Figures (6)  Tables (5)

    • Figure 1. 

      Morphological differences of P. suffruticosa 'Luoyang Hong' plants under drought and salt stress. The numbers indicate days after treatment. The horizontal bar represents 30 cm.

    • Figure 2. 

      Amplified gel electrophoresis of 18 candidate RGs in Table1. M: 2,000 bp Marker; 1−18: miRNAs in Table 1 and the order are kept consistent.

    • Figure 3. 

      Expression levels of miRNAs in 18 candidate parameters. (a) Expression levels under drought stress. (b) Expression levels under salt stress. Box-whisker plot showing the CT variation among 18 test samples. A line across the box depicts the median. In each box, the upper and lower edges indicate the 25th and 75th percentiles. Whiskers go from the minimal to maximal value or, if the distance from the first quartile to the minimum CT value is more than 1.5 times the interquartile range (IQR), from the smallest value included within the IQR to the first quartile.

    • Figure 4. 

      (a) Expression stability of 18 candidate reference miRNAs analyzed in drought stress. The most stable miRNA is shown on the right and the least stable miRNA is shown on the left. (b) Pairwise variations of 18 candidate reference miRNAs in drought stress. (c) Expression stability of 18 candidate reference miRNAs analyzed in salt stress. The most stable miRNA is shown on the right and the least stable miRNA is shown on the left. (d) Pairwise variations of 18 candidate reference miRNAs in salt stress.

    • Figure 5. 

      Normfinder stability values of 18 candidate reference miRNAs. (a) Normfinder stability values under drought stress. (b) Normfinder stability values under salt stress. The abscissa represents different Normfinder stability values, and the ordinate represents different candidate reference miRNAs name.

    • Figure 6. 

      Conceptual figure. The yellow and blue arrows represent drought stress and salt stress, respectively. The right panels show the optimal reference miRNAs identified by Delta CT, GeNorm, NormFinder, and BestKeeper analyses, respectively. The bottom panel displays the top-ranked reference miRNA determined by a comprehensive analysis with RefFinder.

    • miRNAs miRNA sequence (5'–3') Primer F sequence (5'–3')
      csi-miR1515a TCATTTTTGCGTGCAATGATCC CCCGTTTGCGTGCTATGATCC
      PC-5p-15026_548 TGCCCTCTTCTTTTCACCCATT CTGCCCTCTTCTTTTCACCCAT
      gma-miR403a_2ss19TC20TC TTAGATTCACGCACAAACCCG CCAGATTCACGCACAAACCC
      mtr-miR156b-3p TGCTCACTCTCTATCTGTCACC CCGCACTCTCTATCTGTCACC
      ptc-MIR156b-p3 TGCTCACTCTCTATCTGTCACC CCGCACTCTCTATCTGTCACC
      PC-5p-50_56138 TGACGGAGAGAGAGAGCACAC TGACGGAGAGAGAGAGCACAC
      mtr-miR166e-5p_1ss10GT GGAATGTTGTCTGGCTCGAGG GGAATGTTGTCTGGCTCGTGG
      mtr-miR166a_L+1R-1 CTCGGACCAGGCTTCATTCCC GTGTTCGGACCAGGCTTCATT
      PC-5p-209_18822 TTTGCATAGACCATCTAGCGG CCCTTGCATCGACCATCTAGC
      mtr-miR162 TCGATAAACCTCTGCATCCAG CCTCGATAAACCTCTGCGTCC
      mtr-miR166a_L+1 CTCGGACCAGGCTTCATTCCCC GCGCTCGGACCAGGCTTCATT
      PC-3p-160_22001 TTTACGTTGCCTTTCTTCCTC CCTTACCTTGCCTTTCTTCCTC
      mes-miR159a-5p_2ss10GA11CT AGCTGCTGAATTATGGATCCC GGGCTGGCTGCTGAATTATGG
      peu-miR2916_R-1_2ss6AG21TG TGGGGGCTCGAAGACGATCAGA CTGAATTGGGGGCTCGAAGAC
      ath-miR858a_L-1R+1 TTCGTTGTCTGTTCGACCTTG CCCGTTGTCTGTTCTACCTTG
      mdm-MIR482a-p3_2ss11CG20TA TCTTCCCAAGGCCGCCCATACC TTATTCCCAAGGCCGCCCAT
      mtr-miR396b-3p_L-1 TTCAATAAAGCTGTGGGAAG GGTCAATAAGGCTGTGGGAAG
      U6 (snRNA) ACAGAGAAGATTAGCATGGCC

      Table 1. 

      Primers sequence of 18 candidate RGs for qRT-PCR.

    • miRNAs CV (%) SD r Rank
      ptc-MIR156b-p3 1.45 0.37 0.745 1
      mtr-miR156b-3p 1.93 0.47 0.542 2
      PC-3p-160_22001 1.98 0.50 0.711 3
      csi-miR1515a 2.14 0.56 0.723 4
      PC-5p-15026_548 2.60 0.63 0.324 5
      PC-5p-209_18822 2.88 0.73 0.423 6
      peu-miR2916_R-1_2ss6AG21TG 4.72 0.73 0.592 7
      mtr-miR162 3.23 0.74 0.351 8
      PC-5p-50_56138 4.03 0.78 0.631 9
      U6(snRNA) 3.87 0.78 0.847 10
      mtr-miR166e-5p_1ss10GT 4.00 0.79 0.487 11
      mtr-miR166a_L+1R-1 4.25 0.80 0.701 12
      ath-miR858a_L-1R+1 3.62 0.91 0.713 13
      mes-miR159a-5p_2ss10GA11CT 3.86 0.91 0.242 14
      gma-miR403a_2ss19TC20TC 5.65 1.08 0.946 15
      mtr-miR166a_L+1 5.64 1.12 0.844 16
      mtr-miR396b-3p_L-1 5.55 1.21 0.092 17
      mdm-MIR482a-p3_2ss11CG20TA 7.16 1.33 0.881 18

      Table 2. 

      Bestkeeper analyzed the miRNAs sequencing of 18 candidate internal parameters in drought stress.

    • miRNAs CV (%) SD r Rank
      PC-3p-160_22001 1.47 0.36 0.400 1
      mtr-miR166e-5p_1ss10GT 2.21 0.43 0.636 2
      peu-miR2916_R-1_2ss6AG21TG 3.13 0.44 −0.005 3
      csi-miR1515a 2.08 0.52 0.774 4
      ath-miR858a_L-1R+1 2.10 0.52 0.752 5
      gma-miR403a_2ss19TC20TC 2.94 0.53 0.311 6
      mes-miR159a-5p_2ss10GA11CT 2.35 0.57 0.182 7
      mtr-miR166a_L+1R-1 2.96 0.58 0.105 8
      PC-5p-15026_548 2.71 0.64 0.190 9
      PC-5p-50_56138 4.16 0.78 0.933 10
      mtr-miR396b-3p_L-1 4.31 0.97 0.299 11
      mtr-miR162 4.69 1.05 0.846 12
      mtr-miR156b-3p 4.43 1.09 0.910 13
      mdm-MIR482a-p3_2ss11CG20TA 6.21 1.12 0.944 14
      PC-5p-209_18822 4.97 1.16 0.866 15
      mtr-miR166a_L+1 6.43 1.26 0.823 16
      U6(snRNA) 6.67 1.32 0.889 17
      ptc-MIR156b-p3 7.26 1.76 0.446 18

      Table 3. 

      Bestkeeper analyzed the miRNAs sequencing of 18 candidate internal parameters in salt stress.

    • Rank RefFinder Geometric mean
      1 ptc-MIR156b-p3 2.06
      2 mtr-miR156b-3p 2.51
      3 mtr-miR166a_L+1R-1 3.66
      4 PC-5p-50_56138 4.05
      5 csi-miR1515a 4.43
      6 PC-3p-160_22001 5.51
      7 ath-miR858a_L-1R+1 6.36
      8 U6(snRNA) 8.11
      9 peu-miR2916_R-1_2ss6AG21TG 8.82
      10 PC-5p-15026_548 9.2
      11 mtr-miR166a_L+1 10.18
      12 mtr-miR162 10.54
      13 PC-5p-209_18822 10.89
      14 mes-miR159a-5p_2ss10GA11CT 11.56
      15 gma-miR403a_2ss19TC20TC 11.58
      16 mtr-miR166e-5p_1ss10GT 14.34
      17 mdm-MIR482a-p3_2ss11CG20TA 17.24
      18 mtr-miR396b-3p_L-1 17.74

      Table 4. 

      The 18 candidate internal parameters were sorted by comprehensive miRNAs analysis in drought stress.

    • Rank RefFinder Geometric mean
      1 csi-miR1515a 2.83
      2 PC-3p-160_22001 2.94
      3 mtr-miR166e-5p_1ss10GT 3.36
      4 PC-5p-50_56138 3.66
      5 gma-miR403a_2ss19TC20TC 3.83
      6 ath-miR858a_L-1R+1 3.87
      7 mtr-miR166a_L+1R-1 5.94
      8 peu-miR2916_R-1_2ss6AG21TG 6.67
      9 mtr-miR162 8.76
      10 PC-5p-15026_548 9.96
      11 mdm-MIR482a-p3_2ss11CG20TA 9.96
      12 mtr-miR156b-3p 10.89
      13 PC-5p-209_18822 12.01
      14 mes-miR159a-5p_2ss10GA11CT 12.4
      15 mtr-miR166a_L+1 15.19
      16 U6(snRNA) 15.2
      17 mtr-miR396b-3p_L-1 15.25
      18 ptc-MIR156b-p3 18

      Table 5. 

      The 18 candidate internal parameters were sorted by comprehensive miRNAs analysis in salt stress.