FR FB YF Peel Pulp Peel Pulp

M1 AL YFB FG FR FB YF Peel Pulp Peel Pulp M2 300 100 60 40 30 20 25 nt 21 nt 17 nt 10 Supplementary Fig. S1 srna analysis at different stages of prickly pear cactus fruit development. srna analysis in a polyacrylamide gel, the samples were extracted from prickly pear cactus fruit tissues at different stages. 2 µg of LMW RNA of each sample was analyzed by electrophoresis. Lane 1, Ladder 10 bp; Lane 2, Arabidopsis leaves (control); Lane 3, Prickly pear cactus young floral buds; Lane 4, Prickly pear cactus buds before fertilization; Lane 5, Young cactus fruit; Lane 6, Green prickly pear cactus fruit peel; Lane 7, Green prickly pear cactus fruit pulp; Lane 8, Ripe prickly pear cactus fruit peel; Lane 9, Ripe prickly pear cactus fruit pulp; and Lane 10, mirna Marker

Abundance a) AL YFB FG FR FB YF Peel Pulp Peel Pulp U6 mir166 b) Original data YFB FB YF FG peel FG pulp FR peel FR pulp U6 13709189 10200517 16152886 7564431 4813366 8663164 8483123 mir166 1059827 856618 974947 246294 171780 135492 172019 Normalized data Factor N 1.00 1.34 0.85 1.81 2.85 1.58 1.62 U6 normalized 13709189 13709189 13709189 13709189 13709189 13709189 13709189 mir166 normalized 1059827 1151269 827452 446365 489254 214411 277992 c) 1200000 1000000 800000 600000 400000 200000 0 young floral bud early fruit green fruit ripe fruit Supplementary Fig. S2 MiR166 Northern blot normalization. a Northern blot analysis of mir166 at different stages of prickly pear cactus fruit development. 2 µg of LMW RNA of each sample was analyzed by electrophoresis, transferred to a membrane and hybridized with oligonucleotide sequences complementary to mir166, and complementary sequence to U6. b Data signal intensity of U6 and mir166 determined by ImageQuant TL software and then normalized. Briefly, all U6 values were normalized against the value of U6 in the YFB sample. c Expression profile of mir166 after normalization. AL, Arabidopsis leaves; YFB, young floral buds; FB, floral bud; YF, young cactus fruit; GF, green cactus fruit; and RF, ripe cactus fruit

Supplementary Fig. S3 Expression of mirnas in selected fruit crops for analysis in prickly pear cactus fruit development. Each column in the heatmap represents a tissue and each row an expression value of a mirna. The data were obtained of the Comparative Sequencing of Plant Small RNA database http://smallrna.udel.edu (Chavez-Montes et al. 2014). L, leaves; Fl, flower; and Fr, fruit tissue Avocado Banana Grape Orange Chilli Tomato L Fl Fr L Fl Fr L Fl Fr L Fl Fr L Fl Fr L Fl Fr Low expression (0) High expression

Young floral bud Young fruit Pulp green fruit Pulp ripe fruit 1513 1514 1516 1524 156 157 1513 1514 1516 1524 156 157 1513 1514 1516 1524 156 157 1513 1514 1516 1524 156 157 159 160 162 164 165 166 159 160 162 164 165 166 159 160 162 164 165 166 159 160 162 164 165 166 167 168 169 170 172 1857 167 168 169 170 172 1857 167 168 169 170 172 1857 167 168 169 170 172 1857 1916 2119 2950 319 3623 3629 1916 2119 2950 319 3623 3629 1916 2119 2950 319 3623 3629 1916 2119 2950 319 3623 3629 3631 3633 3635 3636 3639 3640 3631 3633 3635 3636 3639 3640 3631 3633 3635 3636 3639 3640 3631 3633 3635 3636 3639 3640 390 393 394 395 3961 3954 390 393 394 395 3961 3954 390 393 394 395 3961 3954 390 393 394 395 3961 3954 396 397 398 399 403 408 396 397 398 399 403 408 396 397 398 399 403 408 396 397 398 399 403 408 413 482 530 535 822 824 413 482 530 535 822 824 413 482 530 535 822 824 413 482 530 535 822 824 827 828 846 858 894 827 828 846 858 894 827 828 846 858 894 827 828 846 858 894 Supplementary Fig. S4 MiRNA expression profiles during prickly pear cactus fruit development. Reverse Northern blot hybridization analysis of mirna expression at different developmental stages. 40 ng of purified srna fraction corresponding to 18-24 nt per sample was used as probe. Numbers correspond to the names of the different mirnas

Young fruit Green fruit Ripe fruit Young fruit Green fruit Ripe fruit Low expression High expression a) b) mir397 mir408 mir156 mir157 mir164 mir2119 mir1524 mir828 mir3636 mir398 mir319 mir1916 mir846 mir399 mir159 mir482 mir822 mir395 mir3954 mir166 mir3635 mir393 mir535 mir165 mir168 mir390 mir824 mir894 mir394 mir3629 mir3633 mir170 mir169 mir3623 mir395 mir394 mir408 mir159 mir535 mir1916 mir3954 mir397 mir824 mir828 mir894 mir390 mir482 mir166 mir822 mir156 mir3635 mir3633 mir399 mir164 mir168 mir165 mir319 mir3636 mir3623 mir1524 mir2119 mir157 mir846 mir398 mir393 mir3629 mir170 mir169 Supplementary Fig. S5 Comparison of cluster analyses of mirna data obtained by the reverse Northern blot hybridization experiment a and the GeneChip mirna 2.0 microarray experiment b of different stages of prickly pear cactus fruit development. Heatmap of mirna expression levels during prickly pear cactus fruit development. Each column represents a stage and the intensity of the red color indicates the relative expression level at that stage of a mirna. Hierarchical clustering was performed using MeV with the following options selected: gene tree, sample tree optimize gene leaf order, Pearson correlation, and complete linkage clustering. A green point indicates mirnas clustered in the same clade in both analyses, an orange point indicates mirnas validated by Northern blot analysis, and a blue point indicates mirnas only detected by reverse Northern blot hybridization. An asterisk indicates mirnas not detected in the same clade but with low abundance by microarray

Whole organ Longitudinal section Tissue print mirna detection by Northern blot Cactus Tomato fruit mirna marker ladder a) b) Tissue print Hybridization c) Hybridization Supplementary Fig. S6 Tissue print analysis for mirnas in different tissues. The different tissue sections were firmly pressed on the nitrocellulose membrane, which were then used for Northern blot hybridization. a Example of a fresh floral bud used for tissue printing and hybridization showing mir164 expression. b Negative control. The oligonucleotide M13 (5 -GTAAAACGACGGCCAGT-3 ) was used as probe to rule out non-specific hybridization. c Positive control. The mirna marker (New England Biolabs) was spotted in the membrane and hybridized with a 21-mer 5 AAATCTCAACCAGCCACTGCT 3 -biotin DNA oligonucleotide probe, complementary to the marker. The assays were performed in duplicate. The red lines indicate the region where the tissue was printed

Supplementary Table S1 Oligonucleotide sequences complementary to mature mirnas. mirna name Probe sequence (5-3 ) mirna name Probe sequence (5-3 ) mir156 CTGGATGCAGAGGTTTATCGA mir535 CCGACGACTCTTTTACATCCT mir157 TGCACGTGCCCTGCTTCTCCA mir822 GTACAACTGTAGTAGGTTATAT mir159 GGGGGATGAGGCCTGGTCCGA mir824 CCTTACCTACCAATCCTCTC mir160 GGGGAATGAAGCCTGGTCCGA mir827 CCGTACACACCCCGTATTATC mir162 CCAGATCATGCTGGCAGCTTCA mir828 CAGACAGCCTCTTCGTTCAGCCTC mir164 TTCCCGACCTGCACCAAGCGA mir846 TAACTGAAGACTTTCCGATTTTCG mir165 CCGGCAAGTCATCCTTGGCTG mir858 TAGCTTTTCCGTAGTAGTTAGTCC mir166 GATATTGGCACGGCTCAATCA mir894 TTTTCTCTCATCTTTATCTA mir167 ATGCAGCATCATCAAGATTCT mir1513 GTGCTCACTCTCTTCTGTCA mir168 GGGAGCTCCCTTCAGTCCAA mir1514 GTGCTCTCTATCTTCTGTCAA mir169 GGCGCTATCCCTCCTGAGCTT mir1516 TAGAGCTCCCTTCAATCCAAA mir170 GATCAATGCGATCCCTTTGGA mir1524 TGGCATACAGGGAGCCAGGCA mir172 GGAGGTGGACAGAATGCCAA mir1857 GAGTTCCCCCAAACACTTCAG mir319 CAGGGCAACTCTCCTTTGGCA mir1916 CAGTTCAAGAAAGCTGTGGAA mir390 AATCTACTGGTAGTTGTTTGT mir2119 CATCAACGCTGCACTCAATGA mir393 ACCACGAACCTGCTTAAACGAT mir2950 CAGGGGCGACCTGAGAACACA mir394 AACTTAACTTCACGAACTTAA mir3623 CGAGTTTGTGCGTGAATCTAA mir395 AAAGCAACAGACAAGCTGGA mir3629 GCCAGGGAAGAGGCAGTGCAT mir396 GTAAGTCATGGCTTTCTCTCA mir3631 TATCAAAGAGAACAAGACGTG mir397 AAGTAAAACTTTTATCCGTAAC mir3633 TGCTTCCCAATCAGCCCATTCC mir398 TACAAGCCATAAGCTCTTTTG mir3635 AAGATGCAGGTGCAAATGCA mir399 GGAGTTCCTTCCTCGGACTCG mir3636 ACTGTTGCTCTCTCTCGTGCG mir403 AGTACGAGGTTCTTTTGGTCC mir3639 ACGCCCTTCGTAAACGTGTAC mir408 TAAAGTGAATCTGTGGAGTT mir3640 ATCTGGTAAACACTCTTCCCT mir413 TTCCCCTACAACTCCCTTTGA mir3951 GCAAAGTGCAGCCCAAGTGG mir482 AAGGTAGAGAACGTGTGACCT mir3954 TGACCGTGATTTCTCTGTCCA mir530 AGTGTTCAAGTAGGTTCGTGGT Probes used to print the mirna membrane array are listed next to the corresponding mirna

Supplementary Table S2 Normalized mirna abundances in different stages of prickly pear cactus fruit development. Young Young Green Ripe mirna floral bud fruit fruit fruit Target family Reference mir156 0.0000 20.4093 19.6711 18.7425 Squamosa promoter-binding protein, CNR Transcription factor Zuo et al., 2011 mir157 0.0000 18.2437 20.1352 18.0760 Squamosa promotor binding-like protein (SPL) Zuo et al., 2011 mir159 21.5987 19.3787 0.0000 18.8826 MYB transcription factors, ACC synthase Zuo et al., 2011 mir164 18.7245 21.8963 21.7085 19.7324 NAC, domain transcription factors Carra et al., 2009 mir165 20.4413 19.7486 19.7295 19.3547 HD-ZIPIII transcription factor Carra et al., 2009 mir166 20.0154 19.6583 18.9002 18.0847 HD-ZIPIII transcription factor Carra et al., 2009 mir168 17.8759 21.1177 20.9776 19.7840 Argonaute protein (AGO1) Zuo et al., 2011 mir169 0.0000 0.0000 18.0561 0.0000 HAP2 transcription factor, CCAAT-binding TF Carra et al., 2009 mir170 0.0000 0.0000 20.3008 0.0000 Scarecrow transcription factor family protein Zuo et al., 2011 mir319 17.4641 19.3402 19.6216 0.0000 GAMYB Transcription factor, TCP transcription factor Zuo et al., 2011 mir390 19.6220 21.8697 20.2151 20.1190 TAS3, Protein phosphatase 2C-related Zuo et al., 2011 mir393 16.1051 0.0000 18.0948 0.0000 Auxin receptors and BHL transcription factors Jagadeeswaran et al., 2012 mir394 19.2995 0.0000 0.0000 0.0000 F-box family protein Zuo et al., 2011 mir395 20.6106 21.3359 21.3904 21.4526 ATP Sulfurylase Zuo et al., 2011 mir397 18.5510 18.5065 0.0000 0.0000 Laccase-7-like Li et al., 2013 mir398 18.4984 0.0000 17.8831 0.0000 Uncharacterized LOC101298296 Li et al., 2013 mir399 17.1853 19.3700 18.9505 0.0000 Probable ubiquitin-conjugating enzyme E2 24-like Li et al., 2013 mir408 22.4851 0.0000 0.0000 0.0000 Cyclin A3, LAC3, LAC12, LAC13 Zuo et al., 2011 mir482 18.1589 21.5892 20.3077 19.5613 Disease resistance protein, Enzyme pectate-lyase Zuo et al., 2012 mir535 18.4266 21.7881 18.0825 18.7427 Translationally controlled tumor protein (TCTP) homolog Li et al., 2013 mir822 19.0354 21.1268 20.6015 20.1359 AGL16 Transcription factor, DCL1 domain-containing proteins Abdel et al., 2008; Shao et al., 2012 mir824 0.0000 20.0283 0.0000 0.0000 AGL16, Ent-kaurene synthase Kutter et al., 2007; Ge et al., 2013 mir828 0.0000 17.9845 0.0000 0.0000 MYB family TF, Ethylene-insensitive 2 (EIN2) Zuo et al., 2012 mir846 0.0000 0.0000 17.3981 0.0000 Jacalin lectin Sun et al., 2012 mir894 0.0000 18.2742 0.0000 0.0000 O-Methyltransferase family Karlova et al., 2013 mir1524 20.2356 20.6637 21.6211 19.9608 Transcription factor AP2-EREBP Valdés-López et al., 2010 mir1916 0.0000 19.8510 0.0000 0.0000 Mitochondrial glycoprotein family protein Zuo et al., 2011 mir2119 18.4154 20.8989 21.7480 20.6218 F-box family protein Valdés-López et al. 2010 mir3623 16.7553 18.0333 19.7443 0.0000 Disease resistance protein (TIR-NBS-LRR class) family Wang et al., 2014 mir3629 0.0000 0.0000 20.3342 0.0000 unknown protein Wang et al., 2014 mir3633 0.0000 19.6789 19.1611 18.0733 GSVIVT01009832001 (AVA-P2) Wang et al., 2014 mir3635 16.5502 21.0750 20.4785 19.0565 unknown protein Wang et al., 2014 mir3636 17.1778 20.1665 21.1443 19.0217 Ribosomal protein S3 Singh et al., 2012 mir3954 0.0000 18.7174 0.0000 0.0000 unknown protein Wang et al., 2014 The relative expression presented is based on the signal intensity of each spot detected by reverse Northern blot hybridization. Abundance is expressed as the base 2 logarithm of pixels