Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (2): 377-388.doi: 10.16420/j.issn.0513-353x.2020-0306
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MA Lulin1,2,*, DUAN Qing1,*, CUI Guangfen1, DU Wenwen1, JIA Wenjie1, WANG Xiangning1, WANG Jihua1,**(), CHEN Fadi2,**(
)
Received:
2020-09-27
Revised:
2020-12-10
Online:
2021-02-25
Published:
2021-03-09
Contact:
WANG Jihua,CHEN Fadi
E-mail:wangjh0505@sohu.com;chenfd@njau.edu.cn
CLC Number:
MA Lulin, DUAN Qing, CUI Guangfen, DU Wenwen, JIA Wenjie, WANG Xiangning, WANG Jihua, CHEN Fadi. Selection and Validation of Reference Genes for qRT-PCR Analysis of the Correlated Genes in Flower Pigments Biosynthesis Pathway of Anemone obtusiloba[J]. Acta Horticulturae Sinica, 2021, 48(2): 377-388.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2020-0306
基因ID Gene ID | FPKM | 比对结果 Blast result | 基因 Gene | |||||
---|---|---|---|---|---|---|---|---|
蓝花 Blue flower | 白花 White flower | |||||||
1 | 2 | 3 | 1 | 2 | 3 | |||
DN35855_c0_g1_i10_1 | 354.26 | 385.74 | 422.52 | 336.35 | 354.00 | 390.89 | Glyceraldehyde 3-phosphate dehydrogenase [Gynura bicolor] (GenBank:AB550243.1) | GAPDH |
DN24449_c0_g1_i2_1 | 314.43 | 331.50 | 366.20 | 272.08 | 300.62 | 327.47 | Elongation factor 1-beta 2-like[Pistacia vera](NCBI reference sequence:XM_031416658.1) | EF-1β |
DN38564_c0_g2_i1_1 | 434.91 | 450.67 | 464.33 | 473.54 | 517.63 | 522.32 | Actin 7 [Prunus dulcis] (GenBank:BBH05534.1) | ACT |
DN25341_c0_g2_i1_2 | 153.63 | 173.57 | 173.87 | 143.18 | 160.60 | 185.16 | Tubulin beta chain[Ricinus communis] (NCBI reference sequence: XM_025156136.1) | β-TUB |
DN24929_c0_g1_i4_2 | 1 016.78 | 975.95 | 933.45 | 1 206.08 | 1 170.53 | 1 067.26 | Polyubiquitin,partial [Wolffia australiana](GenBank:AEZ49160.1) | UBQ |
DN34836_c0_g2_i6_2 | 1 052.09 | 1 172.91 | 1 294.86 | 1 140.09 | 1 193.46 | 1 301.07 | Aquaporin TIP1-2[Brassica rapa] (NCBI reference sequence: XP_009129499.) | AQP |
DN24035_c0_g1_i2_1 | 131.24 | 122.16 | 141.00 | 106.95 | 108.04 | 119.61 | Histone H2A variant 1[Phalaenopsis equestris](NCBI reference sequence:XP_020573071.1) | HIS |
DN25248_c0_g2_i7_1 | 197.63 | 210.20 | 236.21 | 157.42 | 172.19 | 188.42 | 60S ribosomal protein L13-1 [Camellia sinensis](NCBI reference sequence:XP_028086825.1) | RPL13 |
Table 1 Eight candidate reference genes in RNA-seq libraries of Anemone obtusiloba
基因ID Gene ID | FPKM | 比对结果 Blast result | 基因 Gene | |||||
---|---|---|---|---|---|---|---|---|
蓝花 Blue flower | 白花 White flower | |||||||
1 | 2 | 3 | 1 | 2 | 3 | |||
DN35855_c0_g1_i10_1 | 354.26 | 385.74 | 422.52 | 336.35 | 354.00 | 390.89 | Glyceraldehyde 3-phosphate dehydrogenase [Gynura bicolor] (GenBank:AB550243.1) | GAPDH |
DN24449_c0_g1_i2_1 | 314.43 | 331.50 | 366.20 | 272.08 | 300.62 | 327.47 | Elongation factor 1-beta 2-like[Pistacia vera](NCBI reference sequence:XM_031416658.1) | EF-1β |
DN38564_c0_g2_i1_1 | 434.91 | 450.67 | 464.33 | 473.54 | 517.63 | 522.32 | Actin 7 [Prunus dulcis] (GenBank:BBH05534.1) | ACT |
DN25341_c0_g2_i1_2 | 153.63 | 173.57 | 173.87 | 143.18 | 160.60 | 185.16 | Tubulin beta chain[Ricinus communis] (NCBI reference sequence: XM_025156136.1) | β-TUB |
DN24929_c0_g1_i4_2 | 1 016.78 | 975.95 | 933.45 | 1 206.08 | 1 170.53 | 1 067.26 | Polyubiquitin,partial [Wolffia australiana](GenBank:AEZ49160.1) | UBQ |
DN34836_c0_g2_i6_2 | 1 052.09 | 1 172.91 | 1 294.86 | 1 140.09 | 1 193.46 | 1 301.07 | Aquaporin TIP1-2[Brassica rapa] (NCBI reference sequence: XP_009129499.) | AQP |
DN24035_c0_g1_i2_1 | 131.24 | 122.16 | 141.00 | 106.95 | 108.04 | 119.61 | Histone H2A variant 1[Phalaenopsis equestris](NCBI reference sequence:XP_020573071.1) | HIS |
DN25248_c0_g2_i7_1 | 197.63 | 210.20 | 236.21 | 157.42 | 172.19 | 188.42 | 60S ribosomal protein L13-1 [Camellia sinensis](NCBI reference sequence:XP_028086825.1) | RPL13 |
KEGG途径 KEGG pathway | 基因表达情况(白花为对照) Gene expression(White flower was the control group) | 基因ID Gene ID | FPKM | |||||
---|---|---|---|---|---|---|---|---|
蓝花 Blue flower | 白花 White flower | |||||||
1 | 2 | 3 | 1 | 2 | 3 | |||
类黄酮生 物合成 Flavonoid biosynthetic | 上调Up | DN26579_c0_g1_i3_2 | 569.86 | 469.94 | 406.45 | 109.46 | 98.07 | 113.44 |
上调Up | DN27520_c0_g1_i2_2 | 341.41 | 278.90 | 233.47 | 38.08 | 29.97 | 27.08 | |
上调Up | DN27888_c0_g1_i1_1 | 1 023.47 | 839.09 | 694.98 | 89.89 | 74.08 | 65.41 | |
上调Up | DN34928_c0_g1_i4_1 | 237.71 | 182.14 | 158.62 | 19.84 | 21.50 | 18.22 | |
上调Up | DN37635_c0_g1_i7_1 | 390.62 | 309.90 | 252.52 | 27.24 | 22.26 | 25.06 | |
无差异No-difference | DN29460_c0_g1_i3_1 | 1.66 | 2.73 | 1.54 | 1.08 | 0.85 | 0.93 | |
无差异No-difference | DN29383_c0_g1_i2_1 | 16.90 | 15.95 | 20.66 | 9.54 | 11.30 | 13.71 | |
无差异No-difference | DN32877_c0_g1_i3_2 | 64.45 | 55.33 | 49.30 | 53.18 | 40.37 | 68.46 | |
无差异No-difference | DN32877_c0_g2_i5_2 | 132.45 | 137.58 | 136.64 | 119.78 | 124.61 | 149.95 | |
无差异No-difference | DN33447_c0_g1_i2_2 | 99.69 | 101.66 | 84.61 | 93.18 | 87.51 | 108.33 | |
无差异No-difference | DN34032_c0_g1_i1_1 | 100.37 | 101.35 | 92.38 | 92.69 | 86.81 | 93.82 | |
无差异No-difference | DN34871_c2_g3_i1_2 | 34.07 | 41.26 | 40.60 | 34.36 | 36.08 | 45.69 | |
无差异No-difference | DN35887_c0_g1_i1_2 | 133.36 | 129.04 | 121.26 | 96.60 | 102.29 | 115.96 | |
无差异No-difference | DN36570_c0_g2_i4_1 | 81.57 | 91.62 | 87.44 | 81.32 | 80.35 | 109.16 | |
花青素生 物合成 Anthocyanin biosynthetic | 上调Up | DN36200_c0_g1_i3_1 | 589.72 | 479.41 | 394.60 | 57.97 | 47.95 | 45.82 |
无差异No-difference | DN35376_c0_g1_i4_2 | 31.79 | 31.73 | 30.26 | 37.08 | 37.12 | 34.80 |
Table 2 Some genes in the flavonoids/anthocyanins pathways of Anemone obtusiloba
KEGG途径 KEGG pathway | 基因表达情况(白花为对照) Gene expression(White flower was the control group) | 基因ID Gene ID | FPKM | |||||
---|---|---|---|---|---|---|---|---|
蓝花 Blue flower | 白花 White flower | |||||||
1 | 2 | 3 | 1 | 2 | 3 | |||
类黄酮生 物合成 Flavonoid biosynthetic | 上调Up | DN26579_c0_g1_i3_2 | 569.86 | 469.94 | 406.45 | 109.46 | 98.07 | 113.44 |
上调Up | DN27520_c0_g1_i2_2 | 341.41 | 278.90 | 233.47 | 38.08 | 29.97 | 27.08 | |
上调Up | DN27888_c0_g1_i1_1 | 1 023.47 | 839.09 | 694.98 | 89.89 | 74.08 | 65.41 | |
上调Up | DN34928_c0_g1_i4_1 | 237.71 | 182.14 | 158.62 | 19.84 | 21.50 | 18.22 | |
上调Up | DN37635_c0_g1_i7_1 | 390.62 | 309.90 | 252.52 | 27.24 | 22.26 | 25.06 | |
无差异No-difference | DN29460_c0_g1_i3_1 | 1.66 | 2.73 | 1.54 | 1.08 | 0.85 | 0.93 | |
无差异No-difference | DN29383_c0_g1_i2_1 | 16.90 | 15.95 | 20.66 | 9.54 | 11.30 | 13.71 | |
无差异No-difference | DN32877_c0_g1_i3_2 | 64.45 | 55.33 | 49.30 | 53.18 | 40.37 | 68.46 | |
无差异No-difference | DN32877_c0_g2_i5_2 | 132.45 | 137.58 | 136.64 | 119.78 | 124.61 | 149.95 | |
无差异No-difference | DN33447_c0_g1_i2_2 | 99.69 | 101.66 | 84.61 | 93.18 | 87.51 | 108.33 | |
无差异No-difference | DN34032_c0_g1_i1_1 | 100.37 | 101.35 | 92.38 | 92.69 | 86.81 | 93.82 | |
无差异No-difference | DN34871_c2_g3_i1_2 | 34.07 | 41.26 | 40.60 | 34.36 | 36.08 | 45.69 | |
无差异No-difference | DN35887_c0_g1_i1_2 | 133.36 | 129.04 | 121.26 | 96.60 | 102.29 | 115.96 | |
无差异No-difference | DN36570_c0_g2_i4_1 | 81.57 | 91.62 | 87.44 | 81.32 | 80.35 | 109.16 | |
花青素生 物合成 Anthocyanin biosynthetic | 上调Up | DN36200_c0_g1_i3_1 | 589.72 | 479.41 | 394.60 | 57.97 | 47.95 | 45.82 |
无差异No-difference | DN35376_c0_g1_i4_2 | 31.79 | 31.73 | 30.26 | 37.08 | 37.12 | 34.80 |
用途 Purpose | 基因 Gene | 引物序列(5′-3′) Primer sequence | 产物长度/bp Product length |
---|---|---|---|
内参基因 Reference gene | GAPDH | F:ATGGGTGTGAATGAGAAGGA;R:CGTGGACGGTTGTCATAAG | 142 |
EF1-β2 | F:GAAAGAGAGTGGCAAATCCT;R:GCATCTCGATGCTGCGAA | 101 | |
ACT | F:GCCGTGCTTTCTCTCTATG;R:TTGGGACTGTGTGGCTAAC | 82 | |
β-TUB | F:ATATCCCACCAACTGGACT;R:ACTGTTCTGAAACTCTGCG | 90 | |
UBQ | F:CTTTGCCGGGAAACAATTAG;R:GACGGAGCACCAAGTGAA | 86 | |
AQP | F:CCGGCGGAAAGTCTTATAAT;R:AATGTTTCCACCGAGAAGAG | 135 | |
HIS | F:AGCTTGATACACTGATCAAAGG;R:GGTAGACTTGTTGATGAGTGAC | 83 | |
RPL13 | F:TGCAGTTACTCAGCTCACAG;R:CTTTGAAGGCTTTCATCTCCTC | 98 | |
鉴定基因 Identification gene | DN26579_c0_g1_i3_2 | F:AAGACGTGGATCACGGTTA;R:CTTGAACCTCCCATTGCT | 90 |
DN27520_c0_g1_i2_2 | F:GCACCGAAGAGGAATGCAA;R:GTCGATAACTGGAATCTGCG | 86 | |
DN27888_c0_g1_i1_1 | F:GTGGTGGCGGAGATGTTA;R:CCTTTGGTGAGAAACACTCG | 83 | |
DN34928_c0_g1_i4_1 | F:CCTAGTGACAAAGGTGCTG;R:TTAGTAAGTCGTCCATGCCG | 95 | |
DN37635_c0_g1_i7_1 | F:ATGCCTCCTAGCCTGCTAA;R:TACCAGTTGAACCTGTTTCAGA | 84 | |
DN29460_c0_g1_i3_1 | F:ATCAACCTCTCCCTTATAGACC;R:TTTCGTTCTTCTTCCCTGAGT | 82 | |
DN29383_c0_g1_i2_1 | F:ACCAAGATCAAGAATGGCG;R:TTGATATTTGTGTCGGTGAGC | 122 | |
DN32877_c0_g1_i3_2 | F:GAACGGATCTGTTGTCGC;R:GGTAGCTGGCAAATCTCAAT | 126 | |
DN32877_c0_g2_i5_2 | F:TGGCAATGGACATAAACCG;R:CTTTGAACTCAATCTTGTGACC | 87 | |
DN33447_c0_g1_i2_2 | F:TGACCACTGGTACAACGC;R:GACTGTAGTCCGATGAAACAC | 115 | |
DN34032_c0_g1_i1_1 | F:GGAGGAGGAATCATTTGCC;R:TCAACGAAATGTTGCTTAGCG | 117 | |
DN34871_c2_g3_i1_2 | F:ACCAAGATCAAGAATGGCG;R:TTGATATTTGTGTCGGTGAGC | 122 | |
DN35887_c0_g1_i1_2 | F:CACCATGATCTTGCCATTAAC;R:CATTAGCTTTCCAGTAGGCG | 80 | |
DN36570_c0_g2_i4_1 | F:GAACGGATCTGTTGTCGC;R:AGCCAAAGCCTTGTTAAGT | 94 | |
DN36200_c0_g1_i3_1 | F:CAGGAATGTCATCTGTACGG;R:ATGCAACAAGTTACCCAAGTAG | 80 | |
DN35376_c0_g1_i4_2 | F:TGCTTCTTCCCTCACCTG;R:TTCTCGTGGCAAATCCCG | 130 |
Table 3 Primers in this study
用途 Purpose | 基因 Gene | 引物序列(5′-3′) Primer sequence | 产物长度/bp Product length |
---|---|---|---|
内参基因 Reference gene | GAPDH | F:ATGGGTGTGAATGAGAAGGA;R:CGTGGACGGTTGTCATAAG | 142 |
EF1-β2 | F:GAAAGAGAGTGGCAAATCCT;R:GCATCTCGATGCTGCGAA | 101 | |
ACT | F:GCCGTGCTTTCTCTCTATG;R:TTGGGACTGTGTGGCTAAC | 82 | |
β-TUB | F:ATATCCCACCAACTGGACT;R:ACTGTTCTGAAACTCTGCG | 90 | |
UBQ | F:CTTTGCCGGGAAACAATTAG;R:GACGGAGCACCAAGTGAA | 86 | |
AQP | F:CCGGCGGAAAGTCTTATAAT;R:AATGTTTCCACCGAGAAGAG | 135 | |
HIS | F:AGCTTGATACACTGATCAAAGG;R:GGTAGACTTGTTGATGAGTGAC | 83 | |
RPL13 | F:TGCAGTTACTCAGCTCACAG;R:CTTTGAAGGCTTTCATCTCCTC | 98 | |
鉴定基因 Identification gene | DN26579_c0_g1_i3_2 | F:AAGACGTGGATCACGGTTA;R:CTTGAACCTCCCATTGCT | 90 |
DN27520_c0_g1_i2_2 | F:GCACCGAAGAGGAATGCAA;R:GTCGATAACTGGAATCTGCG | 86 | |
DN27888_c0_g1_i1_1 | F:GTGGTGGCGGAGATGTTA;R:CCTTTGGTGAGAAACACTCG | 83 | |
DN34928_c0_g1_i4_1 | F:CCTAGTGACAAAGGTGCTG;R:TTAGTAAGTCGTCCATGCCG | 95 | |
DN37635_c0_g1_i7_1 | F:ATGCCTCCTAGCCTGCTAA;R:TACCAGTTGAACCTGTTTCAGA | 84 | |
DN29460_c0_g1_i3_1 | F:ATCAACCTCTCCCTTATAGACC;R:TTTCGTTCTTCTTCCCTGAGT | 82 | |
DN29383_c0_g1_i2_1 | F:ACCAAGATCAAGAATGGCG;R:TTGATATTTGTGTCGGTGAGC | 122 | |
DN32877_c0_g1_i3_2 | F:GAACGGATCTGTTGTCGC;R:GGTAGCTGGCAAATCTCAAT | 126 | |
DN32877_c0_g2_i5_2 | F:TGGCAATGGACATAAACCG;R:CTTTGAACTCAATCTTGTGACC | 87 | |
DN33447_c0_g1_i2_2 | F:TGACCACTGGTACAACGC;R:GACTGTAGTCCGATGAAACAC | 115 | |
DN34032_c0_g1_i1_1 | F:GGAGGAGGAATCATTTGCC;R:TCAACGAAATGTTGCTTAGCG | 117 | |
DN34871_c2_g3_i1_2 | F:ACCAAGATCAAGAATGGCG;R:TTGATATTTGTGTCGGTGAGC | 122 | |
DN35887_c0_g1_i1_2 | F:CACCATGATCTTGCCATTAAC;R:CATTAGCTTTCCAGTAGGCG | 80 | |
DN36570_c0_g2_i4_1 | F:GAACGGATCTGTTGTCGC;R:AGCCAAAGCCTTGTTAAGT | 94 | |
DN36200_c0_g1_i3_1 | F:CAGGAATGTCATCTGTACGG;R:ATGCAACAAGTTACCCAAGTAG | 80 | |
DN35376_c0_g1_i4_2 | F:TGCTTCTTCCCTCACCTG;R:TTCTCGTGGCAAATCCCG | 130 |
内参基因 Reference gene | 最大值 Maximum value | 最小值 Minimum value | 平均值 Mean | 标准差 SD | 变异系数/% CV |
---|---|---|---|---|---|
GAPDH | 22.86 | 21.81 | 22.315 | 0.381 | 1.709 |
EF1-β2 | 31.99 | 30.38 | 31.269 | 0.614 | 1.963 |
ACT | 21.77 | 20.60 | 21.075 | 0.349 | 1.658 |
β-TUB | 27.02 | 23.03 | 24.289 | 1.523 | 6.270 |
UBQ | 22.96 | 21.51 | 22.184 | 0.586 | 2.641 |
AQP | 22.06 | 20.51 | 21.150 | 0.599 | 2.831 |
HIS | 27.82 | 26.03 | 27.006 | 0.714 | 2.645 |
RPL13 | 25.66 | 23.04 | 24.303 | 1.091 | 4.490 |
Table 4 Analysis of the Ct values of candidate reference genes
内参基因 Reference gene | 最大值 Maximum value | 最小值 Minimum value | 平均值 Mean | 标准差 SD | 变异系数/% CV |
---|---|---|---|---|---|
GAPDH | 22.86 | 21.81 | 22.315 | 0.381 | 1.709 |
EF1-β2 | 31.99 | 30.38 | 31.269 | 0.614 | 1.963 |
ACT | 21.77 | 20.60 | 21.075 | 0.349 | 1.658 |
β-TUB | 27.02 | 23.03 | 24.289 | 1.523 | 6.270 |
UBQ | 22.96 | 21.51 | 22.184 | 0.586 | 2.641 |
AQP | 22.06 | 20.51 | 21.150 | 0.599 | 2.831 |
HIS | 27.82 | 26.03 | 27.006 | 0.714 | 2.645 |
RPL13 | 25.66 | 23.04 | 24.303 | 1.091 | 4.490 |
排序 Ranking | geNorm | NormFinder | BestKeeper | |||||
---|---|---|---|---|---|---|---|---|
内参基因 Reference gene | M值 M | 内参基因 Reference gene | 稳定值 SV | 内参基因 Reference genes | 标准差 SD | |||
1 | UBQ | 0.413 | UBQ | 0.068 | ACT | 0.282 | ||
2 | EF1-β2 | 0.425 | AQP | 0.071 | GAPDH | 0.356 | ||
3 | AQP | 0.441 | EF1-β2 | 0.113 | AQP | 0.531 | ||
4 | HIS | 0.460 | HIS | 0.160 | UBQ | 0.567 | ||
5 | GAPDH | 0.535 | ACT | 0.287 | EF1-β2 | 0.574 | ||
6 | ACT | 0.543 | GAPDH | 0.311 | HIS | 0.678 | ||
7 | RPL13 | 0.644 | RPL13 | 0.325 | RPL13 | 1.055 | ||
8 | β-TUB | 1.128 | β-TUB | 0.784 | β-TUB | 1.283 |
Table 5 Stability analysis of candidate reference genes
排序 Ranking | geNorm | NormFinder | BestKeeper | |||||
---|---|---|---|---|---|---|---|---|
内参基因 Reference gene | M值 M | 内参基因 Reference gene | 稳定值 SV | 内参基因 Reference genes | 标准差 SD | |||
1 | UBQ | 0.413 | UBQ | 0.068 | ACT | 0.282 | ||
2 | EF1-β2 | 0.425 | AQP | 0.071 | GAPDH | 0.356 | ||
3 | AQP | 0.441 | EF1-β2 | 0.113 | AQP | 0.531 | ||
4 | HIS | 0.460 | HIS | 0.160 | UBQ | 0.567 | ||
5 | GAPDH | 0.535 | ACT | 0.287 | EF1-β2 | 0.574 | ||
6 | ACT | 0.543 | GAPDH | 0.311 | HIS | 0.678 | ||
7 | RPL13 | 0.644 | RPL13 | 0.325 | RPL13 | 1.055 | ||
8 | β-TUB | 1.128 | β-TUB | 0.784 | β-TUB | 1.283 |
排序 Ranking | 内参基因 Reference gene | 几何平均数 Geomean of ranking values |
---|---|---|
1 | UBQ | 1.587 |
2 | AQP | 2.621 |
3 | EF1-β2 | 3.107 |
3 | GAPDH | 3.107 |
5 | ACT | 3.915 |
6 | HIS | 4.579 |
7 | RPL13 | 7.000 |
8 | β-TUB | 8.000 |
Table 6 The comprehensive ranking of stability of candidate reference genes
排序 Ranking | 内参基因 Reference gene | 几何平均数 Geomean of ranking values |
---|---|---|
1 | UBQ | 1.587 |
2 | AQP | 2.621 |
3 | EF1-β2 | 3.107 |
3 | GAPDH | 3.107 |
5 | ACT | 3.915 |
6 | HIS | 4.579 |
7 | RPL13 | 7.000 |
8 | β-TUB | 8.000 |
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