Stability of Nuclear and Mitochondrial Reference Genes in Selected Tissues of the Ambrosia Beetle Xylosandrus germanus
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Insect Specimens and Tissue Types
2.2. RNA Isolation, Reverse Transcription, and Primer Design
2.3. Real-Time PCR
2.4. Stability and Statistical Analysis of Candidate Reference Genes
3. Results and Discussion
3.1. Primer Specificity and Efficiency of Reference Genes
3.2. Expression Profiling of Reference Genes
3.3. Stability Analysis of Reference Genes
4. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene Symboland Name | Primer Sequence (5′–3′) | Length (bp) | Efficiency (%) | R2 Regression |
---|---|---|---|---|
28S rRNA 28S Ribosomal RNA | F: ACTCCTTGGTCCGTGTTTCA R: CTCTGGTGACTGTTGGCGA | 120 | 099.0 | 0.998 y = −3.346 + 31.033 |
AK Arginine kinase | F: ACAAGTCTACCGTCGTCTGG R: GTTGGTTGGGCAGAAAGTGA | 103 | 105.8 | 0.999 y = −3.190 + 30.798 |
CAD carbamoyl-phosphate synthetase 2-aspartate transcarbamylase-dihydroorotase | F: GAACCACCGCCATAAACGTT R: TGGACAGTCGGGCATTAACT | 120 | 108.7 | 0.998 y = −3.129 + 38.058 |
CO1 Cytochrome oxidase subunit 1 | F: TTCCTCCTGCTAAAACTGGC R: CCTCAATCCTTGGAGCAATCA | 150 | 104.0 | 0.999 y = −3.229 + 25.483 |
EF1α Elongation factor-1 alpha | F: CCAACCAGAAATAGGCACGA R: CCACCGAACCACCCTACAG | 119 | 108.3 | 0.999 y = −3.138 + 28.178 |
Tissue | Source | Gene | GeNorm | NormFinder | BestKeeper | ΔCt | RefFinder | |||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Stability | Rank | Stability | Rank | Stability | Rank | Stability | Rank | Stability | Final Rank | |||
Whole Body | LR | CO1 | 0.137 | 1 | 0.069 | 1 | 0.24 | 2 | 0.40 | 1 | 1.19 | 1 |
EF1α | 0.137 | 1 | 0.069 | 2 | 0.16 | 1 | 0.40 | 2 | 1.41 | 2 | ||
CAD | 0.215 | 2 | 0.109 | 3 | 0.31 | 4 | 0.44 | 3 | 3.22 | 3 | ||
AK | 0.305 | 3 | 0.516 | 4 | 0.30 | 3 | 0.58 | 4 | 3.72 | 4 | ||
28S | 0.544 | 4 | 0.886 | 5 | 0.91 | 5 | 0.90 | 5 | 5.00 | 5 | ||
FC | CO1 | 0.136 | 1 | 0.068 | 1 | 0.16 | 2 | 0.78 | 1 | 1.19 | 1 | |
AK | 0.136 | 1 | 0.389 | 2 | 0.14 | 1 | 0.81 | 2 | 1.41 | 2 | ||
CAD | 0.870 | 3 | 0.908 | 3 | 1.00 | 4 | 1.06 | 3 | 3.46 | 3 | ||
EF1α | 0.380 | 2 | 1.051 | 4 | 0.52 | 3 | 1.12 | 4 | 3.46 | 4 | ||
28S | 0.985 | 4 | 1.088 | 5 | 1.12 | 5 | 1.16 | 5 | 5.00 | 5 | ||
Head + Pronotum | LR | CO1 | 0.784 | 2 | 0.468 | 1 | 0.84 | 3 | 1.19 | 1 | 1.73 | 1 |
CAD | 0.181 | 1 | 1.068 | 3 | 0.21 | 1 | 1.32 | 3 | 1.73 | 2 | ||
28S | 0.181 | 1 | 1.264 | 4 | 0.26 | 2 | 1.42 | 4 | 2.38 | 3 | ||
AK | 1.148 | 3 | 0.759 | 2 | 1.62 | 4 | 1.32 | 2 | 2.83 | 4 | ||
EF1α | 1.411 | 5 | 1.710 | 5 | 2.19 | 5 | 1.81 | 5 | 5.00 | 5 | ||
FC | CO1 | 1.308 | 2 | 0.849 | 1 | 1.83 | 3 | 1.85 | 1 | 1.73 | 1 | |
CAD | 0.279 | 1 | 1.708 | 3 | 0.32 | 2 | 2.09 | 2 | 1.86 | 2 | ||
28S | 0.279 | 1 | 2.095 | 4 | 0.12 | 1 | 2.28 | 4 | 2.00 | 3 | ||
AK | 1.958 | 3 | 1.565 | 2 | 2.60 | 4 | 2.12 | 3 | 3.13 | 4 | ||
EF1α | 2.165 | 4 | 2.295 | 5 | 3.17 | 5 | 2.48 | 5 | 5.00 | 5 | ||
Inflated Mycangium + Scutellum | LR | AK | 0.359 | 1 | 0.179 | 1 | 2.43 | 3 | 1.47 | 1 | 1.32 | 1 |
CO1 | 0.359 | 1 | 0.610 | 2 | 2.71 | 4 | 1.56 | 2 | 2.00 | 2 | ||
28S | 1.546 | 3 | 1.251 | 3 | 1.25 | 2 | 1.98 | 3 | 2.91 | 3 | ||
CAD | 1.985 | 4 | 2.512 | 5 | 0.55 | 1 | 2.64 | 5 | 3.34 | 4 | ||
EF1α | 0.887 | 2 | 2.136 | 4 | 3.57 | 5 | 2.27 | 4 | 3.94 | 5 | ||
FC | CAD | 0.315 | 1 | 0.989 | 2 | 0.13 | 1 | 1.21 | 2 | 1.41 | 1 | |
CO1 | 0.527 | 2 | 0.296 | 1 | 0.60 | 3 | 1.01 | 1 | 1.73 | 2 | ||
28S | 0.315 | 1 | 1.092 | 4 | 0.21 | 2 | 1.26 | 3 | 2.21 | 3 | ||
AK | 1.090 | 3 | 1.063 | 3 | 1.70 | 4 | 1.35 | 4 | 3.72 | 4 | ||
EF1α | 1.275 | 4 | 1.456 | 5 | 1.90 | 5 | 1.55 | 5 | 5.00 | 5 | ||
Inflated Mycangium | LR | 28S | 0.494 | 1 | 1.467 | 3 | 0.36 | 1 | 1.98 | 2 | 1.57 | 1 |
CO1 | 1.161 | 2 | 0.702 | 1 | 0.84 | 3 | 1.71 | 1 | 1.73 | 2 | ||
CAD | 0.494 | 1 | 1.854 | 4 | 0.50 | 2 | 2.16 | 3 | 2.21 | 3 | ||
EF1α | 1.696 | 3 | 1.435 | 2 | 1.92 | 4 | 2.24 | 4 | 3.36 | 4 | ||
AK | 2.216 | 4 | 2.798 | 5 | 2.66 | 5 | 3.00 | 5 | 5.00 | 5 | ||
FC | CAD | 0.371 | 1 | 0.185 | 1 | 0.40 | 2 | 5.32 | 1 | 1.19 | 1 | |
AK | 0.371 | 1 | 0.185 | 2 | 0.30 | 1 | 5.38 | 2 | 1.41 | 2 | ||
CO1 | 0.832 | 2 | 0.338 | 3 | 1.13 | 3 | 5.43 | 3 | 3.00 | 3 | ||
28S | 1.029 | 3 | 3.140 | 4 | 1.52 | 4 | 5.81 | 4 | 4.00 | 4 | ||
EF1α | 8.160 | 4 | 18.846 | 5 | 16.11 | 5 | 18.86 | 5 | 5.00 | 5 | ||
Deflated Mycangium + Scutellum | LR | AK | 0.361 | 1 | 0.181 | 1 | 0.69 | 3 | 0.75 | 1 | 1.32 | 1 |
28S | 0.361 | 1 | 0.588 | 2 | 0.38 | 1 | 0.85 | 2 | 1.41 | 2 | ||
CO1 | 0.560 | 2 | 0.902 | 4 | 0.45 | 2 | 1.04 | 4 | 3.13 | 3 | ||
CAD | 0.844 | 3 | 0.723 | 3 | 1.33 | 4 | 0.94 | 3 | 3.46 | 4 | ||
EF1α | 0.927 | 4 | 0.934 | 5 | 1.37 | 5 | 1.05 | 5 | 5.00 | 5 | ||
Mycangium + Thorax (without pronotum) + Scutellum + Abdomen 1 | LR | EF1α | 0.121 | 1 | 0.207 | 1 | 0.38 | 3 | 0.52 | 1 | 1.32 | 1 |
AK | 0.121 | 1 | 0.375 | 3 | 0.31 | 1 | 0.55 | 2 | 1.57 | 2 | ||
CO1 | 0.138 | 2 | 0.379 | 4 | 0.32 | 2 | 0.56 | 3 | 2.91 | 3 | ||
CAD | 0.331 | 3 | 0.243 | 2 | 0.77 | 4 | 0.64 | 4 | 3.36 | 4 | ||
28S | 0.707 | 4 | 1.253 | 5 | 1.52 | 5 | 1.27 | 5 | 5.00 | 5 | ||
FC | AK | 0.118 | 1 | 0.047 | 1 | 0.38 | 2 | 0.65 | 1 | 1.19 | 1 | |
CAD | 0.118 | 1 | 0.144 | 2 | 0.40 | 3 | 0.66 | 2 | 1.86 | 2 | ||
EF1α | 0.309 | 2 | 0.188 | 3 | 0.29 | 1 | 0.69 | 3 | 2.28 | 3 | ||
28S | 0.650 | 3 | 1.053 | 4 | 0.81 | 5 | 1.15 | 4 | 4.23 | 4 | ||
CO1 | 0.869 | 4 | 1.117 | 5 | 0.64 | 4 | 1.20 | 5 | 4.73 | 5 |
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Patwa, N.; Ranger, C.M.; Lehenberger, M.; Biedermann, P.H.; Reding, M.E. Stability of Nuclear and Mitochondrial Reference Genes in Selected Tissues of the Ambrosia Beetle Xylosandrus germanus. Insects 2021, 12, 1125. https://doi.org/10.3390/insects12121125
Patwa N, Ranger CM, Lehenberger M, Biedermann PH, Reding ME. Stability of Nuclear and Mitochondrial Reference Genes in Selected Tissues of the Ambrosia Beetle Xylosandrus germanus. Insects. 2021; 12(12):1125. https://doi.org/10.3390/insects12121125
Chicago/Turabian StylePatwa, Nisha, Christopher M. Ranger, Maximilian Lehenberger, Peter H. Biedermann, and Michael E. Reding. 2021. "Stability of Nuclear and Mitochondrial Reference Genes in Selected Tissues of the Ambrosia Beetle Xylosandrus germanus" Insects 12, no. 12: 1125. https://doi.org/10.3390/insects12121125