A Selection of Reliable Reference Genes for Gene Expression Analysis in the Female and Male Flowers of Salix suchowensis
Abstract
:1. Introduction
2. Results
2.1. RNA Quality and Primer Specificity Analysis
2.2. Reference Gene Expression Levels
2.3. Expression Stability Analysis of the Candidate Reference Genes
2.3.1. geNorm Analysis
2.3.2. NormFinder Analysis
2.3.3. ΔCt Analysis
2.3.4. BestKeeper Analysis
2.3.5. RefFinder Analysis
2.4. Reference Gene Stability Verification
3. Discussion
4. Materials and Methods
4.1. Plant Material
4.2. Extraction of Total RNA from Plant Tissues and cDNA Aynthesis
4.3. Selection of Reference Genes and Primer Design
4.4. qRT-PCR of Candidate Reference Genes
4.5. Establishment of Reference Gene Primer Standard Curve
4.6. Data Processing
4.7. Reference Gene Verification
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Gene Description | Locus Name | Primer Sequence F/R(5′-3′) | Product Size (bp) | Efficiency (%) | R2 |
---|---|---|---|---|---|---|
α-TUB1 | alpha-tubulin1 | EVM0011215.1 | CTCTTGGAGCGTTTGTCAGT | 175 | 100.25 | 0.968 |
TCGTAGATGGCTTCATTGTC | ||||||
α-TUB2 | alpha-tubulin2 | EVM0036214.1 | TTTCAAGTGCGGTATCAACT | 250 | 96.45 | 0.981 |
CTTCCTCGTAGTCTTTCTCAA | ||||||
β-TUB | β-Tubulin | EVM0022575.1 | AATGCGGCAACCAAATAGGA | 164 | 86.66 | 0.996 |
TAGAACAGCACGAGGGACAA | ||||||
DnaJ | chaperone protein DnaJ 49 | EVM0005127.1 | GAGCCTATTTATGCCCTTTC | 162 | 95.45 | 0.990 |
GACGTAGTCCTTATCCACCTT | ||||||
CDC2 | Cyclin Dependent Kinase-putative | EVM0002694.1 | GATGTATGGTCAGTGGGATG | 158 | 106.62 | 0.983 |
TTGAAGTCAGGCAAAGAAGT | ||||||
ACT | actin | EVM0032804.1 | CCAAGGAGGCTGCTGGTAAT | 189 | 98.90 | 0.993 |
CCAAGGCCACCATGTCTGTC | ||||||
TIP41 | Type 2A phosphatase activator | EVM0041976.1 | GCTAATGAGATTAAGGGACA | 142 | 101.06 | 0.985 |
TGAAGCAGAGTCAGCAGGAT | ||||||
GAPDH | glyceraldehyde-3-phosphate dehydrogenase | EVM0035312.1 | CGAAGAAGGGCATTACAGCA | 121 | 96.08 | 0.970 |
GAGCATCGGAAGTCAACAGA | ||||||
H2A | histone H2A | EVM0033846.1 | GCAAGTATGCTGAGCGTGTT | 123 | 97.91 | 0.985 |
TACGGGTCTTCTTGTTGTCTC | ||||||
Target gene | ||||||
CFS | cop/fus-specific | EVM0013916.1 | AAGTTGGGAAACAGGTGGTC | 194 | 97.57 | 0.993 |
GTCTCCATTTGGTCGGTTGT |
Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
---|---|---|---|---|---|---|---|---|---|---|
♀ | Gene | DnaJ | α-TUB2 | TIP41 | GAPDH | H2A | α-TUB1 | β-TUB | ACT | CDC2 |
Stability | 0.022 | 0.023 | 0.257 | 0.315 | 0.377 | 0.573 | 0.612 | 0.624 | 0.823 | |
♂ | Gene | ACT | α-TUB2 | α-TUB1 | TIP41 | H2A | DnaJ | GAPDH | β-TUB | CDC2 |
Stability | 0.038 | 0.074 | 0.213 | 0.215 | 0.303 | 0.536 | 0.563 | 0.631 | 0.951 | |
Total | Gene | ACT | DnaJ | TIP41 | H2A | α-TUB1 | β-TUB | α-TUB2 | GAPDH | CDC2 |
Stability | 0.068 | 0.093 | 0.174 | 0.259 | 0.283 | 0.575 | 0.585 | 0.616 | 0.861 |
Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
---|---|---|---|---|---|---|---|---|---|---|
♀ | Gene | ACT | α-TUB1 | TIP41 | GAPDH | H2A | DnaJ | β-TUB | α-TUB2 | CDC2 |
STEDV | 0.68 | 0.68 | 0.75 | 0.76 | 0.83 | 0.96 | 1.01 | 1.03 | 1.23 | |
♂ | Gene | ACT | DnaJ | α-TUB1 | TIP41 | H2A | α-TUB2 | GAPDH | β-TUB | CDC2 |
STEDV | 0.63 | 0.66 | 0.68 | 0.77 | 0.79 | 0.90 | 0.94 | 1.06 | 1.40 | |
Total | Gene | ACT | DnaJ | TIP41 | α-TUB2 | H2A | α-TUB1 | GAPDH | β-TUB | CDC2 |
STEDV | 0.64 | 0.65 | 0.68 | 0.72 | 0.73 | 0.94 | 0.98 | 0.98 | 1.27 |
Rank | ♀ | ♂ | Total | ||||||
---|---|---|---|---|---|---|---|---|---|
Gene | SD | CV | Gene | SD | CV | Gene | SD | CV | |
1 | ACT | 0.42 | 2.05 | ACT | 0.55 | 2.68 | ACT | 0.50 | 2.42 |
2 | DnaJ | 0.56 | 2.84 | DnaJ | 0.56 | 2.86 | DnaJ | 0.56 | 2.85 |
3 | α-TUB1 | 0.77 | 3.88 | α-TUB1 | 0.89 | 4.40 | α-TUB1 | 0.84 | 4.21 |
4 | α-TUB2 | 1.01 | 4.84 | α-TUB2 | 1.05 | 4.67 | GAPDH | 1.11 | 4.94 |
5 | GAPDH | 1.05 | 4.64 | GAPDH | 1.17 | 5.21 | α-TUB2 | 1.22 | 5.63 |
6 | TIP41 | 1.32 | 5.44 | H2A | 1.19 | 5.94 | TIP41 | 1.30 | 5.28 |
7 | H2A | 1.47 | 7.07 | TIP41 | 1.24 | 4.95 | H2A | 1.34 | 6.57 |
8 | β-TUB | 1.85 | 10.28 | β-TUB | 1.88 | 10.16 | β-TUB | 1.88 | 10.28 |
9 | CDC2 | 2.08 | 8.66 | CDC2 | 2.16 | 8.64 | CDC2 | 2.15 | 8.8 |
Method | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
---|---|---|---|---|---|---|---|---|---|
Ranking order under female flower bud (Better-Good-Average) | |||||||||
geNorm | ACT/α-TUB1 | GAPDH | DnaJ | α-TUB2 | TIP41 | H2A | β-TUB | CDC2 | |
NormFinder | DnaJ | α-TUB2 | TIP41 | GAPDH | H2A | α-TUB1 | β-TUB | ACT | CDC2 |
Delta CT | ACT | α-TUB1 | DnaJ | GAPDH | α-TUB2 | TIP41 | H2A | β-TUB | CDC2 |
BestKeeper | ACT | DnaJ | α-TUB1 | α-TUB2 | GAPDH | TIP41 | H2A | β-TUB | CDC2 |
Comprehensive | ACT | DnaJ | α-TUB1 | GAPDH | α-TUB2 | TIP41 | H2A | β-TUB | CDC2 |
Ranking order under male flower bud (Better-Good-Average) | |||||||||
geNorm | ACT/DnaJ | α-TUB1 | α-TUB2 | GAPDH | TIP41 | H2A | β-TUB | CDC2 | |
NormFinder | GAPDH | α-TUB2 | α-TUB1 | TIP41 | H2A | DnaJ | GAPDH | β-TUB | CDC2 |
Delta CT | ACT | DnaJ | α-TUB1 | α-TUB2 | GAPDH | TIP41 | H2A | β-TUB | CDC2 |
BestKeeper | ACT | DnaJ | α-TUB1 | α-TUB2 | GAPDH | H2A | TIP41 | β-TUB | CDC2 |
Comprehensive | ACT | DnaJ | α-TUB1 | α-TUB2 | GAPDH | TIP41 | H2A | β-TUB | CDC2 |
Ranking order under total samples (Better-Good-Average) | |||||||||
geNorm | DnaJ/TIP41 | H2A | ACT | α-TUB1 | α-TUB2 | GAPDH | β-TUB | CDC2 | |
NormFinder | ACT | DnaJ | TIP41 | H2A | α-TUB1 | β-TUB | α-TUB2 | GAPDH | CDC2 |
Delta CT | ACT | DnaJ | TIP41 | α-TUB2 | H2A | α-TUB1 | GAPDH | β-TUB | CDC2 |
BestKeeper | ACT | DnaJ | α-TUB1 | GAPDH | α-TUB2 | TIP41 | H2A | β-TUB | CDC2 |
Comprehensive | ACT | DnaJ | TIP41 | H2A | α-TUB1 | α-TUB2 | GAPDH | β-TUB | CDC2 |
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Zhou, F.; Chen, Y.; Wu, H.; Yin, T. A Selection of Reliable Reference Genes for Gene Expression Analysis in the Female and Male Flowers of Salix suchowensis. Plants 2022, 11, 647. https://doi.org/10.3390/plants11050647
Zhou F, Chen Y, Wu H, Yin T. A Selection of Reliable Reference Genes for Gene Expression Analysis in the Female and Male Flowers of Salix suchowensis. Plants. 2022; 11(5):647. https://doi.org/10.3390/plants11050647
Chicago/Turabian StyleZhou, Fangwei, Yingnan Chen, Huaitong Wu, and Tongming Yin. 2022. "A Selection of Reliable Reference Genes for Gene Expression Analysis in the Female and Male Flowers of Salix suchowensis" Plants 11, no. 5: 647. https://doi.org/10.3390/plants11050647