A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets

Size: px

Start display at page:

Download "A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets"

Silas Wilkins
5 years ago
Views:

1 In the format provided by the authors and unedited. SUPPLEMENTARY INFORMATION VOLUME: 3 ARTICLE NUMBER: A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets Shinsuke Yasuda 1, Yuko Wada 1, Tomohiro Kakizaki 2, Yoshiaki Tarutani 1, Eiko Miura-Uno 1, Kohji Murase 1, Sota Fujii 1, Tomoya Hioki 1, Taiki Shimoda 1, Yoshinobu Takada 3, Hiroshi Shiba 1, Takeshi Takasaki-Yasuda 4, Go Suzuki 5, Masao Watanabe 3 * and Seiji Takayama 1,6 * In diploid organisms, phenotypic traits are often biased by methylation inducer (Smi) derived from an inverted repeat sequence 1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara , Japan. 2 Division of Vegetable Breeding, Institute of Vegetable and Floriculture Science, NARO, Tsu, Mie , Japan. 3 Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi , Japan. 4 Graduate School of Agricultural Science, Kobe University, Kobe, Hyogo , Japan. 5 Division of Natural Science, Osaka Kyoiku University, Kashiwara, Osaka , Japan. 6 Department of Applied Biological Chemistry, The University of Tokyo, Tokyo , Japan. Present address: Department of Integrated Genetics, National Institute of Genetics, Mishima, Shizuoka , Japan (Y.T.); Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki , Japan (H.S.). These authors contributed equally to this work. * nabe@ige.tohoku.ac.jp; takayama@bs.naist.jp NATURE PLANTS DOI: /nplants

2 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 S 60 -haplotype (AB097116) 16. We determined the entire S 60 -genomic sequence by obtaining the lacking SP11 downstream sequence by PCR amplification using primers designed from S 60 -SP11 and the flanking S-locus region of the class-i S 46 -haplotype (AB257128). The kb S 44 -, S 40 - and S 29 -genomic sequences between SP11 and a partial SRK sequence were previously reported 17. The sequence from SP11 downstream to the flanking S-locus region of the S 40 - haplotypes were also PCR-amplified using the primer designed from the S 46 -flanking region and specific S 40 -SP11 primer. For the S 44 -haplotype, a partial SP11 downstream sequence was obtained using Universal GenomeWalker TM 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 S 46 -flanking region and obtained partial S 44 -SP11 downstream sequence. For the S 29 -haplotype, a partial SP11 downstream sequence was PCR-amplified using the primers designed from S 29 -SMI and S 29 -SP11. Then, about 1 kb of downstream of SP11 was sequenced. Full-length SRK genomic fragments of S 44 -, S 40 - and S 29 -haplotypes were amplified using specific primers designed from SRK cdna sequences (AB211198, AB and AB008191, respectively). In the S 44 -, S 40 - and S 29 -haplotypes, each SRK NATURE PLANTS DOI: /nplants

3 SMI2 region was amplified using an SRK primer and SMI2 primers specific for each S-haplotype. For the S 29 -haplotype, the full-length SMI2 genomic fragment was amplified using primers designed based on the S 60 -SMI2 sequence. The S 60 -SMI2 genomic region was independently sequenced. The SMI2 SLG regions of S 44 - and S 40 -haplotypes were amplified using specific SMI2 primers and the primers designed based on each SLG cdna sequence (AB 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 S 60 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 package 30 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 program 31 with the default scoring matrix. We then assessed the secondary NATURE PLANTS DOI: /nplants

4 structures of the predicted inverted repeats using the RNAfold program 32 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 Omega 33. Conserved selection blocks from the alignment were selected using Gblocks 34 with default parameters, yielding a 330 amino acid alignment without gaps. The phylogenetic tree was constructed based on Bayesian inference using the MrBayes program 35 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. NATURE PLANTS DOI: /nplants

5 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. NATURE PLANTS DOI: /nplants

6 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. NATURE PLANTS DOI: /nplants

Supplementary Figure 3 Small RNA processing pattern from Smi2 precursors. srna sequencing analysis of anther srna from each class-ii homozygote.

7 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 S 44 -Smi2 (a), S 60 -Smi2 (b), S 40 -Smi2 (c) and S 29 -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. NATURE PLANTS DOI: /nplants

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.

8 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 S 9 -Smi2 (class-i) and S 60 -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 NATURE PLANTS DOI: /nplants

9 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. NATURE PLANTS DOI: /nplants

10 Supplementary Figure 5 Self-incompatibility phenotype of stigma of homozygotes with S 60 - SMI2 transgene. (a) Stigmas from S 40 S 40 homozygotes with S 60 -SMI2 transgene were pollinated with pollen from S 40 S 40 and S 60 S 60 homozygotes, respectively. (b) Stigmas from S 29 S 29 homozygotes with S 60 -SMI2 transgene were pollinated with pollen from S 29 S 29 and S 60 S 60 homozygotes, respectively. Bundle of pollen tubes (PT) indicates compatible pollination (arrow). NATURE PLANTS DOI: /nplants

11 Supplementary Table 1 Summary of inverted repeat regions from the S 44 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 S 44 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 E-04* ,803-7,936 - E, G ,692-8,799 + G E-04* ,692-8,799 - G E ,772-8,842 + G ,041-14,132 (SMI) + E, G, M ,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 ,873-28,935 - E, G, M ,183-46,291 + M ,410-46,491 + E, G N. H. N. H. N. H ,415-46,482 - E, G, M N. H ,493-46,548 + G ,495-46,546 - G ,286-51,457 (SMI2) + E, M E E-08 51,295-51,448 - E E-05* 0.001* 5.00E-08* NATURE PLANTS DOI: /nplants

12 Supplementary Table 2 Summary of inverted repeat regions from the S 60 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 S 60 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 ,417-11,508 (SMI) + E, G, M ,417-11,508 - E, G, M ,098-37,225 (SMI2) + E, G, M E E-10 37,098-37,225 - E, G E E-10 42,424-42,498 - G 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 ,044-43,123 - G, M ,091-43,208 + G ,184-43,231 - G N. H. N. H ,204-43,299 + G ,204-43,299 - G ,514-45,625 - M ,869-58,955 + M ,289-71,390 - M NATURE PLANTS DOI: /nplants

13 Supplementary Table 3 Summary of inverted repeat regions from the S 40 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 S 40 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 ,769-5,876 + M ,404-7,556 + E ,404-7,556 - E, M ,713-10,804 (SMI) + E, G, M ,768-26,870 + G E-24 26,773-26,865 - G E-23 36,180-36,333 + G E ,180-36,333 - G E ,261-46,434 (SMI2) + E, G, M E E-11 46,271-46,424 - E, G E E-11 47,644-47,805 + E 0.001* 1.00E-04* ,644-47,805 - E E NATURE PLANTS DOI: /nplants

14 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 nt reads Unique (18-45 nt ) Smi2 S44S44 17,257,506 8,142,282 2,520, S60S60 12,417,899 4,535,608 1,821,768 2 S40S40 16,628,424 7,443,950 2,766, S29S29 11,384,786 3,225,969 1,294,497 0 NATURE PLANTS DOI: /nplants

15 Supplementary Table 5 Primer sequences. Primers for genome sequencing S46 S-flanking F S60 SP11 downstream R S40 SP11 downstream R S44 SP11 downstream 5 -CGGTACCAAGATCAAGCACATTCCAG-3 5 -CTGAGGTAACTCAAGCAGATGTGATCTG-3 5 -CACCAAATCTTCCAATTTGTGATCTGAG-3 5 -GGGAGGATTAATTGCTACTGTTGCAAAG-3 GenomeWalk R S44 SP11 downstream 5 -CTATGCAATATACGGCGGCAGTGGATC-3 GenomeWalk R nested S44 SP11 downstream R S29 SMI F S29 SP11 downstream R S44 full SRK 5 F S40 full SRK 5 F S29 full SRK 5 F Class-II full SRK 3 R Class-II SRK 3 F S44 SMI2 R S40 SMI2 R S29 SMI2 R S29 full SMI2 F S29 full SMI2 R S44 SMI2 F S44 SLG R S40 SMI2 F S40 SLG R S44 SLG F S40 SLG F S-flanking region R 5 -TGGGTTCATGCATGTACCTGAGAGAAC-3 5 -CCTCGATTTGGTACATACAAGTACAACTG-3 5 -CACTAGATGTGGGAGCTAGGAAC-3 5 -TACACCTTCTCGTTCTTGCTAGTC-3 5 -AAAGGGTACATAACATTTACCAC-3 5 -TTGTCGGGGAGCGATGAAAAG-3 5 -TGGTGATTTGGTTCACTGTCC-3 5 -GAACCAAATCACCATGTCGATCATTGACG-3 5 -ACTACATGCGAGTCTATCAGTCACGAAG-3 5 -ACACGTTATAGACTACATGTGAGTCTATC-3 5 -TATCAACACGTTATAGACTACTTGTGAGTCTATCAG-3 5 -GAAGCTTCCTCTCTTTTCTTTATTCTCC-3 5 -TTGGTAAAATATATATTTATGYTC-3 5 -TTCGTGACTGATAGACTCGCATGTAGTC-3 5 -TTATTACTAGAGTTAACCGGTGCATGTGC-3 5 -TCTTTGTGACCGATAGACTCACATGTAGTC-3 5 -CCTTATTAGAGTTAACCGGTGCATGTGC-3 5 -GCACATGCACCGGTTAACTCTAGTAAT-3 5 -GCACATGCACCGGTTAACTCTAATAAGG-3 5 -ATCTTTTGCTGGAACTTGGGTTCAC-3 NATURE PLANTS DOI: /nplants

16 Supplementary Table 5 Primer sequences. Continued. SUPPLEMENTARY INFORMATION Primers for stem-loop RT-PCR S Smi2-RT S44 Smi2-RT mir166-rt mir166 F S60 Smi2 F S40 Smi2 F S29 Smi2 F universal RT 5 -GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACAAGATA-3 5 -GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACAAGACA-3 5 -GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACGGGGAA-3 5 -CAGCATCGGACCAGGCTTCA-3 5 -CGGCGGACACACCTTATTTGTGTA-3 5 -CGCCGTACACACTTTATTCGTGTA-3 5 -CGCGACACACGTTATTCGTGTA-3 5 -GTGCAGGGTCCGAGGT-3 Primers for S60-Smi2 expression constructs S60 SMI2 Hind F S60 SMI2 BamH R 5 -CCATGTCGATCATTGAAGCTTGGTAA-3 5 -CTAGGCCCGTCAGTATCACCGCTATTTTG-3 Primers for quantitative real-time PCR S44 SP11-RT-F S44 SP11-RT-R S60 SP11-RT-F S60 SP11-RT-R S40 SP11-RT-F S40 SP11-RT-R S29 SP11-RT-F S29 SP11-RT-R GAPDH-F GAPDH-R 5 -TTGACATATGTTCAAGCTCTAGATGTGG-3 5 -TCGTGGAGTTTAAGCATGATCCTCTG-3 5 -TGACATCTGTTCAAGCACTAGATGTGG-3 5 -TTACACTCTGTGCTCCTGGAATTAATGC-3 5 -TTGACATATGTTCAAGCACTAGATGTGG-3 5 -TAGACAGTCTTCGCTCACTGAATTTACG-3 5 -TGACATCTGTTCAAGCACTAGATGTG-3 5 -TGACAGTCTCTGCTCTTGGTATTTAAG-3 5 -GACCTTACTGTCAGACTCGAG-3 5 -CGGTGTATCCAAGGATTCCCT-3 Primers for bisulphite sequencing S40 SP11 F S40 SP11 R S40 SP11 F nested S40 SP11 R nested S29 SP11 F S29 SP11 R S29 SP11 F nested S29 SP11 R nested 5 -TTTATTAATTAAAATTTAAAGTGTATTT-3 5 -AATCCTAAATCCTCAACAAAAAAAA-3 5 -GTATTTTGAAGAAATATGAGAGGAG-3 5 -CTATATATATATTTTTCCTTCACATATC-3 5 -TGTGAAATTATTTTTAAAATGTTATTTTGT-3 5 -AAACAATTCCTAACTCCCACATCTA-3 5 -ATGTTATTTTGTTATTATGTAAGG-3 5 -CTCTAAATATATATATATTTTTTTCTTCAC-3 NATURE PLANTS DOI: /nplants

17 Supplementary References 30. Rice, P., Longden, I. & Bleasby, A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16, (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, (2007). 35. Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, (2012). NATURE PLANTS DOI: /nplants

Sperm cells are passive cargo of the pollen tube in plant fertilization

In the format provided by the authors and unedited. SUPPLEMENTARY INFORMATION VOLUME: 3 ARTICLE NUMBER: 17079 Sperm cells are passive cargo of the pollen tube in plant fertilization Jun Zhang 1, Qingpei