Supplementary Figure 1. Number of CC- and TIR- type NBS- LRR genes and presence of mir482/2118 on sequenced plant genomes.

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1 Number of CC- NBS and CC- NBS- LRR R- genes Number of TIR- NBS and TIR- NBS- LRR R- genes mir482 and mir2118 Cajanus cajan Glycine max Hevea brasiliensis Malus domes;ca Manihot esculenta Medicago truncatula Phaseolus vulgaris Prunus mume Prunus persica Pyrus bretschneideri Rehd. Solanum lycopersicum Solanum tuberosum Only mir482 Aquilegia coerulea Arabidopsis lyrata Arabidopsis thaliana Brassica rapa Capsella rubella Citrus sinensis Eucalyptus grandis Gossypium raimondii Jatropha curcas L. Picea abies Populus trichocarpa Ricinus communis Thelungiella parvula Theobroma cacao Vi;s vinifera Only mir2118 Brachypodium dystachion Cicer arie;num Musa acuminata Oryza sa;va L. ssp. Japonica Phyllostachys heterocycla Setaria italica Sorghum bicolor Zea mays No mir482/2118 Amborella trichopoda Carica Papaya Chlamydomonas reinhard;i Citrullus lanatus Cucumis melo L. Cucumis sa;vus L. Fragaria vesca Linum usita;ssimum Lotus japonicus Mimulus guqatus Phoenix dactylifera L. Physcomitrella patens Selaginella moellendorffii Thelungiella halophilla Supplementary Figure 1. Number of CC- and TIR- type NBS- LRR genes and presence of mir482/2118 on sequenced plant genomes.

2 mirna 482 a e b f P. persica, chr1 ppa004208m-ppa005754m 29,608,060 29,742, C. melo, scaffold00016, LG X MELO3C MELO3C ,153,667 4,227,336 Supplementary Figure 2. Overview of microsyntey between a 174 kb peach(prunus persica) sequence containing a cluster of four mirna 482 genes and its homologous sequence in melon(cucumis melo). Black boxes represent protein-coding genes. The source of the genome sequences can be found in the supplementary Table 1. Figure drawn to scale.

3 Supplementary File 1. Methods Datasets The sources of the plant genome assemblies analysed and their sets of predicted protein sequences can be found in the Supplementary Table 1. Identification of C. melo, Cucumis sativus, Citrullus lanatus, Prunus persica, and Fragaria vesca syntenic regions In order to detect syntenic regions among the genomes the MCScanX toolkit was employed (Wang et al., 2012). The genomes were processed according to the software instructions as follows: Files with the predicted proteins and their.gff3 annotation files were downloaded from the sources described in supplementary table 1. Then, for all pair of species (A,B), BLASTP was used with e-value 1e-10, database: Species A proteins, and query: Species B proteins. The results from these A vs. B and B vs. A analysis were combined to produced a single A-B blast file. The.gff3 files were modified so that they contained only gene annotations in the following format: 'sp# gene_name starting_position ending_position', where 'sp#' is 'csn', 'cln',..., and N is the number of the corresponding scaffold/chromosome. These modified.gff3 files were combined to produce A-B gff3 files. The combined blast and gff3 files were processed by MCScanX ( scanning the different genomes to identify putative homologous regions and to align those regions using genes as anchors. Identification of putative NBS-LRR resistance genes A list of 84 putative melon R- genes containing TIR, CC, NBS, and/or LRR domains were obtained from the Plant Resistance Gene Data Base ( BLASTP analyses were then performed using as database those 84 proteins and as query the complete set of predicted protein sequences of every plant species analysed. But for three cases, the protein files didn't contain alternative protein sequences for a given gene, in order to avoid overestimation of the

4 number of NBS-LRR genes; also, in one instance, the protein file was not available and the identification of NBS-LRR genes was performed using BLASTX with e-value 1e-30 against the corresponding genome assembly (see Supplementary Table 1). The search for CC and TIR domains on the candidate R-genes was done using, respectively, programs MARCOIL (default parameters, threshold 90) and HMMSCAN (using the TIR.hmm profile from PFAM and MSV, bias, Vit, and Fwd filters). Identification of putative mir482 genes A comprehensive search for mir482 homologs was performed for the genomes of three cucurbits (melon, cucumber, and watermelon), some species that have not undergone recent genome duplication (grapevine and two species in the Rosaceae group: Peach and strawberry) as well as some species that have undergone genome duplications (soybean in the legumes, apple in the Rosaceae, and Arabidopsis as a reference). Finally, papaya was included since it is a distantly related species that seems not to have undergone recent genome duplications and contains few NBS-LRR genes. The homology search was done using the entire data set of known mir482. Both hairpin sequences (53) and mature mirnas (64) were obtained from mirbase Release 20 ( Two searches were performed, against the nine genome sequences analysed. Firstly a relaxed (sensitive) search based on the mature mirnas as templates (choosing a BLAST word size of 7 and a E-value cut-off of 10 which increases sensitivity for short/near exact matches) to find all possible mirnas, at the cost of a high false positive rate (specificity), and secondly a stringent (specific) search using the pre-mirna as templates which however is likely to miss out mirna due to poor conservation outside the mature-mirna stretches (reduced sensitivity). The results were visually inspected to identify regions showing significant homology. The lower/higher-stringency homology searches provide an estimation of, respectively, mir482 absence/presence in the nine genomes analysed. In addition, strict homology searches were done using the available assemblies of forty-nine further plant genomes (see Figure 1 and Supplementary Table 1). The secondary structure of selected candidate mirnas were manually analysed using the RNAfold webserver ( Further

5 analyses were done by using the sequences available for mirna of the 482 family. They were used to search specifically with low stringency the melon genome. A number of similar sequences were found essentially corresponding to fragments within the NBS-LRR genes. All the homologous sequences found were used to produce a dataset of putative target sequences in the melon genome that were used to search for other homologous sequences in the genome with no result that could indicate the presence of genes precursors of mirnas of the 482 family in the melon. Mature forms of mir482 and mir2118 family members, both belonging to the mir482 superfamily that regulates the expression of NBS-LRR disease resistance genes, are very similar (Shivaprasad et al., 2012). Therefore, extra care was taken to avoid misidentification of mir482 and mir2118 hits when analysing the BLAST results. In particular, homology searches using as query the pre-mature sequences of known mir482 and 2118 showed that, although the mature forms are quite similar, the premirna sequences are different enough as to give different results under the stringent search criteria. Identification of mir482/2118 targets in NBS-LRR mrnas The software psrnatarget ( with Maximum Expectation 5 and hpsize 17 was used to find the target position, if any, of mir482/2118s on TIR- and CC-NBS-LRR genes. For this, the complete set of superfamily mir482 members (mature forms) of each selected species (as found in mirbase release 20), together with the nucleotide CDS sequences of that species NBS- LRR genes were used. For each species, the best CC- and TIR-NBS-LRR hits were translated to protein and a motiv detection with Interpro ( was run in search of p- loop domains. The coordinates of the predicted target sites were found in all cases to be contained in the regions coding for the predicted domains. Finally, the target regions from all target site/mirna pairs for a given species were extracted and aligned with clustalw to create a consensus motiv. The variable residues were then found to correspond exactly to those described in Shivaprasad et al.

6 Supplementary File 2. Target prediction of mir482/2118 in TIR- and CC-NBS resistance genes of four representative plant species. The complete set of superfamily mir482/2118 members (mature forms, as found in mirbase release 20) of four selected species (Glycine max, representative of legumes; Malus domestica, species with most CC/TIR-NBS genes; Prunus persica, representative of Rosaceae; Vitis vinifera, outgroup for species that have not undergone recent genome duplications), togethe with the nucleotide CDS sequences of that species NBS-LRR genes were used to predict the target position, if any, of mir482/2118 on CC- and TIR-NBS-LRR genes using the software psrnatarget (Maximum Expectation, hpsize 17). The results are shown in the Table 1 below. For each species, the best CC- and TIR-NBS-LRR hits were translated to protein and a motiv detection with Interpro was run in search of p-loop domains. The coordinates of the predicted target sites were found in all cases to be contained in the regions coding for the predicted domains. The target regions from all target site/mirna pairs for a given species were extracted and aligned with clustalw to create a consensus motiv. The variable residues were then found to correspond exactly to those described in (Shivaprasad et al., 2012, Plant Cell 24(3):859-74). The alignments are shown in Figures 1-8. Table 1. mirn482/2118 target prediction in CC/TIR-NBS genes Species TIR-NBS genes 1, 2 CC-NBS genes 1, 2 mirna482/ Glycine max 143 (48) 122 (40) 13 (6/6) Malus domestica 287 (131) 335 (89) 8 (8/8) Prunus persica 130 (78) 140 (87) 10 (10/10) Vitis vinifera 20 (8) 98 (17) 1 (1/1) 1Source of CC/TIR nucleotide coding sequences: 2Total number of genes and, in parentheses, number of genes being targeted. 3Total number as found in mirbase release 20 and, in parentheses, X/Y, with X and Y number of mirnas with at least, respectively, one CC- and one TIR- NBS target.

7 Figure 1. ClustalW alignment of mir482/2118 target sites in CC-NBS genes of Glycine max.

8 Figure 2. ClustalW alignment of mir482/2118 target sites in TIR-NBS genes of Glycine max.

9 Figure 3. ClustalW alignment of mir482/2118 target sites in CC-NBS genes of Malus domestica.

10 Figure 4. ClustalW alignment of mir482/2118 target sites in TIR-NBS genes of Malus domestica.

11 Figure 5. ClustalW alignment of mir482/2118 target sites in CC-NBS genes of Prunus persica.

12 Figure 6. ClustalW alignment of mir482/2118 target sites in TIR-NBS genes of Prunus persica.

13 Figure 7. ClustalW alignment of mir482/2118 target sites in CC-NBS genes of Vitis vinifera. Figure 8. ClustalW alignment of mir482/2118 target sites in TIR-NBS genes of Vitis vinifera.

14 Sheet1 Supplementary Table 1. Number of NBS-LRR genes and mir482 presence on sequenced plant genomes. Organism Source of genome assembly Assembly version Genome size (Mb) Assembly size (Mb) Gene number Recent genome duplication events b, c d e d j No. of NBS-LRR Genes No. of mirna No. of mir482/2118 mi2118k mirbase k mir482 j,l Published Estimated CC-typee TIR-typee Published mirbase f Amborella trichopoda ,6846 No n n Aquilegia coerulea Phytozome ,823 No y n y/- Arabidopsis lyrata Phytozome ,657 2 x WGD 187 [1] y n y/- Arabidopsis thaliana Phytozome ,416 2 x WGD 159 [1] y n y/- _g _g _g Beta vulgaris RefBeet No n n _g _g _g Betula nana No y n Brachypodium dystachion Phytozome ,552 3 x WGD n y -/y Brassica rapa Phytozome ,374 2 x WGD + 1 x WGT y n Cajanus cajan ,680 1 x WGD 406 [2] [2] y y -/y Cannabis sativa CanSat ,074 No 155 h n n Capsella rubella Phytozome ,521 2 x WGD [26] y n Carica Papaya Phytozome ,584 No 55 [3] n n Chlamydomonas reinhardtii Phytozome v ,737 No n n Cicer arietinum ,255 1 x WGD 187 [4] [4] n y -/y Citrullus lanatus ,44 No [27] n n Citrus sinensis Phytozome ,379 No 741 [5] [5] 60 y n y/- Cucumis melo L ,424 No 81 [6] [6] 120 n n Cucumis sativus L. Phytozome Gy14 line ,548 No 61 [7] [7] n n Eucalyptus grandis Phytozome ,376 No y? n Fragaria vesca Phytozome ,831 No 94 [8] [28] n n Glycine max Phytozome ,175 2 x WGD 506 [4] y y y/y Gossypium hirsutum 2,830 Unclear polyploidy 78 y y/- Gossypium raimondii Phytozome ,505 No [29] 4 y n y/- _g Hevea brasiliensis 1 2,150 1,110 68,955 No 618 [9] 179 [9] 42 [9] 28 y y y/y _g _g _g Hordeum vulgare L v2 5,300 3 x WGD 191 [10] 67 n y -/y a Jatropha curcas L ,437 No 92 [11] 197i y n Linum usitatissimum Phytozome ,471 1 x WGD [30] 124 n n Lotus japonicus 2, ,971 1 x WGD n n Malus domestica Phytozome ,386 1 x WGD 992 [12] [12] 206 y y y/y Manihot esculenta Phytozome Cassava ,666 No [31] 153 y y y/y Medicago truncatula Phytozome ~ ,135 1 x WGD 705 [13] [13] 672 y y y/y Mimulus guttatus Phytozome ,140 No n n Musa acuminata ,549 4 x WGD 117 [14] [14] n y Oryza sativa L. ssp. Japonica Phytozome ,049 3 x WGD 535 [15] n y -/y Phaseolus vulgaris Phytozome ,197 1 x WGD y y y/y Phoenix dactylifera L ,889 2 x WGD 144 [16] n n Phyllostachys heterocycla ,052 2,052 31,987 3 x WGD [32] n y -/y Physcomitrella patens Phytozome 3 (annotation done on v1.6) ,273 No n n Picea abies ,600 12,301 26,437 No [33] 40 y n y/- Populus trichocarpa Phytozome ,335 1 x WGD 302 [17] [17] 352 y n y/- Prunus mume ,390 No 223 [18] [18] y y -/y Prunus persica Phytozome ,864 No [34] 180 y y y/y _g Pyrus bretschneideri Rehd ,812 1 x WGD 396 [19] 207 [19] 60 [19] 297 [19] y y -/y Ricinus communis Phytozome ,221 No 97 [20] y n Selaginella moellendorffii Phytozome ,273 No [35] 58 n n Setaria italica Phytozome ,471 3 x WGD [36] n y -/y Solanum lycopersicum Phytozome ITAG ,727 1 x WGT 266 [21] [21] y y y/y Solanum tuberosum Phytozome ,119 1 x WGT 408 [22] [37] 224 y y y/y Sorghum bicolor Phytozome ,032 3 x WGD 211 [23] [23] 205 n y -/y Thelungiella halophilla Phytozome ,351 2 x WGD [38] n n Thelungiella parvula ,132 2 x WGD y n Theobroma cacao Phytozome ,452 No 297 [24] [24] 82 y n Triticum aestivum 17,000 3 x WGD 43 Vitis vinifera Phytozome 12x March 2010 release ,346 No 535 [25] y n y/- Zea mays Phytozome 2 2,300 2,066 39,656 4 x WGD n y -/y a Number of sequences in the downloaded protein file. The paper describing the sequencing of the Jatropha genome, however, reports 40,929 protein-coding genes. b Source: c WGD: Genome doubling; WGT: Genome tripling. d Bibliographic source in square brackets. e As described in the Methods section. f The downloaded protein file may lists all protein transcripts. Therefore, the number of NBS-LRR genes may be overestimated. g Unnanotated genome assembly or annotation not accessible. h Protein file not available. Detection of putative TIR-NBS-LRR genes was done using blastx with e-value 1e-30 against the genome assembly. i The downloaded protein file probably lists all protein transcripts (see footnote a ). Therefore, the number of NBS-LRR genes may be overestimated. In fact, the published number of NBS-LRR genes for this species is much lower. j k Pressence (y: found, n: not found) as described in the Methods section. l Pressence (y: found in mirbase) of mir482/mir2118. Other species with mir482 listed in mirbase but with no published genome assemblies: Glycine soja, Nicotiana tabacum, Panax ginseng, Pinus densata, Pinus tadea, and Vigna unguiculata. [37] Zhang, R. et al. Iden1fica1on and characteriza1on of mirna transcriptome in potato by high- throughput sequencing. PLoS ONE 2013; 8(2):e [38] Yang, R. et al. The reference genome of the halophy1c plant Eutrema salsugineum. Front Plant Sci. 2013; 4:46. Page 1