Table 2.
Candidate reference genes tested and primer sequences
| Reference gene1 (accession number) | Primer sequences (5'-3')2 | Amplicon lenght (bp) | Tm (°C) | Manual threshold3 | PCR efficiency (%) | Regression coefficient (R2) | Average Cq value |
|---|---|---|---|---|---|---|---|
| CYC | F: AGCTTTGAAGTTGGCAGTAG | ||||||
| R: GATCGCGTATTCATGGACTTTAG | Discarded (aspecific amplification) | ||||||
| SPS | F: CATTGCAAGAACTATTTGTCACG | ||||||
| R: GCAGAGATCAAATGGTTCAAATC | |||||||
| ACT | F: TGTGCTTGACTCTGGTGATG | 220 | 58 | ||||
| R: TTCATAATCAAGGGCAACGTAAGC | Discarded (aspecific amplification) | ||||||
| RUBISCO | F: CATTATCAAGAAGGGCAAGATGTG | 169 | 60 | ||||
| R: TGTTGTACATCCCTGGAAGTTG | |||||||
| GAPDH | F: GTATTGTTGAGGGACTGATGACC | 182 | 57 | 0.047 | 92 | 0.999 | 23.31 |
| (JN599100) | R: AGTAAGCTTGCCATTGAACTCAG | ||||||
| TUB | F: TACTGTGGTTGAGCCATACAATG | 162 | 60 | 0.069 | 98 | 0.993 | 23.87 |
| (JN599101) | R: GCAGAGATCAAATGGTTCAAATC | ||||||
| EF1 | F: CAAGTACTACTGCACCGTCATTG | 199 | 57 | 0.025 | 89 | 0.984 | 25.10 |
| (JN599095) | R: GATCATCTGCTTCACTCCAAGAG | ||||||
| UBQ | F: GCAAGAAGAAGACCTACACCAAG | 225 | 60 | 0.054 | 100 | 0.991 | 19.00 |
| (JN599096) | R: CCTTCTGGTTGTAGACGTAGGTG | ||||||
| 18S | F: GTCCAGACATAGGAAGGATTGAC | 245 | 63 | 0.052 | 106 | 0.995 | 15.08 |
| (JN599097) | R: GAACATCTAAGGGCATCACAGAC | ||||||
| 25S | F: GCATGAATGGATTAACGAGATTC | 165 | 63 | 0.098 | 95 | 0.998 | 17.00 |
| (JN599099) | R: GGCTCCCACTTATCCTACAC | ||||||
| 26S | F: GATAGCGTACAAGTACCGTGAGG | 238 | 63 | 0.102 | 94 | 0.993 | 20.45 |
| (JN599098) | R: GTTTCGGGTCAAATAGGAAGAAC | ||||||
1CYC cyclophilin, SPS sucrose phosphate synthase, ACT actin, RUBISCO ribulose biphosphate carboxylase, GAPDH glyceraldehyde-3-phosphate dehydrogenase, TUB beta-tubulin, EF1 elongation factor 1α, UBQ ubiquitin, 18S 18S ribosomal RNA (nuclear gene), 25S 25S ribosomal RNA (nuclear gene), 26S 26S ribosomal RNA (mitochondrial gene)
2F: forward primer and R: reverse primer
3The threshold for fluorescence detection was set using the logarithmic amplification plot so that it is above the background fluorescence, below the linear region and at the beginning of the region of exponential amplification (i.e. the linear portion of the plot)