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Additional File (.zip) included Figs A-J and Tables A-J. Fig A. Percentage of expressed miRNAs in each family. (A) The expressed miRNAs of different families in leaves. (B) The expressed miRNAs of different families in roots. Fig B. Distribution of length of known miRNAs that expressed in the present study. Fig C. The conservation analysis of predicted novel miRNAs. The mature sequence of predicted novel miRNAs in the present study and known miRNAs in plants were aligned. (A) Novel_1_44595. (B) Novel_1_47179. (C) Novel_3_27520. (D) Novel_3_29234. (E) Novel_9_5030. (F) Novel_1-_3438. zma: Zea mays. osa: Oryza sativa. bdi: Brachypodium distachyon. tae: Triticum aestivum. mtr: Medicago truncatula. ath: Arabidopsis thaliana. ptc: Populus trichocarpa. vvi: Vitis vinifera. rco: Ricinus communis. Fig D. The phylogenetic tree and sequence of three novel miRNAs. The phylogenetic trees of Novel-2-38 (A), Novel-10-88 (B) and Novel-10-147 (C) were shown. The sequence of relative miRNAs in different species of Novel-2-38 (D), Novel-10-88 (E) and Novel-10-147 (F). ath: Arabidopsis thaliana, osa: Oryza sativa, sbi: Sorghum bicolor, zma: Zea mays. (G) The novel mature sequence of miRNAs were cloned using stem-loop methods in B73 (B), H082183 (H) and Lv28 (L). The maker (M) was 100-bp. Fig E. Correlation result of miRNome in each replicate. The TPM value of all known miRNAs in each replicate was calculated as log10(TPM+1) for the correlation analysis. The correlation coefficient r and the P value are labelled in each replicate sample. H and L indicate the two maize inbred lines H082183 and Lv28, respectively. MD and SD indicate moderate and severe drought, respectively. MC and SC indicate well-watered controls of moderate and severe drought, respectively. 1 and 2 indicate the two replicates. Fig F. The T-plot of drought responsive miRNAs and their target genes. (A) GRMZM2G065451, (B) GRMZM2G113779, (C) GRMZM2G414805, (D) GRMZM2G371033, (E) AC207656.3_FGT002, (F) GRMZM2G390641, (G) GRMZM2G159399, (H) GRMZM2G096358, (I) GRMZM2G114850, (J) GRMZM2G113779, (K) GRMZM5G806833, (L) GRMZM2G061734, (M) GRMZM2G004090, (N) GRMZM2G028054, (O) GRMZM2G304745, (P) GRMZM2G103812. Fig G. The cluster analysis of genes. (A) GRMZM2G096358 (MYB) and GRMZM2G114850 (NAC) in leaves of H082183. (B) GRMZM2G390641 (ARF) in leaves of Lv28. (C) GRMZM2G159399 (ARF) in leaves of Lv28. (D) AC207656.3_FG002 (ARF), GRMZM2G065451 (SPL) and GRMZM2G113779 (SPL) in leaves of Lv28. (E) GRMZM2G067624 (SPL) and GRMZM2G371033 (SPL) in leaves of Lv28. (F) GRMZM2G004090 (MYB) in roots of H082183. (G) GRMZM2G028054 (MYB) in roots of H082183. (H) GRMZM2G061734 (SPL) and GRMZM2G160917 (SPL) in roots of Lv28. (I) GRMZM2G460544 (SPL) and GRMZM5G806833 (SPL) in roots of Lv28. Fig H. Co-expressed genes of miR156 targeted MYB and NAC genes that had over-presented transcription factor binding sequence in promoter regions. The co-expressed genes of NAC and MYB genes that had significant over-presented MYB (A) or NAC (B) binding motif sequence in promoter regions were listed. The MYB (A) and NAC (B) binding sequences in promoter regions were plotted. Fig I. The expression of miR159 and target genes in roots under ABA treatment. (A) miR159. (B) GRMZM2G004090 (MYB). (C) GRMZM2G028054 (MYB). Fig J. Correlation result of miRNA expression by qRT-PCR and sequencing. Five miRNAs in leaves and five miRNAs in roots were selected to perform qRT-PCR validation under moderate drought and severe drought in H082183 and Lv28. Table A. Primers used in the present study. Table B. Sequencing data of all 32 small RNA libraries. a H: H082183; L: Lv28; MD: moderate drought; SD: severe drought; MC: well-watered control of moderate drought; SC: well-watered control of severe drought; 1 and 2: two replicates. Table C. Result of reference genome alignment of small RNA reads. a H: H082183; L: Lv28; MD: moderate drought; SD: severe drought; MC: well-watered control of moderate drought; SC: well-watered control of severe drought; 1 and 2: two replicates. Table D. Alignment result of different small RNA databases. a H: H082183; L: Lv28; MD: moderate drought; SD: severe drought; MC: well-watered control of moderate drought; SC: well-watered control of severe drought; 1 and 2: two replicates. Table E. Numbers of expressed miRNAs in each sample. a H: H082183; L: Lv28; MD: moderate drought; SD: severe drought; MC: well-watered control of moderate drought; SC: well-watered control of severe drought; 1 and 2: two replicates. Table F. The data output of degradome sequencing. Table G. Predicted novel miRNAs. a The number of libraries that had both miRNA and miRNA* reads. Table H. Expression of all miRNA-mRNA pairs in leaves and roots. Table I. Drought responsive miRNAs in different species. a miR156e-3p was down-regulated under moderate drought, while miR156b-5p and miR156d-5p were up-regulated under severe drought in Lv28. b miR164h-5p was down-regulated under moderate drought in H082183, but miR164c-3p and miR164h-3p were up-regulated under both moderate and severe drought in H082183. c miR171g-3p was down-regulated under moderate drought, but miR171h-3p and miR171k-3p were up-regulated under severe drought in Lv28. d miR156d-3p, miR156f-3p and miR156g-3p were down-regulated, while miR156e-3p was up-regulated under severe drought in H082183. e miR159a-3p, miR159b-3p, miR159f-3p, miR159j-3p and miR159k-3p were up-regulated under moderate drought in H082183, but miR159c-3p, miR159c-5p, miR159d-3p and miR159d-5p were down-regulated under moderate drought in Lv28. f miR167a-5p, miR167b-3p, miR167b-5p, miR167c-5p and miR167d-5p were up-regulated under severe drought in H082183, while miR167j-3p was down-regulated under moderate drought in Lv28. Table J. Drought responsive miRNAs by using different drought treatment methods in maize. a This line present the maize inbred lines used in experiment, in which 81565, 87–1, HKI-1532 and H082183 were drought tolerant lines, and 21ES, Dan340, V-372 and Lv28 were drought sensitive lines. b This line present the sampling time point after drought. The h and d present hours and days after drought treatment. c miR171h-3p and miR171k-3p were up-regulated, while miR171g-3p was down-regulated under drought. d miR156e-3p was up-regulated, while miR156d-3p, miR156f-3p and miR156g-3p were down-regulated under drought.
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