Kaur, Rajbir2026-03-202026-03-202026-02-202026-03-192026-03-19http://hdl.handle.net/1993/39667Brassica napus L. (rapeseed or canola) is a major oilseed crop, but pre-harvest seed loss due to pod shatter continues to limit the productivity and profitability of its cultivation. This study aimed to identify natural genetic variation associated with the pod shatter in the parental genotypes of a B. napus doubled haploid (DH) population from the B. napus breeding program using long-read sequencing data. A total of 37 genes, previously linked to pod shatter, were analyzed to identify genetic differences that might influence phenotypic diversity in this trait. The sequencing data were aligned to the B. napus reference genome ZS11 and used to call structural variants (SVs), single-nucleotide polymorphisms (SNPs), and insertions/deletions (InDels). Unique SVs were identified in each parent and confirmed through Polymerase Chain Reaction within candidate genes AGAMOUS, ARABIDOPSIS DEHISCENCE ZONE POLYGALACTUROSE2, NAC SECONDARY WALL THICKENING PROMOTING FACTOR1, and SHATTERPROOF1, indicating that allelic diversity at these loci could affect pod shatter tolerance between the parents. Additionally, moderate-impact SNPs were found within ABA-INSENSITIVE 5, ARABIDOPSIS DEHISCENCE ZONE POLYGALACTUROSE1, and REPLUMLESS genes, suggesting their possible regulatory roles in the expression of this trait. These results establish a foundation for developing molecular markers associated with pod shatter tolerance and for functional validation of the identified polymorphisms in other B. napus populations. To functionally validate the roles of genes identified through sequence variation analysis, the CRISPR/Cas9 system was used to knockout SHATTERPROOF1, along with two other genes involved in pod shatter, ALCATRAZ and INDEHISCENT. Additionally, to address the limitations of tissue culture-based transformation in B. napus, an alternative spray-based transformation method was proposed. However, screening of putative transformants revealed transient T-DNA presence, as confirmed by whole-genome sequencing, indicating that the approach was ineffective under the tested conditions. These findings highlight the need to identify more responsive genotypes for transformation, to improve the spray-based method, and to explore new transformation techniques to achieve effective and reliable genome-editing in B. napus. Overall, this study establishes a foundation for exploring genetic diversity and integrating genomics techniques into breeding programs, offering promising avenues to enhance key agronomic traits in B. napus.engBrassica napusPod shatterLong-read sequencing