Brassica napus is the most important oil crop plant for edible oil and renewable energy source worldwide. Yield loss caused by pod shattering is a main problem during B. napus harvest. In this study, six BnSHP1 and two BnSHP2 homoeologs were targeted by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) genome editing system and multiple SHP1 and SHP2 mutated lines were identified for evaluating the contribution for pod-shattering resistance. Our data suggest that BnSHP1A09 is probably a promising homoeolog for controlling lignin contents at dehiscence zone. Simultaneous mutation of BnSHP1A09/C04-B/A04 and BnSHP2A05/C04-A exhibited reduced lignified layer and separation layer adjacent to valves and replum. The pod-shattering resistance index (SRI) subsequently increased to 0.31 in five homoeolog mutation lines compared with the wild type (SRI = 0.036), which provide the theoretical basis for breeding of commercial pod-shattering resistance variety.
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