Peach is one of the most economically valuable fruit trees. Haploid peach trees occur spontaneously at very low frequencies and they are usually highly sterile. Therefore, the haploid with partial fertility is an extremely rare germplasm, which is highly valuable to genetic research and breeding programs. In this study, we investigated the cytological mechanism underlying the fertility of a peach haploid mutant ‘9-D’ derived from a spontaneous mutation. Cytologic evaluation and flow cytometry analysis demonstrated that ‘9-D’ is a pure haploid. Scanning electron microscope analysis revealed a considerable proportion of abnormal pollen grains in ‘9-D’. Pollen viability assay by Alexander staining showed that 50.4% of pollen grains from ‘9-D’ were viable. However, the pollen germination assay showed that only 7.6% of the pollen grains could germinate normally. Investigation of the chromosomal behavior of pollen mother cells at different stages of meiosis showed that pollen mother cells of ‘9-D’ lacked the process between anaphase I and prophase II of meiosis. Various types of sporophyte morphology were observed in haploid pollen mother cells at the tetrad stage. Measurement of the diameter of pollen grains indicated the presence of pollen with 2x ploidy in ‘9-D’. The offspring of ‘9-D’ were predominantly triploid or triploid aneuploid. The triploid offspring were more likely derived from the 2x male gametophyte combined with the haploid female gametophyte, which may explain the reason why ‘9-D’ has fertility. This study not only expands our understanding of haploid fertility mechanisms, but is also useful for ploid breeding programs in peach.

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