Onion is one of the major vegetable crops in terms of production as well as consumption. In the current research, available onion genetic stock was evaluated to identify male‐sterile lines and produce high‐yielding F1 hybrids for future breeding programmes. A mitochondrial DNA‐based marker was mapped and correlated with phenotypic traits to isolate male‐sterile plants. Based on the floral and pollen structure, nine putative male‐sterile lines were identified. On the other hand, for nuclear marker identification at Ms locus, two sets of primers were used, one for Ms dominant allele and another for sterile and maintainer plants. Results revealed that 70% of open pollinated varieties (OPVs) possess plants with sterile cytoplasm coupled with genetic sterility at Ms locus, called sterile “A” line. Approximately 20% of plants in some genotypes were identified with normal (N) cytoplasm having recessive fertility gene at Ms locus, called maintainer “B” line. Based on the present findings, “A”, “B” and “R” (restorer line), future F1 hybrid seed production systems in onion is discussed.
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