| Summary: | Vacuolar H<sup>+</sup>-ATPases (V-ATPase) are multi-subunit complexes that function as ATP hydrolysis-driven proton pumps. They play pivotal roles in physiological processes, such as development, metabolism, stress, and growth. However, there have been very few studies on the characterisation of V-ATPase (VHA) genes in Rosaceae species. Therefore, in the present study, we performed a genome-wide analysis and identified <i>VHA</i> gene family members in five Rosaceae species (<i>Pyrus bretschneideri</i>, <i>Malus domestica</i>, <i>Prunus persica</i>, <i>Fragaria vesca,</i> and <i>Prunus mume</i>). A total of 159 <i>VHA</i> genes were identified, and were classified into 13 subfamilies according to the phylogenetic analysis. The structure of VHA proteins revealed high similarity among different <i>VHA</i> genes within the same subgroup. Gene duplication event analysis revealed that whole-genome duplications represented the major pathway for expansion of the <i>Pyrus bretschneideri VHA</i> genes (<i>PbrVHA</i> genes). The tissue-specific expression analysis of the pear showed that 36 <i>PbrVHA</i> genes were expressed in major tissues. Seven <i>PbrVHA</i> genes were significantly downregulated when the pollen tube growth stopped. Moreover, many <i>PbrVHA</i> genes were differentially expressed during fruit development and storage, suggesting that <i>VHA</i> genes play specific roles in development and senescence. The present study provides fundamental information for further elucidating the potential roles of <i>VHA</i> genes during development and senescence.
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