Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data
Abstract
:1. Introduction
2. Results
2.1. Primer Specificity and PCR Amplification Efficiency
2.2. Threshold Cycle (Ct) Values of Candidate RGs
2.3. Expression Stability of Candidate RGs
2.3.1. GeNorm analysis
2.3.2. NormFinder Analysis
2.3.3. BestKeeper Analysis
2.4. Unified Rank Lists by RankAggreg
2.5. Validation of Candidate RGs
3. Discussion
4. Materials and Methods
4.1. Plant Material
4.2. Selection of Candidate RGs and Primer Design
4.3. RNA Isolation, cDNA Synthesis, and qRT-PCR
4.4. Data Analysis
4.5. Statistical Method for Rank Aggregation
4.6. Validation of the Candidate RGs
4.7. Library Construction, Sequencing, and Assembly of Transcriptomes
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
cDNA | Complementary DNA |
CLO | Caleosin |
Ct | Cycle threshold |
DAS | Day after sowing |
DGAT | Diacylglycerol acyltransferase |
FPKM | Fragments per kilobase of exon model per million mapped reads |
MUFA | Monounsaturated fatty acid |
qRT-PCR | Quantitative real-time reverse transcription-polymerase chain reaction |
RG | Reference gene |
RT | Room temperature |
Tm | Melting temperature |
References
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Gene | Description | Primer Sequence (5′-3′; F, forward; R, Reverse) | Amplicon Length (bp) | Primers Tm (°C) | E (%) | R2 |
---|---|---|---|---|---|---|
18S | 18S Ribosomal RNA | F: GGAAGTTTGAGGCAATAACAGG | 140 | 53.71/55.18 | 97.29 | 0.9931 |
R: TATCCCCATCACGATGAAATTTCTC | ||||||
ACT | Actin | F: CTCAACCCCAAGGCCAACA | 146 | 53.52/53.81 | 115.7 | 0.9923 |
R: CCATCACCAGAGTCAAGAACAATA | ||||||
ADF7 | Actin-depolymerizing factor 7 | F: GACACCGCAAGGGTAAGG | 118 | 52.84/52.25 | 103.59 | 0.9942 |
R: CAAGCCCCATCTCAGTAGG | ||||||
CYC | Cyclophilin | F: GGTGAAAAGGGTATCGGTC | 140 | 51.73/52.41 | 105.16 | 0.9998 |
R: TTTCCGTAGATGGACTCGC | ||||||
EF1α | Elongation factor | F: CTGGTATGCTTGTGACATTTGG | 175 | 54.07/53.96 | 98.64 | 0.9931 |
1-alpha | R: TCGTCCTTGGAGTTGGAGG | |||||
EF2 | Elongation factor2 | F: TGTCCTTCCGTGAGACCGTA | 203 | 55.85/53.17 | 103.93 | 0.9959 |
R: TCCTTGTCCCATCCGAACT | ||||||
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase | F: ATTCCCAGCAGCACTGGTG | 93 | 52.75/51.93 | 110.61 | 0.9962 |
R: AGTTGGCACACGGAAAGCC | ||||||
MDH | Malate | F: ACCCTCTTGTGTCGGTTCTT | 189 | 54.44/52.28 | 95.78 | 0.9944 |
Dehydrogenase | R: TTGTCATGCCTGGTTTACG | |||||
PGK | Phosphoglycerate | F: AGAAACCAAGGCTTCGTCA | 168 | 52.24/52.98 | 92.54 | 0.992 |
Kinase | R: AAGGGAGTCACAACCATCATT | |||||
RPL11 | Ribosomal protein | F: CTGGATGCTTTGGATTCGG | 174 | 51.82/53.5 | 96.39 | 0.9965 |
L11 | R: CCTTGGTAACTCTGTGCTGGA | |||||
Rubisco | Ribulose bisphosphate carboxylase | F: ATGTCTACGTGGTGGACTTGAT | 124 | 52.23/51.92 | 108.65 | 0.9973 |
R: TGTTTCGGCTTGTGCTTTAT | ||||||
TUB4 | Tubulin beta-4 | F: CAGGAAGGAGGCTGAAAAT | 156 | 52.06/53.6 | 118.22 | 0.9931 |
R: GAGGGGAAGACAGAGAAGGT | ||||||
UCE2 | Ubiquitin-conjugating enzyme 2 | F: ATCATCAAGGAGACCCAGCG | 183 | 55.14/53.39 | 98.21 | 0.9998 |
R: CTTAGGGGCAGCCATAGGA | ||||||
UBL5 | Ubiquitin-like | F: ATAATCCCCGTATTTCCACTGC | 96 | 54.34/55.54 | 109.54 | 0.9952 |
protein 5 | R: GAATCTATCCTATCCACGCTCTCT | |||||
CLO | Caleosin | F: ACGGCATTGTTTATCCCTGG | 178 | 53.86/54.32 | 117.02 | 0.9914 |
R: TGTTTGGCTCTGTGTATGTTGTGT | ||||||
DGAT2-2 | Diacylglycerol | F: CAGGTGGTGTTCAAGAGATGCT | 126 | 54.85/53.26 | 102.45 | 0.9953 |
O-acyltransferase 2-2 | R: CAAAGGAGAAAACAGGGACAAGT |
geNorm Rank | Tissues of YN | Tissues of XJ | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Aboveground | Underground | Tuber | Total | Aboveground | Underground | Tuber | Total | |||||||||
Gene | M | Gene | M | Gene | M | Gene | M | Gene | M | Gene | M | Gene | M | Gene | M | |
1 | UCE2 | 0.316 | UCE2 | 0.048 | UCE2 | 0.041 | UCE2 | 0.403 | UCE2 | 0.292 | UCE2 | 0.215 | TUB4 | 0.220 | UCE2 | 0.267 |
2 | MDH | 0.320 | TUB4 | 0.062 | TUB4 | 0.053 | MDH | 0.409 | UBL5 | 0.292 | TUB4 | 0.233 | UBL5 | 0.241 | ADF7 | 0.296 |
3 | UBL5 | 0.398 | ACT | 0.120 | ACT | 0.115 | TUB4 | 0.422 | EF1α | 0.303 | ACT | 0.270 | UCE2 | 0.529 | UBL5 | 0.382 |
4 | ADF7 | 0.414 | EF2 | 0.380 | EF2 | 0.275 | UBL5 | 0.593 | CYC | 0.341 | UBL5 | 0.285 | PGK | 0.589 | 18S | 0.474 |
5 | ACT | 0.463 | MDH | 0.514 | MDH | 0.373 | CYC | 0.665 | GAPDH | 0.345 | ADF7 | 0.310 | ADF7 | 0.591 | CYC | 0.510 |
6 | CYC | 0.560 | RPL11 | 0.598 | GAPDH | 0.461 | EF2 | 0.722 | ACT | 0.346 | 18S | 0.348 | CYC | 0.591 | EF2 | 0.534 |
7 | TUB4 | 0.591 | CYC | 0.657 | CYC | 0.542 | RPL11 | 0.756 | MDH | 0.367 | EF1α | 0.375 | RPL11 | 0.679 | GAPDH | 0.551 |
8 | EF2 | 0.593 | EF1α | 0.724 | RPL11 | 0.608 | GAPDH | 0.818 | ADF7 | 0.382 | PGK | 0.386 | MDH | 0.711 | TUB4 | 0.574 |
9 | EF1α | 0.623 | GAPDH | 0.775 | Rubisco | 0.655 | 18S | 0.869 | TUB4 | 0.384 | RPL11 | 0.397 | EF1α | 0.770 | EF1α | 0.576 |
10 | RPL11 | 0.641 | UBL5 | 0.814 | EF1α | 0.684 | ACT | 0.969 | 18S | 0.394 | CYC | 0.419 | EF2 | 0.831 | MDH | 0.612 |
11 | GAPDH | 0.654 | 18S | 0.878 | UBL5 | 0.727 | ADF7 | 1.014 | EF2 | 0.446 | EF2 | 0.438 | ACT | 0.855 | RPL11 | 0.643 |
12 | 18S | 0.740 | PGK | 0.940 | 18S | 0.791 | EF1α | 1.089 | RPL11 | 0.485 | MDH | 0.448 | 18S | 0.975 | PGK | 0.777 |
13 | PGK | 1.163 | ADF7 | 0.995 | PGK | 0.865 | PGK | 1.365 | PGK | 0.513 | GAPDH | 0.485 | GAPDH | 1.320 | ACT | 0.790 |
14 | Rubisco | 1.304 | Rubisco | 1.182 | ADF7 | 0.954 | Rubisco | 1.552 | Rubisco | 0.956 | Rubisco | 0.660 | Rubisco | 1.409 | Rubisco | 0.963 |
Norm- Finder Rank | Tissues of YN | Tissues of XJ | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Aboveground | Underground | Tuber | Total | Aboveground | Underground | Tuber | Total | |||||||||
Gene | M | Gene | M | Gene | M | Gene | M | Gene | M | Gene | M | Gene | M | Gene | M | |
1 | UCE2 | 0.152 | TUB4 | 0.211 | TUB4 | 0.221 | UCE2 | 0.359 | UCE2 | 0.233 | TUB4 | 0.190 | TUB4 | 0.191 | UBL5 | 0.231 |
2 | UBL5 | 0.320 | RPL11 | 0.290 | RPL11 | 0.236 | UBL5 | 0.409 | EF1α | 0.292 | UBL5 | 0.233 | PGK | 0.241 | UCE2 | 0.345 |
3 | MDH | 0.225 | MDH | 0.335 | UCE2 | 0.264 | EF2 | 0.494 | CYC | 0.475 | UCE2 | 0.409 | ADF7 | 0.772 | ADF7 | 0.358 |
4 | ADF7 | 0.227 | UCE2 | 0.438 | MDH | 0.308 | MDH | 0.545 | PGK | 0.500 | CYC | 0.476 | CYC | 0.837 | CYC | 0.413 |
5 | EF1α | 0.444 | ACT | 0.454 | EF2 | 0.324 | ACT | 0.557 | ACT | 0.550 | EF1α | 0.493 | UBL5 | 0.862 | EF2 | 0.430 |
6 | ACT | 0.450 | EF2 | 0.458 | EF1α | 0.350 | ADF7 | 0.592 | MDH | 0.606 | RPL11 | 0.549 | UCE2 | 0.865 | 18S | 0.442 |
7 | GAPDH | 0.591 | GAPDH | 0.473 | GAPDH | 0.355 | GAPDH | 0.606 | RPL11 | 0.632 | 18S | 0.598 | RPL11 | 0.884 | ACT | 0.455 |
8 | CYC | 0.635 | CYC | 0.476 | ACT | 0.473 | CYC | 0.627 | TUB4 | 0.636 | ACT | 0.604 | MDH | 1.003 | GAPDH | 0.479 |
9 | EF2 | 0.639 | EF1α | 0.531 | UBL5 | 0.479 | EF1α | 0.643 | EF2 | 0.667 | EF2 | 0.646 | EF1α | 1.003 | EF1α | 0.485 |
10 | TUB4 | 0.691 | UBL5 | 0.563 | CYC | 0.496 | TUB4 | 0.698 | GAPDH | 0.724 | MDH | 0.773 | ACT | 1.037 | TUB4 | 0.714 |
11 | RPL11 | 0.694 | PGK | 0.679 | Rubisco | 0.527 | RPL11 | 0.722 | UBL5 | 0.817 | PGK | 0.787 | EF2 | 1.154 | MDH | 0.951 |
12 | 18S | 0.833 | 18S | 0.725 | PGK | 0.753 | 18S | 0.754 | 18S | 0.982 | GAPDH | 1.181 | 18S | 1.163 | RPL11 | 1.054 |
13 | PGK | 1.419 | ADF7 | 0.800 | 18S | 0.807 | PGK | 1.753 | Rubisco | 1.327 | ADF7 | 1.194 | GAPDH | 1.260 | Rubisco | 1.277 |
14 | Rubisco | 1.702 | Rubisco | 1.316 | ADF7 | 0.989 | Rubisco | 1.936 | ADF7 | 1.619 | Rubisco | 1.818 | Rubisco | 2.045 | PGK | 1.494 |
Best- Keeper Rank | Tissues of YN | Tissues of XJ | ||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Aboveground | Underground | Tuber | Total | Aboveground | Underground | Tuber | Total | |||||||||||||||||
Gene | SD | CV | Gene | SD | CV | Gene | SD | CV | Gene | SD | CV | Gene | SD | CV | Gene | SD | CV | Gene | SD | CV | Gene | SD | CV | |
1 | MDH | 0.27 | 1.05 | UCE2 | 0.42 | 1.35 | TUB4 | 0.10 | 1.16 | UCE2 | 0.44 | 1.94 | MDH | 0.09 | 0.96 | UCE2 | 0.55 | 1.31 | UCE2 | 0.23 | 1.13 | UCE2 | 0.57 | 1.31 |
2 | CYC | 0.30 | 1.36 | MDH | 0.43 | 1.93 | UCE2 | 0.28 | 1.54 | MDH | 0.48 | 2.23 | EF2 | 0.23 | 0.96 | MDH | 0.63 | 1.52 | TUB4 | 0.29 | 1.24 | 18S | 0.65 | 1.70 |
3 | UCE2 | 0.39 | 1.85 | ACT | 0.45 | 5.27 | EF2 | 0.40 | 1.80 | EF2 | 0.50 | 2.23 | UCE2 | 0.27 | 1.37 | UBL5 | 0.78 | 3.04 | PGK | 0.45 | 2.12 | UBL5 | 0.67 | 2.06 |
4 | ADF7 | 0.42 | 1.82 | TUB4 | 0.48 | 2.20 | MDH | 0.41 | 1.92 | 18S | 0.55 | 3.07 | GAPDH | 0.31 | 1.28 | TUB4 | 0.84 | 3.02 | ACT | 0.51 | 2.37 | ADF7 | 0.68 | 2.55 |
5 | UBL5 | 0.60 | 3.28 | ADF7 | 0.49 | 2.16 | 18S | 0.52 | 2.29 | UBL5 | 0.61 | 2.23 | CYC | 0.36 | 1.72 | 18S | 0.84 | 3.23 | MDH | 0.53 | 2.86 | EF2 | 0.72 | 2.70 |
6 | EF2 | 0.60 | 3.15 | EF2 | 0.56 | 3.10 | GAPDH | 0.55 | 2.38 | CYC | 0.64 | 2.52 | 18S | 0.36 | 1.38 | ADF7 | 0.88 | 3.54 | CYC | 0.55 | 2.20 | EF1α | 0.83 | 3.20 |
7 | 18S | 0.66 | 3.73 | GAPDH | 0.62 | 2.70 | CYC | 0.59 | 3.29 | RPL11 | 0.74 | 3.96 | ADF7 | 0.40 | 2.04 | EF1α | 0.92 | 3.44 | RPL11 | 0.56 | 2.19 | MDH | 0.85 | 3.17 |
8 | RPL11 | 0.69 | 4.46 | PGK | 0.63 | 3.21 | RPL11 | 0.70 | 3.56 | GAPDH | 0.77 | 4.24 | EF1α | 0.52 | 2.61 | ACT | 0.96 | 4.00 | GAPDH | 0.62 | 3.08 | CYC | 0.90 | 2.42 |
9 | ACT | 0.78 | 5.09 | UBL5 | 0.67 | 3.65 | Rubisco | 0.74 | 2.90 | ADF7 | 0.86 | 4.54 | UBL5 | 0.52 | 2.70 | RPL11 | 1.00 | 3.88 | UBL5 | 0.65 | 4.07 | GAPDH | 0.97 | 3.60 |
10 | GAPDH | 0.96 | 5.26 | RPL11 | 0.81 | 3.22 | EF1α | 0.79 | 4.57 | ACT | 0.88 | 5.67 | RPL11 | 0.75 | 3.40 | PGK | 1.07 | 4.01 | ADF7 | 0.80 | 4.24 | RPL11 | 1.22 | 4.76 |
11 | EF1α | 1.06 | 5.99 | CYC | 0.88 | 5.54 | UBL5 | 0.81 | 4.37 | TUB4 | 1.26 | 6.17 | TUB4 | 0.83 | 4.31 | EF2 | 1.09 | 4.47 | 18S | 0.89 | 4.66 | PGK | 1.46 | 6.17 |
12 | TUB4 | 1.10 | 5.59 | 18S | 0.98 | 4.96 | ACT | 0.92 | 4.55 | EF1α | 1.40 | 7.43 | PGK | 1.08 | 4.85 | CYC | 1.14 | 3.37 | EF1α | 0.90 | 4.63 | TUB4 | 1.69 | 7.09 |
13 | Rubisco | 2.31 | 7.59 | EF1α | 1.05 | 4.98 | PGK | 0.92 | 4.37 | Rubisco | 2.34 | 7.50 | ACT | 2.35 | 5.72 | GAPDH | 1.25 | 4.98 | EF2 | 1.01 | 4.83 | ACT | 1.81 | 7.79 |
14 | PGK | 2.35 | 8.03 | Rubisco | 1.58 | 8.53 | ADF7 | 0.96 | 4.75 | PGK | 2.41 | 8.22 | Rubisco | 2.35 | 6.30 | Rubisco | 2.74 | 6.33 | Rubisco | 1.21 | 6.15 | Rubisco | 2.60 | 7.83 |
Rank | Tissues of YN | Tissues of XJ | Tissues of Two Cultivars | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Above | Under | Tuber | Total | Above | Under | Tuber | Total | Above | Under | Tuber | Total | |
1 | UCE2 | UCE2 | TUB4 | UCE2 | UCE2 | UCE2 | TUB4 | UCE2 | UCE2 | UCE2 | TUB4 | UCE2 |
2 | MDH | TUB4 | UCE2 | MDH | EF1α | UBL5 | PGK | UBL5 | MDH | TUB4 | UCE2 | UBL5 |
3 | UBL5 | MDH | EF2 | UBL5 | CYC | TUB4 | UCE2 | ADF7 | EF1α | ACT | MDH | EF2 |
4 | ADF7 | ACT | MDH | EF2 | MDH | ADF7 | CYC | 18S | UBL5 | UBL5 | CYC | CYC |
5 | ACT | EF2 | RPL11 | CYC | EF2 | 18S | UBL5 | EF2 | CYC | MDH | RPL11 | MDH |
6 | EF2 | RPL11 | GAPDH | GAPDH | UBL5 | ACT | ADF7 | CYC | EF2 | RPL11 | EF2 | ADF7 |
7 | CYC | GAPDH | ACT | TUB4 | GAPDH | EF1α | MDH | EF1α | ACT | ADF7 | PGK | 18S |
8 | EF1α | CYC | CYC | ACT | ACT | RPL11 | ACT | GAPDH | ADF7 | 18S | ACT | GAPDH |
9 | TUB4 | UBL5 | EF1α | RPL11 | 18S | MDH | RPL11 | MDH | GAPDH | EF1α | UBL5 | TUB4 |
10 | GAPDH | EF1α | Rubisco | 18S | PGK | CYC | EF1α | TUB4 | 18S | EF2 | GAPDH | EF1α |
11 | RPL11 | PGK | UBL5 | ADF7 | RPL11 | PGK | EF2 | ACT | RPL11 | CYC | EF1α | ACT |
12 | 18S | 18S | 18S | EF1α | ADF7 | EF2 | GAPDH | RPL11 | TUB4 | GAPDH | ADF7 | RPL11 |
13 | PGK | ADF7 | PGK | PGK | TUB4 | GAPDH | 18S | PGK | PGK | PGK | 18S | PGK |
14 | Rubisco | Rubisco | ADF7 | Rubisco | Rubisco | Rubisco | Rubisco | Rubisco | Rubisco | Rubisco | Rubisco | Rubisco |
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Bai, X.; Chen, T.; Wu, Y.; Tang, M.; Xu, Z.-F. Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data. Int. J. Mol. Sci. 2021, 22, 2569. https://doi.org/10.3390/ijms22052569
Bai X, Chen T, Wu Y, Tang M, Xu Z-F. Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data. International Journal of Molecular Sciences. 2021; 22(5):2569. https://doi.org/10.3390/ijms22052569
Chicago/Turabian StyleBai, Xue, Tao Chen, Yuan Wu, Mingyong Tang, and Zeng-Fu Xu. 2021. "Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data" International Journal of Molecular Sciences 22, no. 5: 2569. https://doi.org/10.3390/ijms22052569