Table 3.
Candidate genes and corresponding primers used for qPCR experiments Coffee sequences were selected (E-value ≤1e−10) from public databases with the BLAST programs (Altschul et al., 1990). Gene names were assigned based on the best BLASTX hit obtained by comparing the selected coffee ESTs with public databases. GenBank (GB) accession numbers of coffee EST sequences are also given. Primers were designed using the Primer Express software (Applied Biosystems).
| Protein name | Gene name | GB numbers | Primer name | Primer sequence | bp | CG |
| Ubiquitin | CcUBQ10 | GW488515 | BUBI-F | 5′-AAGACAGCTTCAACAGAGTACAGCAT-3′ | 104 | R* |
| BUBI-R | 5′-GGCAGGACCTTGGCTGACTATA-3′ | |||||
| Glyceraldehyde-3-phosphate dehydrogenase | CcGAPDH | GW445811 | GAPDH-F | 5′-TTGAAGGGCGGTGCAAA-3′ | 59 | R* |
| GAPDH-R | 5′-AACATGGGTGCATCCTTGCT-3′ | |||||
| Cathepsin | CcCTS1 | GW442860 | CATHEP-F | 5′-CCCATTATCGTTCTGGTGTTTACAA-3′ | 80 | R |
| CATHEP-R | 5′-ACCCCATCCAATCAGCTTTACA-3′ | |||||
| Actin | CcACT1 | GW428479 | ACTIN-F | 5′-GGATTTCCAGGGCTGAATATGA-3′ | 80 | R |
| ACTIN-R | 5′-CGATACGATAGTAGCAGCTTAAAAGC-3′ | |||||
| ADP-ribosylation factor (ARF) | CcADP1 | GW450736 | ADP-F | 5′-ATGAATGCTGCTGAAATAACTGATAAG-3′ | 80 | R |
| ADP-R | 5′-GCACAGGTGCTTTGGATATACCA-3′ | |||||
| Cyclophilin | CcCYN1 | GW488988 | CYCLO-F | 5′-TCTTCATCTGCACCGACAAGA 3′5′-CACATCCATACCCTCCGTGAT-3′ | 80 | R |
| CYCLO-R | ||||||
| WRKY transcription factor | CcWRKY2 | GT647873 | 18089-F | 5′-CATGATCCCAAAGCAGTTATTACG-3′ | 67 | 1 |
| 18089-R | 5′-TTCTGGCAGTTGGTACATCATGAT-3′ | |||||
| Photosystem I subunit XI | CcPSAL1 | GT646615 | ND | 1 | ||
| Early light-induced protein (ELIP) | CcELIP1 | GT647659 | ** | 1 | ||
| Early light-induced protein (ELIP) | CcELIP2 | GT647647 | ** | 1 | ||
| Unknown protein 1 | CcUNK1 | DV689820 | 182052-F | 5′-TATAGTGTTTATGGTGTGGCTTTCAGT-3′ | 79 | 1 |
| 182052-R | 5′-GTACCACCGTAGGGAGACGTATG-3′ | |||||
| Catalase | CcCAT1 | GT649488 | 18297-F | 5′-TGACAGAACCTGCAGTAAAGACAGTATT-3′ | 146 | 2 |
| 18297-R | 5′-AACCAACCAAATGAACGAACAAT-3′ | |||||
| Catalase | CcCAT2 | GT648692 | SH3055G111-F | 5′-GATACCCAGCGACACCGTCTT-3′ | 70 | 5 |
| SH3055G111-R | 5′-GATGAGCACACTTGGGAGCAT-3′ | CcCAT1 | ||||
| Acyl-CoA-binding protein | CcACBP1 | GT646045 | 9158-F | 5′-AATACTACCAATGCAAGCAAGCTTA-3′ | 128 | 2 |
| 9158-R | 5′-CCTTCCATGCATCCCACTTT-3′ | |||||
| Chlorophyll a/b-binding protein | CcCAB1 | GT647358 | 18230-F | 5′-CTCTGAACTTCACCAGCTCTTCAA-3′ | 140 | 2 |
| 18230-R | 5′-GAGCTCGTCGTCCAATGAAGA-3′ | |||||
| Metallothionein like protein | CcMET1 | GT650680 | ** | 2 | ||
| GLB2-like non-symbiotic haemoglobin | CcNSH1 | GT649198 | RBCS3'-F | 5′-CTAAGGGATACGGATGAAATTCCA-3′ | 102 | 2 |
| RBCS3'-R | 5′-CTCTCGCAGCTGCACTACTGATT-3′ | |||||
| Glycine-rich protein | CcGRP1 | GT650953 | GC182-F | 5′-AAGCCAGCTTCTAGCTATGTCATGA-3′ | 100 | 2 |
| GC182-R | 5′-GATCCAGCACTTATTTGAGATCACA-3′ | |||||
| Mannose 6-phosphate reductase | CcMPR1 | GT648734 | LPSH3069F05-F | 5′-AATCAGCAATTACAGCGTTTTGC-3′ | 100 | 2,3 |
| LPSH3069F05-R | 5′-AGTGACACAGATGCCGTGCTT-3′ | |||||
| Aldose-phosphate reductase | CcAPR1 | GT649483 | LPSH3060G02-F | 5′-TGAAGCCAGCTGTGAATCAACT-3′ | 100 | 5 |
| LPSH3060G02-R | 5′-GTGTGGGCAGTGACACAGATG-3′ | CcMPR1 | ||||
| Unknown protein 10 | CcUNK10 | GT648004 | D18240-F | 5′-TAGCCTTGTTCTTTTAGGGAGTCTTATC-3′ | 134 | 2 |
| D18240-R | 5′-AGAGCTTCGTCCAGGAAGAAGA-3′ | |||||
| TNF-associated factor (TRAF) | CcTRAF1 | GT652792 | LP-12677-FLP-12677-R | 5′-GACTCTGGGCACATCGTGATAG-3′5′-TGCGAAGGGAAAGAATGGAA-3′ | 100 | 3 |
| Prephenate dehydrogenase | CcPDH1 | GT655248 | LP175502-F | 5′-GCAGGGACATGGACGAATTT-3′ | 100 | 3 |
| LP175502-R | 5′-TGAAACGGCATGGACTTGAC-3′ | |||||
| Unknown protein 8 | CcUNK8 | DV695331 | LP18100-F | 5′-CTCGCGTGGCCGAGATT-3′ | 100 | 3 |
| LP18100-R | 5′-CCCTCACATTTCCACGTGAAT-3′ | |||||
| Dehydrin | CcDH3 | DV689895 | 18390-F | 5′-TTAATAGCAGCTTTCCAGTGTGTCA-3′ | 100 | 3 |
| 18390-F | 5′-GATCCCCCAAAAAGCAGAAAA-3′ | |||||
| EDR1-like MAPKK kinase | CcEDR1 | GW457961 | LPSH3054B02-F | 5′-TGTCAAATTGATGAAAAGCGAAGA-3′ | 100 | 3 |
| LPSH3054B02-R | 5′-AAGGAAAAGTAGGAAATCAGCCAAA-3′ | |||||
| EDR1-like MAPKK kinase | CcEDR2 | DV681462 | EDR13-FEDR13-R | 5′ CGGCATAAGAGCGAGTGGAA 3′5′ ATGCAATCGCTGGTGTAGAAAA 3′ | 70 | 6 |
| Small heat shock protein (sHSP) | CcHSP1 | GW447897 | LPSH3056B04-F | 5′-GGTAGGACGCCATGGGAGAT-3′ | 100 | 3, 4 |
| LPSH3056B04-R | 5′-CCTCAACCCACACCTTCACAT-3′ | |||||
| Carbonic anhydrase | CcCA1 | GT008701 | HRAM1-F | 5′-AAGGCCATTGTCGGACTTCA-3′ | 100 | 4 |
| HRAM1-R | 5′-TTGTTTGCAACTCTGCAGTGATT-3′ | |||||
| Type-2C protein phosphatase (PP2C) | CcPP2C | GW435032 | HRAM2-F | 5′-CGAAGAAATCAGGCGTATCAGAG-3′ | 101 | 4 |
| HRAM2-R | 5′-TAAACCGCACGTCACCAAAAG-3′ | |||||
| Oxygen-evolving complex PSII (psbO) | CcPSBO | GT647517 | HRAM12-F | 5′-TGAATTTCTCGTGCCATCATACA-3′ | 101 | 4 |
| HRAM12-R | 5′-CTCCAGCAGGCAATGCAACT-3′ | |||||
| Oxygen-evolving complex PSII (psbP) | CcPSBP | GW488995 | HRAM7-F | 5′-AGCTGTCGATTCCCTCAAAATG-3′ | 100 | 4 |
| HRAM7-R | 5′-GAGACACTGCTGTTGCTATCGAA-3′ | |||||
| Oxygen-evolving complex PSII (psbQ) | CcPSBQ | GT645658 | HRAM36-F | 5′-CAGGGCAATCAAGGTTGGA-3′ | 102 | 4 |
| HRAM36-R | 5′-CGGTCCTTGAGTGGCAAATC-3′ | |||||
| Rubisco small chain (RbcS) | CcRBCS1 | GT649534 | 18244-F | 5′-CACCAACTGGAAAGTTGAAGAA-3′ | 169 | 6 |
| 18244-R | 5′-TATCCCGGTGACCTGTGGTATT-3′ | |||||
| Subtilisin-like serine protease | CcSDD1 | GT659840 | SDD1-F | 5′-GAGCCCCGATTGATCTTCTG-3′ | 101 | 6 |
| SDD1-R | 5′-ACTCAGCCCCAAAAGGGTTAA−3′ | |||||
| Glucosyltransferase arbutin synthase | CcGAS1 | GT721173 | ARBU09-F | 5′-AAAGCGTTGCAGGAGCAAGA-3′ | 80 | 6 |
| ARBU09-R | 5′-CTGTCCCCTGAGCCCATCT-3′ | |||||
| Abscisic acid receptor | CcPYL3 | GT720590 | PYL31-F | 5′-CGGTGACGACTGTCCATGAG-3′ | 80 | 6 |
| PYL31-R | 5′-CGGCACGTCAACGATATACG-3′ | |||||
| Abscisic acid receptor | CcPYL7 | DV704550 | PYL72-F | 5′-GAGAAGCACATTCTTGGGATCAA-3′ | 80 | 6 |
| PYL72-R | 5′-GGATGCACGGTAAGGATGGA-3′ | |||||
| Amidase (IAA synthesis) | CcAMI1 | GT651356 | HD141612-F | 5′-AGATGTCTGCCACAGTGTGAAGA-3′ | 80 | 6 |
| HD141612-R | 5′-GGAGTGCAAGGATGCCAAAA-3′ | |||||
| RD29-like protein | CcRD29 | GT660256 | RD29-F | 5′-TGATGATCAAGATCCCCAACAC-3′ | 100 | 6 |
| RD29-R | 5′-CTTCGCTTTCGCCTTCACTT-3′ | |||||
| AP2/ERF DREB-like | CcDREB1 | GW463524 | DREBA09-F | 5′-CAATGCCTGCAAAGCCAATTA-3′ | 80 | 6 |
| DREBA09-R | 5′-TTTTCCTGCCTGCACGTTTC-3′ | |||||
| NAC-RD26-like | CcRD26 | GT003652 | NACRD26-F | 5′-TTTGGCCCTGCGCTCTAGT-3′ | 98 | 6 |
| NACRD26-R | 5′-AAGCGGGTCAGTTTCTCGAA-3′ | |||||
| MYB-type 2 transcription factor | CcMYB1 | GT689406 | MYB61-F | 5′-CCCGGCAATCTTCCAGCTA−3′ | 100 | 6 |
| MYB61-R | 5′-TCAAGCGTGGCAACTTCACT−3′ | |||||
| Caffeoyl-CoA 3-O methyltransferase | CcCCoAOMT1 | GT715419 | CCOAOMT-F | 5′-GACACACCAGCCCTTTCGAT-3′ | 100 | 6 |
| CCOAOMT-R | 5′-CAGCTCTCGCTCTTCCAGAAG-3′ | |||||
| Clp protease ATP-binding subunit | CcCLP1 | GT651512 | CLP1-F | 5′-CCACCCCAGTAGGACCAGAAA-3′ | 80 | 6 |
| CLP1-R | 5′-CATTCGTCGTGCTCGTGTTG-3′ | |||||
| Ascorbate peroxidase | CcAPX1 | GT697455 | ASCPER1-F | 5′-GACCTGAACAATGCCCAGAAG-3′ | 70 | 6 |
| ASCPER1-R | 5′-CGTAAATGAGCAGCAGGTGATG-3′ | |||||
| Ascorbate peroxidase | CcAPX2 | EE193467 | ASCPER6-F | 5′-AGACCGTGTCTCAAACCGACTAC-3′ | 80 | 5 CcAPX1 |
| ASCPER6-R | 5′ GTTGATCTGTTGGCCCAAAGA 3′ | |||||
| Leucine zipper (ABA signalling) | CcABI5 | GT645781 | ABI52-F | 5′-GCCGCTGCAGCCTCTATTT-3′ | 99 | 6 |
| ABI52-R | 5′-AGCTGAGCGTTGTTCCCTATCT-3′ | |||||
| Leucine zipper (ABA signalling) | CcAREB1 | EE198828 | AREB11-F | 5′-TGTTGTAAGGGAGGATGCTCAA-3′ | 100 | 6 |
| AREB11-R | 5′-TCCAAACCCAAAAGCCTGAT-3′ | |||||
| Homeobox leucine zipper hypothetical protein | CcHDZ1 | GT724869 | HD68272-F | 5′-GACGTTGACGGACGAGAACA−3′ | 100 | 6 |
| HD68272-R | 5′-TGTAGCCGCTGGCAACTG−3′ | |||||
| Homeobox leucine zipper hypothetical protein | CcHDZ2 | GW447493 | HD111162-F | 5′-AGTGCCAGGGAAAAGAATTCC-3′ | 100 | 6 |
| HD111162-R | 5′-TCCGGCTGTCTCTTTCTCATG-3′ |
R, reference genes tested, with CcUBQ10 and CcGAPDH being used as reference genes in qPCR experiments (*, **. gene expression tested by northern blot but not by qPCR); ND, gene expression not determined.
The size of the amplicon in bp is also indicated. The selected candidate genes (CGs) corresponded to (1) genes exclusively overexpressed in the leaf SH3 cDNA library of drought-tolerant clone 14 of C. canephora var. Conilon subjected to drought (Fig. 1A); (2) genes mainly overexpressed in the SH3 cDNA library (Fig. 1B); (3) genes identified by the macroarray experiment (Fig. 2); (4) genes encoding proteins showing differential accumulation by 2-DE (Fig. 3); (5) genes homologous to other CGs; and (6) genes selected from the literature. CGs are ordered as presented in the text.