Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), poses a severe threat to global banana production, and breeding resistant cultivars remains the most effective control strategy. Mutation breeding, including radiation mutagenesis and somaclonal variation, has become a primary approach for developing resistant germplasm in triploid Cavendish bananas. However, whether secondary bud-sport selection from resistant somaclonal lines inadvertently compromises original resistance mechanisms at the molecular level remains poorly understood. In this study, we generated 44 mutants from Baxi jiao via ⁶⁰Co γ-irradiation and selected five lines with distinct phenotypic variations. We also collected somaclonal variant lines GCTCV-218, GCTCV-119, GCTCV-105, their bud-sport derivatives (NK_No.1, NTH, RK_No.1), and the radiation-induced resistant mutant ‘Zhongre No.1’. Using whole-genome resequencing and transcriptome analysis, we systematically compared the genetic and transcriptomic outcomes of these breeding strategies. Radiation mutagenesis induced substantial genomic structural variations and generated novel expression patterns of defense-related genes. In contrast, while bud-sport derivatives of GCTCV-218 remained genetically similar to their parent, they exhibited significant downregulation or loss of key resistance gene expression, particularly PR-1 family members. Our findings reveal that phenotype-driven somaclonal selection can inadvertently erode original resistance mechanisms, and we recommend prioritizing radiation mutagenesis for developing banana cultivars with stable and durable resistance to Foc TR4.

The accurate identification of hybrid progeny is essential for improving the efficiency of banana (Musa spp.) crossbreeding, yet traditional morphological methods are time-consuming, unstable, and ineffective for distinguishing hybrids at early developmental stages. In this study, the highly fertile and stress-tolerant landrace 'Dongguan Dajiao' (Musa × paradisiaca, DGDJ) was used as the maternal parent and crossed with two wild diploid species, Musa acuminata (XGYJ, AA) and Musa balbisiana (HNYJ, BB), as the paternal parents, producing the progeny lines X1701, X1702, X1931, and X1932. PacBio HiFi sequencing was performed using the Revio platform, followed by k-mer analysis, genome assembly, and parental read coverage analysis to systematically evaluate the genome size, heterozygosity, and genomic composition in both parents and the progeny. Flow cytometry confirmed that the maternal parent DGDJ is triploid (3x), the paternal parents XGYJ and HNYJ are diploid (2x), and all four progeny lines are tetraploid (4x). The results revealed that DGDJ possesses a complex genomic constitution comprising the A, B, and S genomes with a heterozygosity level of 5.77%. All progeny were identified as tetraploids, with paternal contributions ranging from approximately 14% to 17% and maternal inheritance exceeding 62%, confirming their status as true hybrids. Collectively, this study establishes a k-mer-based method for identifying banana hybrid progeny using HiFi sequencing data, offering critical technical support for germplasm innovation and crossbreeding in banana.