| Summary: | The genus <i>Populus</i> is composed of dioecious woody plants and adult females produce large numbers of seed hairs that can affect public health and pose a potential fire risk. However, it is difficult to distinguish between males and females based on their morphology at the seedling stage. Therefore, developing a technology that identifies the gender of poplar seedlings is crucial for controlling seed hairs. In this study, we developed an approach for the early gender identification of <i>Tacamahaca</i> and <i>Aigeiros</i> species based on the male-specific sequence in <i>Populus simonii</i>. The gender of <i>Tacamahaca</i> and <i>Aigeiros</i> species can be accurately identified by PCR. The sequencing results showed that the male-specific sequence was conserved in <i>P. simonii</i> and its F<sub>1</sub> progenies. Interestingly, there were three nucleobase differences between <i>Tacamahaca</i> and <i>Aigeiros</i> species. Sequence alignment showed that the male-specific sequence had not been assembled on the pseudochromosome. Subsequently, fluorescence in situ hybridization (FISH) was used to locate this specific sequence at the short arm end of chromosome 19 in male <i>P. simonii</i>. This study provides an efficient and convenient method for early gender determination of <i>Tacamahaca</i> and <i>Aigeiros</i> species and lays the groundwork for exploring key sex-determination genes.
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