FR FB YF Peel Pulp Peel Pulp

Transcription

1 M1 AL YFB FG FR FB YF Peel Pulp Peel Pulp M 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

2 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 U mir Normalized data Factor N U6 normalized mir166 normalized c) 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

3 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 (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

4 Young floral bud Young fruit Pulp green fruit Pulp ripe fruit 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 nt per sample was used as probe. Numbers correspond to the names of the different mirnas

5 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

6 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

7 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

8 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 mir Squamosa promoter-binding protein, CNR Transcription factor Zuo et al., 2011 mir Squamosa promotor binding-like protein (SPL) Zuo et al., 2011 mir MYB transcription factors, ACC synthase Zuo et al., 2011 mir NAC, domain transcription factors Carra et al., 2009 mir HD-ZIPIII transcription factor Carra et al., 2009 mir HD-ZIPIII transcription factor Carra et al., 2009 mir Argonaute protein (AGO1) Zuo et al., 2011 mir HAP2 transcription factor, CCAAT-binding TF Carra et al., 2009 mir Scarecrow transcription factor family protein Zuo et al., 2011 mir GAMYB Transcription factor, TCP transcription factor Zuo et al., 2011 mir TAS3, Protein phosphatase 2C-related Zuo et al., 2011 mir Auxin receptors and BHL transcription factors Jagadeeswaran et al., 2012 mir F-box family protein Zuo et al., 2011 mir ATP Sulfurylase Zuo et al., 2011 mir Laccase-7-like Li et al., 2013 mir Uncharacterized LOC Li et al., 2013 mir Probable ubiquitin-conjugating enzyme E2 24-like Li et al., 2013 mir Cyclin A3, LAC3, LAC12, LAC13 Zuo et al., 2011 mir Disease resistance protein, Enzyme pectate-lyase Zuo et al., 2012 mir Translationally controlled tumor protein (TCTP) homolog Li et al., 2013 mir AGL16 Transcription factor, DCL1 domain-containing proteins Abdel et al., 2008; Shao et al., 2012 mir AGL16, Ent-kaurene synthase Kutter et al., 2007; Ge et al., 2013 mir MYB family TF, Ethylene-insensitive 2 (EIN2) Zuo et al., 2012 mir Jacalin lectin Sun et al., 2012 mir O-Methyltransferase family Karlova et al., 2013 mir Transcription factor AP2-EREBP Valdés-López et al., 2010 mir Mitochondrial glycoprotein family protein Zuo et al., 2011 mir F-box family protein Valdés-López et al mir Disease resistance protein (TIR-NBS-LRR class) family Wang et al., 2014 mir unknown protein Wang et al., 2014 mir GSVIVT (AVA-P2) Wang et al., 2014 mir unknown protein Wang et al., 2014 mir Ribosomal protein S3 Singh et al., 2012 mir 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