Table 1.
Primers used in this work.
| Sequence | Accessions/reference | Forward primer 5′–3′ | Reverse primer 5′–3′ |
|---|---|---|---|
| Cloning control and/or transgenic status control | |||
| Elongation Factor Malus domestica | AJ223969 | CTCTTGGTGTCAGGCAAATG (1) | TCAAGGTTGGTGGACCTCTC (2) |
| 23S ribosomal RNA Agrobacterium tumefaciens | CP014260.1 gene locus_tag = “AWN88_17620” 1310643..1313449 | GTAAGAAGCGAACGCAGGGAACT (3) | GACAATGACTGTTCTACGCGTAA (4) |
| nptII plasmid pK7WG2D | Karimi et al., 2002 | ATCGGGAGCGGCGATACCGTA (5) | GAGGCTATTCGGCTATGACTG (6) |
| Cloning box in pDONR207 plasmid | Thermo Fisher (Invitrogen) | TCGCGTTAACGCTAGCATGGATCTC (7) | GTAACATCAGAGATTTTGAGACAC (8) |
| gRNAs box in pDEcas9Kr plasmid | This work | AGCTCCCTAGGCCTGTTATC (9) | CTAGGCTGGATCGGAATTATCG (10) |
| Cas9 coding sequence in pDEcas9Kr plasmide | Fauser et al., 2014$ | TGAGTTGGTGAAGGTGATGGG (11) | TAACGATGTTCACCTGTGGCA (12) |
| pDEcas9Kr backbone at LB border | Fauser et al., 2014$ | TTGCTGCTCCATAACATCAAA (29) | ATACAGGCAGCCCATCAGTC (30) |
| pDEcas9Kr backbone at RB border | Fauser et al., 2014$ | TTTAAAAGGGCGTGAAAAGG (31) | CTTCTCGGAAAACAGCTTGG (32) |
| Target sequences cloning and analysis | |||
| MdPDS | MD04G0021400∗ | AGTGGGCTTGTGTCTCCG (13) | CCGCCTAAAACATCTCTCGC (14) |
| MdTFL1 | AB052994 | GGGAGGTTTGGGACTAGCAA (15) | TAGACGGCAGCGACAGGAAGA (16) |
| PcTFL1 | PCP025869.1 | ATGAAAAGAGCATCGGAGC (17) | CTCTGCGCGTTGAAGTAGAC (18) |
| Cloning box in pJET 1.2 plasmid | Thermo Fisher (Fermentas) | CGACTCACTATAGGGAGAGCGGC (19) | AAGAACATCGATTTTCCATGGCAG (20) |
| Off-target genes analysis | |||
| PEBPMD12 | MD12G1023900∗ | TGAGTTATGAGATGCCGAAGC (21) | TGGGAAACAAAAGTTACAATGG (22) |
| PEPBMD14-1 | MD14G1021100∗ | ACAAGGATTCCACTTCCAAGC (23) | AGCATTTATACCAGTGCAGGTG (24) |
| PEPBMD14-2 | MD14G1021100∗ | AAGAGAGGCGCTGAGCTATG (25) | GCACTTTCTCTCTGCGCATT (26) |
| 2-oxoglutarate | MD01G1193900∗ | GACGGAAAACGCACACATTA (27) | ATGTGCAGAAGAGCCATTCC (28) |
$Replacement of DL-Phosphinothricin resistance by kanamycine resistance in this work. ∗Available athttps://iris.angers.inra.fr/gddh13(): primer code.