, the S-haplotypes in B. rapa have been categorized asclass-I (for example, S8, S9, S12 and S52) or class-II (for example,S44, S60, S40 and S29), with class-I S-haplotypes dominating overclass-II S-haplotypes1–3. When class-I and class-II S-haplotypesare heterozygous, a 24 nucleotide (nt) sRNA called SP11

methylation inducer (Smi) derived from an inverted repeat sequenceof class-I S-haplotype induces DNA methylation of the recessiveclass-II SP11 promoter and results in mono-allelic gene silencing4,15.Interestingly, class-II S-haplotypes exhibit linear dominancerelationships, resulting in a complicated dominance hierarchy:class-I S > (S44 > S60 > S40 > S29)

3. However, the molecular mechan-ism underlying the linear dominance hierarchy within class-IIS-haplotypes remains unknown.

To elucidate the molecular mechanism underlying the lineardominance hierarchy of class-II S-haplotypes in B. rapa, wesequenced the full S-locus regions of three class-II S-haplotypes,S44, S60 and S40, for which partial S-locus sequences were previouslypublished16,17. We also determined the partial S-locus sequence ofS29 haplotype. The sRNA gene candidates in the S44, S60 and S40S-locus regions were identified using three de novo sRNA predictionprograms. Predicted stem-loops encoding sequences homologous tothe four S-haplotypes of SP11 sequences ±1 kb were further selectedusing a BLAST search. As a result, we identified two inverted repeatsequences in all of the S-haplotypes (Supplementary Tables 1–3):the previously identified SMI3 and a novel SMI-like sequence,SP11 METHYLATION INDUCER 2 (SMI2) (SupplementaryFig. 1). In each S-haplotype, SMI2 was located 1.9–8.6 kb down-stream of SRK but was not observed in the SP11 genomic regionof the class-I S-haplotype in B. rapa8,18,19 (Fig. 1a). SMI2 was onlyobserved in class-II S-haplotypes, which formed a distinctcluster from class-I S-haplotypes in the phylogenetic analysis(Supplementary Fig. 2 and Supplementary Methods).

To determine whether SMI2 in each S-haplotype is expressed andprocessed into sRNAs at the relevant developmental stage, wesequenced sRNAs from early stage (stages 1–3) anthers10, duringwhich SP11 DNA methylation occurs15. We obtained 11–17million sRNA sequences for each class-II S-homozygote(Supplementary Table 4). The mapping of sRNA sequences tocognate SMI2 transcripts revealed that 24 nt sRNAs (designatedSmi2) with sequence similarity to class-II SP11 promoters were pro-cessed from the stem structures of S44-, S60- and S40-Smi2 precursors(Fig. 1b). Although we observed multiple distinct sRNA fragmentsfrom the precursor of the most recessive S29-haplotype, we didnot detect mature Smi2 (Supplementary Fig. 3). To accuratelymeasure the amount of processed Smi2, we performed stem-loopPCR with reverse transcription (RT–PCR)20 using sRNA extractedfrom early stage anthers from S44-, S60-, S40- and S29-homozygotes.

1Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan. 2Division of Vegetable Breeding, Instituteof Vegetable and Floriculture Science, NARO, Tsu, Mie 514-2392, Japan. 3Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8577,Japan. 4Graduate School of Agricultural Science, Kobe University, Kobe, Hyogo 657-8501, Japan. 5Division of Natural Science, Osaka Kyoiku University,Kashiwara, Osaka 582-8582, Japan. 6Department of Applied Biological Chemistry, The University of Tokyo, Tokyo 113-8657, Japan. †Present address:Department of Integrated Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan (Y.T.); Graduate School of Life and EnvironmentalSciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan (H.S.). ‡These authors contributed equally to this work. *e-mail: [email protected];[email protected]

LETTERSPUBLISHED: XX XX 2016 | VOLUME: 3 | ARTICLE NUMBER: 16206

NATURE PLANTS 3, 16206 (2016) | DOI: 10.1038/nplants.2016.206 | www.nature.com/natureplants 1

A complex dominance hierarchy is controlled bypolymorphism of small RNAs and their targetsShinsuke Yasuda1‡, Yuko Wada1‡, Tomohiro Kakizaki2‡, Yoshiaki Tarutani1†, Eiko Miura-Uno1,Kohji Murase1, Sota Fujii1, Tomoya Hioki1, Taiki Shimoda1, Yoshinobu Takada3, Hiroshi Shiba1†,Takeshi Takasaki-Yasuda4, Go Suzuki5, Masao Watanabe3* and Seiji Takayama1,6*

In diploid organisms, phenotypic traits are often biased byeffects known as Mendelian dominant–recessive interactionsbetween inherited alleles. Phenotypic expression of SP11alleles, which encodes the male determinants of self-incompat-ibility in Brassica rapa, is governed by a complex dominancehierarchy1–3. Here, we show that a single polymorphic 24nucleotide small RNA, named SP11 methylation inducer 2(Smi2), controls the linear dominance hierarchy of the fourSP11 alleles (S44 > S60 > S40 > S29). In all dominant–recessiveinteractions, small RNA variants derived from the linkedregion of dominant SP11 alleles exhibited high sequence simi-larity to the promoter regions of recessive SP11 alleles andacted in trans to epigenetically silence their expression.Together with our previous study4, we propose a new model;sequence similarity between polymorphic small RNAs andtheir target regulates mono-allelic gene expression, whichexplains the entire five-phased linear dominance hierarchy ofthe SP11 phenotypic expression in Brassica.

Most Mendelian dominance relationships are explained by loss-of-function mutations in haplosufficient genes, which make thewild-type allele dominant over the mutant allele. In a few cases,however, dominance is thought to be controlled by other geneticelements called ‘dominance modifiers’5,6. In one such case, a smallRNA (sRNA) controls the dominance of pollen self-incompatibilityphenotypes in Brassica rapa4. Self-incompatibility is a geneticmechanism, controlled by multiple S-haplotypes at the S-locus,for avoiding self-fertilization by rejecting self-pollen to maintaingenetic diversity in the species7. For the Brassicaceae self-incompat-ibility, each S-haplotype encodes a male determinant, S-locusprotein 11 (SP11, also called SCR)8–10, and a female determinant,S-locus receptor kinase (SRK)11,12; the S-haplotype-specific inter-action between these determinants triggers a self-incompatibilityresponse in the stigma epidermis13,14.

SP11 is sporophytically expressed in the anther tapetum, and theself-incompatibility phenotypes in pollen are determined by domi-nance relationships between the two S-haplotypes that the plantcarries. According to the dominance of pollen self-incompatibilityphenotypes, the S-haplotypes in B. rapa have been categorized asclass-I (for example, S8, S9, S12 and S52) or class-II (for example,S44, S60, S40 and S29), with class-I S-haplotypes dominating overclass-II S-haplotypes1–3. When class-I and class-II S-haplotypesare heterozygous, a 24 nucleotide (nt) sRNA called SP11

methylation inducer (Smi) derived from an inverted repeat sequenceof class-I S-haplotype induces DNA methylation of the recessiveclass-II SP11 promoter and results in mono-allelic gene silencing4,15.Interestingly, class-II S-haplotypes exhibit linear dominancerelationships, resulting in a complicated dominance hierarchy:class-I S > (S44 > S60 > S40 > S29)

3. However, the molecular mechan-ism underlying the linear dominance hierarchy within class-IIS-haplotypes remains unknown.

To elucidate the molecular mechanism underlying the lineardominance hierarchy of class-II S-haplotypes in B. rapa, wesequenced the full S-locus regions of three class-II S-haplotypes,S44, S60 and S40, for which partial S-locus sequences were previouslypublished16,17. We also determined the partial S-locus sequence ofS29 haplotype. The sRNA gene candidates in the S44, S60 and S40S-locus regions were identified using three de novo sRNA predictionprograms. Predicted stem-loops encoding sequences homologous tothe four S-haplotypes of SP11 sequences ±1 kb were further selectedusing a BLAST search. As a result, we identified two inverted repeatsequences in all of the S-haplotypes (Supplementary Tables 1–3):the previously identified SMI3 and a novel SMI-like sequence,SP11 METHYLATION INDUCER 2 (SMI2) (SupplementaryFig. 1). In each S-haplotype, SMI2 was located 1.9–8.6 kb down-stream of SRK but was not observed in the SP11 genomic regionof the class-I S-haplotype in B. rapa8,18,19 (Fig. 1a). SMI2 was onlyobserved in class-II S-haplotypes, which formed a distinctcluster from class-I S-haplotypes in the phylogenetic analysis(Supplementary Fig. 2 and Supplementary Methods).

To determine whether SMI2 in each S-haplotype is expressed andprocessed into sRNAs at the relevant developmental stage, wesequenced sRNAs from early stage (stages 1–3) anthers10, duringwhich SP11 DNA methylation occurs15. We obtained 11–17million sRNA sequences for each class-II S-homozygote(Supplementary Table 4). The mapping of sRNA sequences tocognate SMI2 transcripts revealed that 24 nt sRNAs (designatedSmi2) with sequence similarity to class-II SP11 promoters were pro-cessed from the stem structures of S44-, S60- and S40-Smi2 precursors(Fig. 1b). Although we observed multiple distinct sRNA fragmentsfrom the precursor of the most recessive S29-haplotype, we didnot detect mature Smi2 (Supplementary Fig. 3). To accuratelymeasure the amount of processed Smi2, we performed stem-loopPCR with reverse transcription (RT–PCR)20 using sRNA extractedfrom early stage anthers from S44-, S60-, S40- and S29-homozygotes.

1Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan. 2Division of Vegetable Breeding, Instituteof Vegetable and Floriculture Science, NARO, Tsu, Mie 514-2392, Japan. 3Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8577,Japan. 4Graduate School of Agricultural Science, Kobe University, Kobe, Hyogo 657-8501, Japan. 5Division of Natural Science, Osaka Kyoiku University,Kashiwara, Osaka 582-8582, Japan. 6Department of Applied Biological Chemistry, The University of Tokyo, Tokyo 113-8657, Japan. †Present address:Department of Integrated Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan (Y.T.); Graduate School of Life and EnvironmentalSciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan (H.S.). ‡These authors contributed equally to this work. *e-mail: [email protected];[email protected]

LETTERSPUBLISHED: XX XX 2016 | VOLUME: 3 | ARTICLE NUMBER: 16206

NATURE PLANTS 3, 16206 (2016) | DOI: 10.1038/nplants.2016.206 | www.nature.com/natureplants 1

© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONVOLUME: 3 | ARTICLE NUMBER: 16206

NATURE PLANTS | DOI: 10.1038/nplants.2016.206 | www.nature.com/natureplants 1

http://dx.doi.org/10.1038/nplants.2016.206

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–



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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

using the miRPara program31 with the default scoring matrix. We then assessed the secondary





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*








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


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








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


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’





Supplementary References

30. Rice, P., Longden, I. & Bleasby, A. EMBOSS: the European Molecular Biology Open Software

Suite. Trends Genet. 16, 276–277 (2000).

31. Wu, Y., Wei, B., Liu, H., Li, T. & Rayner, S. MiRPara: a SVM-based software tool for prediction

of most probable microRNA coding regions in genome scale sequences. BMC Bioinformatics 12,

107 (2011).

32. Lorenz, R. et al. ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26 (2011).

33. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments

using Clustal Omega. Mol. Syst. Biol. 7, 539 (2014).

34. Talavera, G. & Castresana, J. Improvement of phylogenies after removing divergent and

ambiguously aligned blocks from protein sequence alignments. Syst. Biol. 56, 564–577 (2007).

35. Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice

across a large model space. Syst. Biol. 61, 539–542 (2012).





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