Table 2. Segregation Analysis of Single T-DNA nip4;1 and nip4;2 Mutants and Double amiRNA Knockdowns.
| Cross | Parent Female × Male | F1 Segregation Genotype or Phenotype | Expected Ratio | Observed Ratio | Observed Ratio | χ2 P Value |
|---|---|---|---|---|---|---|
| Single mutants (self-crosses) | nip4;1-1+/− | NIP4;1+/+:+/−:−/− | 1:2:1 | 3.0:2.8:1 | 86:81:29 | P < 0.0001 |
| nip4;1-3+/− | NIP4;1+/+:+/−:−/− | 1:2:1 | 2.0:2.1:1 | 107:111:53 | P < 0.0001 | |
| nip4;1-2+/− | NIP4;1+/+:+/−:−/− | 1:2:1 | 1.9:2.3:1 | 46:54:24 | P < 0.01 | |
| nip4;2-2+/− | NIP4;2+/+:+/−:−/− | 1:2:1 | 2.0:1.9:1 | 122:110:58 | P < 0.0001 | |
| nip4;2-1+/− | NIP4;2+/+:+/−:−/− | 1:2:1 | 2.4:2.1:1 | 79:68:33 | P < 0.0001 | |
| Double knockdowns (self-crosses) | ami356-1+/− | KanR:KanS | 3:1 | 1.7:1 | 1037:600 | P < 0.0001 |
| ami356-4+/− | KanR:KanS | 3:1 | 1.4:1 | 112:80 | P < 0.0001 | |
| ami260-4+/− | KanR:KanS | 3:1 | 1.6:1 | 546:342 | P < 0.0001 | |
| qrt ami260-1+/− | KanR:KanS | 3:1 | 1.2:1 | 234:201 | P < 0.0001 | |
| Single mutants (reciprocal crosses) | wt × nip4;1-1+/− | KanR:KanS | 1:1 | 0.8:1 | 162:213 | P < 0.01 |
| nip4;1-1+/− × wt | KanR:KanS | 1:1 | 1.0:1 | 50:48 | P = 0.84 | |
| wt × nip4;1-3+/− | KanR:KanS | 1:1 | 0.7:1 | 95:140 | P < 0.01 | |
| nip4;1-3+/− × wt | KanR:KanS | 1:1 | 1.0:1 | 120:123 | P = 0.85 | |
| wt × nip4;1-2+/− | KanR:KanS | 1:1 | 0.7:1 | 145:200 | P < 0.01 | |
| nip4;1-2+/− × wt | KanR:KanS | 1:1 | 1.0:1 | 97:97 | P = 1.00 | |
| wt × nip4;2-2+/− | BastaR:BastaS | 1:1 | 0.8:1 | 284:367 | P < 0.01 | |
| nip4;2-2+/− × wt | BastaR:BastaS | 1:1 | 1.0:1 | 63:62 | P = 0.93 | |
| Double knockdowns (reciprocal crosses) | wt × ami356-1+/− | KanR:KanS | 1:1 | 0.7:1 | 82:125 | P < 0.01 |
| ami356-1+/− × wt | KanR:KanS | 1:1 | 1.0:1 | 94:98 | P = 0.77 | |
| Complementation lines (self-crosses) | nip4;1-2−/− NIP4;1-9+/− | BastaR:BastaS | 3:1 | 3.7:1 | 246:67 | P = 0.14 |
| nip4;2-1−/− NIP4;2-9+/− | BastaR:BastaS | 3:1 | 3.9:1 | 324:83 | P < 0.05 |
Disruption of NIP4;1 and NIP4;2 genes caused defective male-specific gene transmission. Segregation analysis of complementation lines showed that EGFP-NIP4;1 and EGFP-NIP4;2 rescued the nip4;1 and nip4;2 pollen transmission defect. wt, the wild type.