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Figure 1.
Floral characteristics of 21 early-flowering and 21 late-flowering peony cultivars at full-bloom stage. A: Paeonia suffruticosa 'Taoyan Hong', B: Paeonia suffruticosa 'Huolian Jindan', C: Paeonia suffruticosa 'Pinghu Qiuyue', D: Paeonia suffruticosa 'JiaoHong', E: Paeonia suffruticosa 'Dapeng Zhanchi', F: Paeonia suffruticosa 'CangJiao', G: Paeonia suffruticosa 'LanJu', H: Paeonia suffruticosa 'HaiBo', I: Paeonia suffruticosa 'Lan Hudie', J: Paeonia suffruticosa 'Lan Yueliang', K: Paeonia suffruticosa 'ZhaoFen', L: Paeonia suffruticosa 'Yucui Caidie', M: Paeonia suffruticosa 'Jianshi Fen', N: Paeonia suffruticosa 'Xishi Fen', O: Paeonia suffruticosa 'Mantian Xing', P: Paeonia suffruticosa 'JingYu', Q: Paeonia suffruticosa 'Suxin Bai', R: Paeonia suffruticosa 'Xueyuan Hongxing', S: Paeonia rockii 'Ziban Bai', T: Paeonia suffruticosa 'ErQiao', U: Paeonia ostii 'FengDan', a: Paeonia suffruticosa 'Feiyan Hongzhuang', b: Paeonia suffruticosa 'Shouan Hong', c: Paeonia suffruticosa 'Haitang Zhengrun', d: Paeonia suffruticosa 'Daduo Lan', e: Paeonia suffruticosa 'Doulv', f: Paeonia suffruticosa 'Shenhei Zi', g: Paeonia suffruticosa 'Zihong Zhengyan', h: Paeonia rockii 'MingMou', i: Paeonia rockii 'Xian Emao', j: Paeonia rockii 'Bai Zhangbing', k: Paeonia rockii 'Bai Yanwei', l: Paeonia rockii 'Yuban Xiuqiu', m: Paeonia rockii 'Bingxin Fenhe', n: Paeonia suffruticosa 'Fen Zhouchou', o: Paeonia suffruticosa 'Xiuqiu Hong', p: Paeonia suffruticosa 'Zilou Xiangcui', q: Paeonia suffruticosa 'ZiYan', r: Paeonia suffruticosa 'JinGe', s: Paeonia suffruticosa 'JinZhi', t: Paeonia suffruticosa 'Baiwang Shizi', u: Paeonia suffruticosa 'Lian He'.
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Figure 2.
The flower development of Paeonia ostii 'FengDan' occurred in 16 stages: (a) overwintering dormancy, (b) bud break, (c) bud swelling, (d) bud elongation, (e) erect bud stage, (f) small bell stage, (g) large bell stage, (h) round peach stage, (i) flat peach stage, (j) color exposure, (k) bud cracking, (l) initial flowering, (m) half flowering, (n) full bloom, (o) onset of senescence, (p) full senescence. (a)–(e): bud stage, (f)–(i) flower bud stage, (j)–(p) flowering stage.
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Figure 3.
Electrophoresis detection of total RNA. Marker: DNA marker DL2000; 1–21: flower development period of a tree peony.
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Figure 4.
Amplification results of each primer. Marker: DNA marker DL2000; 1–23: amplification results of the 23 reference genes to be tested.
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Figure 5.
The melting curve of each primer. a–w: Melting curve plots corresponding to each of the 23 tested candidate reference genes, each showing a single specific amplification peak without non-specific products or primer dimers.
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Figure 6.
Expression levels of the 23 candidate reference genes in a tree peony. (a) Expression levels in full-bloom petals of early- and late-flowering cultivars. (b) Expression levels during 16 stages of floral bud differentiation and flower development.
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Figure 7.
Results of early- and late-flowering variety experiments using geNorm software. a: Average expression stability value (M) ranking of 23 candidate reference genes (genes arranged from least stable on the left to most stable on the right); b: Pairwise variation (V) analysis to determine the optimal number of reference genes for normalization.
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Figure 8.
Results of bud differentiation and flower development experiments using geNorm software. a: Average expression stability value (M) ranking of 23 candidate reference genes; genes arranged from left to right represent the least stable to the most stable. b: Pairwise variation (V) analysis to determine the optimal number of internal reference genes for normalization; the threshold value of V is set as 0.15.
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Figure 9.
Results of expression verification analysis of PsFRL5 in different varieties of peony flowers at 7 stages of development. a: PPC; b: GTP; c: PP2A. Notes: MU: mutant of Paeonia ostii 'Fengdan'; FD: Paeonia ostii 'Fengdan'; LH: Paeonia suffruticosa 'Lianhe'.
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Primers Sequences 5'-3' EF1-α-1-F CATCGGACAAGCCACTGCGT EF1-α-1-R CGTTATCACCAGGCAGTCCC Ubi-lp-F CCATCCTGGTGAAGTGGCTAA Ubi-lp-R TCAAGTGGGACAAACCGCAT UBQ-F CATCGGACAAGCCACTGCGT UBQ-R CGTTATCACCAGGCAGTCCC UBC-F TCTTAGGGTTTCTGTGCGGG UBC-R GAAGGCTTGCCAATGGAACA UPL-F TGTTGGCGACATTCCTTTCCT UPL-R TCCTACCGTTTGCTCCTACCTC GAPDH-F GGAAAATCCGTTGCCATCAT GAPDH-R CCTCTGTAACGGCATCTTTGG HIS-F CGAAAGTCTGCTCCTACTACGG HIS-R ATCCTTGGGCATAATGGTGAC CYP-F CCTCCAAACCCTAAAAATCCA CYP-R GAACTGGCATCCCTTGTAACC RPL-F GTGGGTTCGCATTCCTGAGA RPL-R AGCCAACCTCAAACCACCCT CSLD-F TCGTCCGCTACCACTTCCG CSLD-R CCTTGCCTCAACTACATCGTCA KOR-F ACACCGAACAATGCGAGAAC KOR-R AAGAAATGCTCCCATCAGGTT AGD-F TGCCATCGTCACCTGTTGC AGD-R CCCCCCAGCCTTTCCAAT ARP-F CGTTTGGTGTTTGGCGTTAT ARP-R AAACATCCCATCCACATCCTT IWS-F CTCTGCCACTCCACCGAAGC IWS-R AACACGGACTGGGTCGGATG GTP-F TCCTCGCATCGCTTGGTCTA GTP-R TGTCGGCTGGTGCTGAACTAA PP2A-F TGTGAGGGACAAAGCAGTGGA PP2A-R CCAGGTTTGACGCAGCAGAC eIF3-F ATCGCAAAAACCCTACCCGTC eIF3-R CGGGAGTTCTGATTGTAGTTGCG eIF2-F AGCCTGGTTATGAGGTTGACG eIF2-R GACCCACCAACCTATCAGCAC PPR-F CTCGTTCAGGTTATTAGCGGTG PPR-R CAGAAACAGAGACAGCCCAATC HDH-F GGCACTCAGTCACAGTTTCAC HDH-R TGTCCCGCTCGCATAATCTC PPC-F CTTGGTATCGGGTCCTATCGT PPC-R GGGACAACTCGTAATGGCTTC EF1-α-F CCGCCAGAGAGGCTGCTAAT EF1-α-R GCAATGTGGGAAGTGTGGCA Tubulin-α-F CCGTCAACTTTTCCACCCTG Tubulin-α-R CCTCACTCGGTCAAGGCAGA Table 1.
Primer sequences of candidate reference genes.
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Gene name Tm (°C) Slope R2 Efficiency EF1-α-1 64 −3.508 0.987 92.8% UBi-lp 61 −3.283 0.977 101.7% UBQ 61 −3.238 0.987 101.7% UBC 61 −3.392 0.994 97.1% UPL 62 −3.194 0.981 108.2% GAPDH 58 −3.101 0.971 110.1% HIS 59 −3.162 0.983 98.5% CYP 59 −3.111 0.978 97.3% RPL 61 −3.249 0.990 103.7% CSLD2 62 −3.468 0.970 99.4% KOR 58 −3.445 0.980 95.7% AGD 61 −3.144 0.973 102.8% ARP 59 −3.220 0.970 104.2% IWS 59 −3.306 0.980 98.1% GTP 62 −3.528 0.999 97.2% PP2A 61 −3.165 0.990 106.2% eIF2 59 −3.231 0.991 94.65 eIF3 64 −3.125 0.986 95.9% PPR 62 −3.284 0.993 96.9% HDH 61 −3.5755 0.970 99.5% PPC 59 −3.2020 0.996 97.6% EF1-α 62 −3.2965 0.980 99.8% Tubulin-α 62 −3.1160 0.990 102.6% Table 2.
Amplification characteristics of 23 candidate reference gene primers.
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Gene name Stability value SD Rank AGD 0.52 0.09 1 PPC 0.53 0.09 2 eIF2 0.54 0.09 3 GTP 0.62 0.10 4 KOR 0.62 0.10 5 CSLD2 0.65 0.11 6 UBi-lp 0.68 0.11 7 UBQ 0.70 0.11 8 UPL 0.79 0.12 9 HDH 0.79 0.12 10 eIF3 0.81 0.13 11 PPR 0.82 0.13 12 UBC 0.85 0.13 13 GAPDH 0.88 0.14 14 EF1-α 0.97 0.15 15 HIS 1.06 0.16 16 IWS 1.15 0.17 17 ARP 1.20 0.18 18 PP2A 1.21 0.18 19 EF1-α-1 1.33 0.20 20 CYP 1.67 0.25 21 Tubulin-α 1.78 0.26 22 RPL 2.15 0.31 23 Table 3.
Results of early- and late-flowering cultivar experiments using NormFinder software.
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Gene name Cq Stability
rankGeometric mean Mean Min Max SD CV/% eIF3 25.21 25.24 21.87 27.63 0.97 3.83 1 GTP 23.85 23.89 21.27 25.95 1.08 4.54 2 UBQ 22.82 22.87 20.17 26.28 1.11 4.85 3 eIF2 26.73 26.78 22.81 28.79 1.24 4.63 4 KOR 27.67 27.73 22.42 29.76 1.24 4.46 5 UBC 22.23 22.29 19.52 27.67 1.33 5.96 6 UBi-lp 27.35 27.40 23.40 31.88 1.36 4.95 7 AGD 24.55 24.60 21.03 27.7 1.39 5.64 8 PPC 26.34 26.40 21.46 28.65 1.40 5.32 9 CSLD2 26.25 26.32 21.65 29.23 1.43 5.45 10 HIS 25.36 25.45 19.51 28.07 1.47 5.77 11 ARP 27.84 27.91 22.88 31.23 1.47 5.27 12 PP2A 25.70 25.80 18.69 28.48 1.54 5.96 13 HDH 26.66 26.73 23.36 31.39 1.57 5.87 14 EF1-α-1 21.33 21.44 15.19 26.70 1.58 7.39 15 EF1-α 24.62 24.72 19.29 29.89 1.63 6.61 16 GAPDH 25.10 25.18 20.84 28.29 1.68 6.67 17 UPL 25.30 25.39 20.43 28.95 1.68 6.62 18 PPR 29.37 29.47 23.40 34.24 1.73 5.85 19 Tubulin-α 24.42 24.56 20.06 35.03 1.76 7.18 20 CYP 25.84 25.97 19.02 29.11 1.81 6.97 21 IWS 26.00 26.09 21.48 29.81 1.96 7.52 22 RPL 30.79 30.99 20.94 34.19 2.31 7.46 23 Table 4.
Results of early- and late-flowering cultivar experiments using BestKeeper software.
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Gene name Geomean of ranking values Stability rank Gene name Geomean of ranking values Stability rank PPC 2.06 1 PPR 12.26 13 AGD 2.83 2 GAPDH 13.84 14 eIF2 3.87 3 HIS 14.57 15 GTP 3.94 4 EF1-α 15.24 16 KOR 3.94 5 ARP 16.26 17 CSLD2 4.36 6 PP2A 17.28 18 eIF3 6.56 7 IWS 18.13 19 UBQ 6.93 8 EF1-α-1 18.61 20 UBi-lp 7.00 9 CYP 21.00 21 HDH 10.59 10 Tubulin-α 21.48 22 UPL 10.67 11 RPL 23.00 23 UBC 10.72 12 Table 5.
Results of early- and late-flowering cultivar experiments using RefFinder software.
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Gene name Stability value SD Rank GTP 0.27 0.04 1 EF1-α 0.37 0.05 2 GAPDH 0.37 0.05 3 EF1-α-1 0.40 0.06 4 Tubulin-α 0.42 0.06 5 HDH 0.43 0.06 6 PPR 0.44 0.06 7 UBQ 0.45 0.06 8 IWS 0.48 0.07 9 PPC 0.48 0.07 10 UBi-lp 0.48 0.07 11 UBC 0.48 0.07 12 CYP 0.51 0.07 13 AGD 0.51 0.07 14 KOR 0.53 0.07 15 eIF3 0.55 0.07 16 CSLD2 0.66 0.09 17 ARP 0.735 0.096 18 HIS 0.687 0.090 19 UPL 0.757 0.099 20 eIF2 0.810 0.105 21 RPL 1.009 0.130 22 PP2A 1.155 0.148 23 Table 6.
Results of bud differentiation and flower development experiments using NormFinder software.
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Gene name Cq Stability
rankGeometric mean Mean Min Max SD CV/% RPL 28.08 28.10 26.07 29.94 0.81 2.88 1 GTP 20.92 20.95 18.60 24.89 0.82 3.89 2 EF1-α 20.60 20.62 19.24 24.59 0.84 4.05 3 UBC 20.38 20.42 18.47 25.02 0.92 4.49 4 PP2A 21.88 21.92 17.85 24.44 0.95 4.32 5 ARP 24.80 24.83 22.48 27.31 0.97 3.89 6 IWS 23.33 23.37 21.32 28.86 0.97 4.16 7 EF1-α-1 19.55 19.61 17.02 26.17 1.00 5.08 8 CYP 22.41 22.47 20.04 29.22 1.00 4.45 9 AGD 20.71 20.75 18.27 26.63 1.03 4.98 10 HDH 23.53 23.58 21.59 30.15 1.04 4.39 11 UBQ 21.14 21.20 18.04 26.09 1.05 4.97 12 Tubulin-α 19.96 20.01 17.39 25.95 1.06 5.30 13 UBi-lp 23.82 23.88 21.51 31.37 1.06 4.45 14 KOR 24.07 24.12 20.81 28.99 1.11 4.61 15 GAPDH 21.58 21.63 19.12 26.98 1.15 5.34 16 eIF3 21.79 21.84 19.51 27.58 1.16 5.30 17 PPC 22.17 22.23 19.20 29.20 1.16 5.22 18 HIS 20.94 20.99 18.38 24.53 1.19 5.68 19 PPR 23.86 23.92 20.69 29.41 1.25 5.23 20 UPL 22.47 22.53 19.46 26.85 1.33 5.91 21 CSLD2 23.38 23.45 20.20 28.78 1.35 5.76 22 eIF2 22.21 22.28 19.07 28.97 1.44 6.48 23 Table 7.
Results of bud differentiation and flower development experiments using BestKeeper software.
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Gene name Geomean of ranking values Stability rank Gene name Geomean of ranking values Stability rank GTP 2.06 1 PPC 10.32 13 EF1-α-1 2.83 2 UBQ 10.57 14 EF1-α 4.36 3 PPR 10.77 15 HDH 5.30 4 ARP 14.79 16 UBi-lp 5.73 5 KOR 15.24 17 GAPDH 6.31 6 PP2A 15.71 18 Tubulin-α 6.35 7 eIF3 15.71 19 CYP 8.47 8 CSLD2 18.13 20 IWS 8.82 9 HIS 18.49 21 UBC 10.02 10 UPL 19.47 22 RPL 10.16 11 eIF2 21.22 23 AGD 10.22 12 Table 8.
Results of bud differentiation and flower development experiments using RefFinder software.
Figures
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Tables
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