The genome-wide long hairpin RNA interference (lhRNAi) library is an important resource for plant gene function research. Molecularly characterizing lhRNAi mutant lines is crucial for identifying candidate genes associated with corresponding phenotypes. In this study, a dwarf and sterile line named P198 was screened from the Brassica napus (B. napus) RNAi library. Three different methods confirmed that eight copies of T-DNA are present in the P198 genome. However, only four insertion positions were identified in three chromosomes using fusion primer and nested integrated polymerase chain reaction. Therefore, the T-DNA insertion sites and copy number were further investigated using Oxford Nanopore Technologies (ONT) sequencing, and it was found that at least seven copies of T-DNA were inserted into three insertion sites. Based on the obtained T-DNA insertion sites and hairpin RNA (hpRNA) cassette sequences, three candidate genes related to the P198 phenotype were identified. Furthermore, the potential differentially expressed genes and pathways involved in the dwarfism and sterility phenotype of P198 were investigated by RNA-seq. These results demonstrate the advantage of applying ONT sequencing to investigate the molecular characteristics of transgenic lines and expand our understanding of the complex molecular mechanism of dwarfism and male sterility in B. napus.
Keywords: ONT sequencing; RNAi; T-DNA; dwarfism; molecular characteristic; sterility; transcriptome.