Table 1.
Primer sequences and PCR/ qPCR conditions for genotyping, PHYE amiRNA generation, and expression analysis.
| Genotyping primers | ||
|---|---|---|
| phyA dCAPS F (EcoNI cuts WT) | TAACTGAATACACCATTCCCTTAACC | 47⋅C/45 s |
| phyA dCAPS R (EcoNI cuts WT) | ATAATCGCTCTATAGTCACC | WT 215/20; phyA 235 |
| phyB1 dCAPS F (HinF1 cuts WT) | CTAAAATTCAAAGAGGAGGTCAGATT | 58⋅C/20 s |
| phyB1 dCAPS R (HinF1 cuts WT) | GAAGGGGTAAAAAGGGTCCTAA | WT 172/20; phyB1 192 |
| phyB2 F WT specific | CCCTTTTTCCTTTTCTGACC | 62⋅C/45 s |
| phyB2 R non-specific | GACAATATTGAGGATGGGTA | WT 513; phyB2 absent |
| phyB2 F mutant specific | GTCTTGATTTCGTCTGGA | 66.5⋅C/45 s |
| phyB2 R non-specific | GACAATATTGAGGACGGGAGAGTT | WT absent; phyB2 452 |
| phyE amiRNA F | GCTCGGACGCATATTACACA | 58⋅C/30 s |
| phyE amiRNA R | ACCATGATTACGCCAAGCTC | WT absent; PHYE amiRNA 493 |
| positive control F | TGATGTTGATGGGCAGGTTA | 58–67⋅C/30 s |
| positive control R | CACTCAGAACACCAGCCAAA | 676 |
| Genotyping primers for phyA, phyB1, and phyB2 (Weller et al., 2000). | ||
| PHYE amiRNA generation primers | ||
| I miR-s | GATAAATCTGACAGAAGACGCTGTCTCTCTTTTGTATTCC | |
| II miR-a | GACAGCGTCTTCTGTCAGATTTATCAAAGAGAATCAATGA | |
| III miR*s | GACAACGTCTTCTGTGAGATTTTTCACAGGTCGTGATATG | |
| IV miR*a | GAAAAATCTCACAGAAGACGTTGTCTACATATATATTCCT | |
| Oligo A | CTGCAAGGCGATTAAGTTGGGTAAC | |
| Oligo B | GCGGATAACAATTTCACACAGGAAACAG | |
| PCR conditions: http://wmd3.weigelworld.org/downloads/Cloning_of_artificial_microRNAs.pdf | ||
| qPCR primers | ||
| PHYB1 F | CAATGCTCTAAGAGGCGTGGA | 61⋅C/1 min |
| PHYB1 R | CTGGAGCAAGCATTAACCACCA | 2-step protocol, 40 cycles |
| PHYB2 F | GGAAGGGTGGGTAGAAGTCC | 61⋅C/1 min |
| PHYB2 R | GGGCAAACAATCCTGAACTC | 2-step protocol, 40 cycles |
| PHYE F | GGAGACAAGTGAAGCCTGTGAG | 60⋅C/1 min |
| PHYE R | TGCCGTCCTCTATACCTCCAA | 2-step protocol, 40 cycles |
| control F (Solyc03g111090) | CTGACTTCTCAGCAGAACTCCAAT | 60–62⋅C/1 min |
| control R (Solyc03g111090) | TTCACCCTTTTCAATGCTCTTCTC | 2-step protocol, 40 cycles |