Table 1 PCR methods and primers sequences and main conditions used in this study
Gene | Primer | Product size (bp) | Primer sequence (5′-3′) | Amplification conditions | H2O (μl)a | MgCl2 (μl) | dNTP (0.2 mM) (μl) | Primer (μl) | Units Taq DNA polymerase (μl) | DNA (μl) | Total volume (μl) |
|---|---|---|---|---|---|---|---|---|---|---|---|
SSU | R221 | 603 | GGTTCCTTTCCTGATTTACG | 94 °C for 5 min, 94 °C for 30s, 60 °C for 30s, 72 °C for 30s, 72 °C for 5 min (30 cycles) | 30.6 | 5.0 | 1.0 | 1.0 (15 pmol) | 1.4 (1 U/μl) | 10 | 50 |
R332 | GGCCGGTAAAGGCCGAATAG | ||||||||||
R223 | 358 | TCCCATCGCAACCTCGGTT | 94 °C for 5 min, 94 °C for 30s, 65 °C for 30s, 72 °C for 30s, 72 °C for 5 min (30 cycles) | 10.6 | 2.5 | 0.5 | 0.5 (7.5 pmol) | 0.7 (0.5 U/μl) | 10a | 25 | |
R333 | AAAGCGGGCGCGGTGCTG | ||||||||||
0. 25 (3.75Â pmol) | |||||||||||
ITS1 | LITSR | 300–350 | CTGGATCATTTTCCGATG | 94 °C for 5 min, 94 °C for 30s, 53 °C for 30s, 72 °C for 30s, 72 °C for 5 min (30 cycles) | 30.6 | 5.0 | 1.0 | 1.0 (15 pmol) | 1.4 (1 U/μl) | 10 | 50 |
L5.8S | TGATACCACTTATCGCACTT | ||||||||||
SAC | 280–330 | CATTTTCCGATGATTACACC | 94 °C for 5 min, 94 °C for 30s, 57 °C for 30s, 72 °C for 30s, 72 °C for 5 min (30 cycles) | 10.6 | 2.5 | 0.5 | 0.5 (7.5 pmol) | 0.7 (0.5 U/μl) | 10b | 25 | |
VAN2 | CGTTCTTCAACGAAATAGG |