Table 2.
Molecular and genetic analysis of CRISPR/Cas9‐induced mutations in BnCLV3 and their transmission to the T1 and T2 generations
| Analysis of T0 plants | Mutation segregation in T1 plants | Mutation segregation in T2 plants | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Plant ID | Genotype of BnCLV3 | Mutation detected (bp)a | No. of tested plants with editing | WT | Hetero | Homo | Mutation transmissionb | T‐DNA free | No. of tested plants with editing | WT | Hetero | Homo | Mutation transmission | T‐DNA free |
| SCLV3‐3 | Aa | −8 | 5 | 0 (AA) | 0 (Aa) | 5 (aa) | 100.0% | 1/35 (2.9%) | 19 | 0 (AA) | 0 (Aa) | 19 (aa) | 100.0% | 42/137 (0.7%) |
| Cc | +1, −1 | 2 (CC) | 3 (Cc) | 0 (cc) | 60.0% | 17 (CC) | 2 (Cc) | 0 (cc) | 10.5% | |||||
| SCLV3‐6 | Aa | −1, +1 | 4 | 1 (AA) | 2 (Aa) | 1 (aa) | 75.0% | 5/18 (11.1%) | 6 | 2 (AA) | 3 (Aa) | 1 (aa) | 66.7% | 9/9 (100.0%) |
| Cc | −3 | 1 (CC) | 2 (Cc) | 1 (cc) | 75.0% | 0 (CC) | 0 (Cc) | 6 (cc) | 100.0% | |||||
| SCLV3‐16 | Aa | +1, −11 | 3 | 0 (AA) | 0 (Aa) | 3 (aa) | 100.0% | 0/34 (0%) | 20 | 0 (AA) | 0 (Aa) | 20 (aa) | 100.0% | 2/65 (3.1%) |
| Cc | +1 | 0 (CC) | 3 (Cc) | 0 (cc) | 100.0% | 7 (CC) | 13 (Cc) | 0 (cc) | 65.0% | |||||
| SCLV3‐27 | Aa | +1, −10, −39/+83 | 4 | 0 (AA) | 2 (Aa) | 2 (aa) | 100.0% | 12/30 (40.0%) | 10 | 0 (AA) | 0 (Aa) | 10 (aa) | 100.0% | 23/31 (74.2%) |
| Cc | +1 | 4 (CC) | 0 (Cc) | 0 (cc) | 0.0% | 10 (CC) | 0 (Cc) | 0 (cc) | 0.0% | |||||
| SCLV3‐31 | aa | +1, −2 | 4 | 0 (AA) | 0 (Aa) | 4 (aa) | 100.0% | 9/35 (20.0%) | 5 | 0 (AA) | 0 (Aa) | 5 (aa) | 100.0% | 7/20 (35.0%) |
| cc | +1, −1 | 0 (CC) | 0 (Cc) | 4 (cc) | 100.0% | 0 (CC) | 0 (Cc) | 5 (cc) | 100.0% | |||||
| SCLV3‐35 | aa | +1, −91 | 8 | 0 (AA) | 0 (Aa) | 8 (aa) | 100.0% | 2/36 (5.5%) | 5 | 0 (AA) | 0 (Aa) | 5 (aa) | 100.0% | 3/15 (20.0%) |
| cc | +1, −2 | 0 (CC) | 0 (Cc) | 8 (cc) | 100.0% | 0 (CC) | 0 (Cc) | 5 (cc) | 100.0% | |||||
| SCLV3‐167 | Aa | +1 | 2 | 2 (AA) | 0 (Aa) | 0 (aa) | 0.0% | 2/9 (22.2%) | 9 | 9 (AA) | 0 (Aa) | 0 (aa) | 0.0% | 8/41 (19.5%) |
| Cc | −26 | 0 (CC) | 0 (Cc) | 2 (cc) | 100.0% | 0 (CC) | 0 (Cc) | 9 (cc) | 100.0% | |||||
| SCLV3‐184 | aa | +1 | 5 | 0 (AA) | 0 (Aa) | 5 (aa) | 100.0% | 2/35 (5.7%) | 14 | 0 (AA) | 0 (Aa) | 14 (aa) | 100.0% | 5/84 (6.0%) |
| Cc | +1 | 0 (CC) | 2 (Cc) | 3 (cc) | 100.0% | 4 (CC) | 3 (Cc) | 7(cc) | 71.4% | |||||
| UCLV3‐169 | aa | +1, +1 | 2 | 0 (AA) | 0 (Aa) | 2 (aa) | 100.0% | 3/11 (18.2%) | 6 | 0 (AA) | 0 (Aa) | 6 (aa) | 100.0% | 0/15 (0%) |
| Cc | −6 | 0 (CC) | 1 (Cc) | 1 (cc) | 100.0% | (CC) | 3 (Cc) | 3 (cc) | 100.0% | |||||
| UCLV3‐29 | Aa | −9 | 2 | 0 (AA) | 0 (Aa) | 2 (aa) | 100.0% | 9/18 (50.0%) | 2 | 0 (AA) | 0 (Aa) | 2 (aa) | 100.0% | 11/11 100.0%) |
| Cc | +1 | 1 (CC) | 1 (Cc) | 0 (cc) | 50.0% | 2 (CC) | 0 (Cc) | 0 (cc) | 0.0% | |||||
| UCLV3‐214 | aa | +1/−6, −1, −6 | 2 | 0 (AA) | 0 (Aa) | 2 (aa) | 100.0% | 4/18 (22.2%) | 2 | 0 (AA) | 0 (Aa) | 2 (aa) | 100.0% | 7/7 (100.0%) |
| Cc | +1 | 1 (CC) | 1 (Cc) | 0 (cc) | 50.0% | 2 (CC) | 0 (Cc) | 0 (cc) | 0.0% | |||||
Hetero, heterozygous; Homo, homozygous.
a
‘−’ and ‘+’ indicate the deletion and insertion of the indicated number of nucleotides, respectively; ‘−/+’ indicates the simultaneous deletion and insertion of the indicated number of nucleotides; ‘+,…’ indicates multiple types of insertions or deletions occurring in different mutation events at the same target site.
b
Based on the number of plants carrying the observed mutation over the total number of plants tested.