Table 1. Gene-specific primers and amplicon characteristics of candidate reference genes and a target gene used in RT-qPCR analysis.
| Gene name | Target sequence | Primer sequence (5’ → 3’) (forward/reverse) | Amplicon length (bp) | Tm (°C)a | Amplification efficiencyb | R2 |
|---|---|---|---|---|---|---|
| AfEF1a | CL3197 contig1 |
CACCCCCAAGTACTCCAAG GACAAATGGGACCTTGTCAG |
100 | 80.53 | 1.859 | 0.99992 |
| AfGAPDH1 | Unigene 36693 |
TTCTTCCTGAGTTGAACGGC ATGCAGCCTTCTCGATTCTG |
102 | 80.67 | 1.879 | 0.99997 |
| AfGAPDH2 | Unigene 36695 |
GAATACACCTCTGACATCACCA CTCAACAATGCCAAACTTGTCA |
102 | 79.93 | 1.883 | 0.99994 |
| AfTBP2 | CL4896 contig1 |
TGAGCCAGAACTTTTTCCTG CTTTGCACCGGTCAAGACA |
100 | 78.14 | 1.881 | 0.99995 |
| AfTUA1 | Uni36141 |
GTGCCTACCGTCAGCTTTT CGATCTCCTTTCCAACAGTGT |
102 | 83.94 | 1.868 | 0.99997 |
| AfUBC1 | Uni2728 |
ACAGTAACGGGAGCATATGTC GGTCCGTTAGCAGAGAACAG |
102 | 78.43 | 1.867 | 0.99994 |
| AfUBC2 | CL162 contig2 |
GGACATTTTCGAGTGGCAAT GGGTAGTCAGAGGGCAACTG |
99 | 80.81 | 1.860 | 0.99995 |
| AfVP1 | CL7717 contig1 |
TGACTTTATGTTCGCGGAAG AGTTGGAGGAGGATGAGGAG |
101 | 82.62 | 1.807 | 0.99989 |
a The amplicon melting temperature calculated by StepOne software
b The amplification efficiency calculated by LinReg software