Abstract:

Objective: This study aims to establish a high-quality chromosome preparation method for Juglans mandshurica with dispersed and good morphology chromosome using the male inflorescence of J. mandshurica as materials, so as to lay the foundation for further research on the molecular cytogenetics of J. mandshurica. Method: The process of anther development was observed using carbol fuchin staining. Male inflorescences at the vigorous anther cell division stage were collected and pre-treated with 1 MPa nitrous oxide (N₂O), 0.7 mmol?L^?1 cycloheximide, and 2 mmol?L^?1 8-hydroxyquinoline for different durations, respectively. The inflorescence was peeled off to take out the anthers that were enzymatically digested to obtain suspensions. Then suspensions were spread on a slide using a needle on a heater at 55 °C. The mitotic metaphase chromosomes were used for fluorescence in situ hybridization (FISH) with 45S rDNA and 5S rDNA probes. Result: 1) When the male inflorescence became a fresh green color, reached a length of about 1.5 cm, and the tip of the anther turned red, the anther cell division was vigorous and a large number of microsporocyte mother cells and mitotic metaphase chromosomes could be observed. Therefore, the optimal sampling time was when the anthers in the upper part of the male inflorescence turned red, which can ensure that most of the anthers were in the vigorous cell division period. 2) Chromosomes treated with 2 mmol?L^?1 8-hydroxyquinoline for 4, 6, and 8 hours were condensed insufficiently, had unclear margins, and exhibited obvious trailing. Most of the centromeres could not be recognized. Chromosomes treated with 0.7 mmol?L^?1 cycloheximide for 4 hours were long and condensed insufficiently, with severe trailing. Chromosomes treated for 6 hours showed appropriate condensation and good morphology, with most of the centromeres recognizable. Chromosomes treated for 8 hours had clear margins but excessive condensation, with unclear centromeres. Chromosomes treated with 1 MPa N₂O for 2 hours had appropriate length but insufficient condensation, blurry margins. Chromosomes treated with 1 MPa N₂O for 3 and 4 hours had high condensation levels, were thick and short, with no clear morphological differences between chromosomes. In addition, some chromosomes treated with N₂O for 4 hours exhibited sticky stretching. Thus, the optimal pretreatment condition for male inflorescence of J. mandshurica was 0.7 mmol?L^?1 cycloheximide for 6 hours. 3) The chromosome number of J. mandshurica was 32, with a base number of 16 (2n = 2x = 32). The FISH signals of 45S rDNA and 5S rDNA probes appeared bright on the chromosomes of J. mandshurica. The hybridization signals of 45S rDNA were located near the centromeres of one pair of metacentric chromosomes, with similar signal intensities on both chromosomes. The hybridization signals of 5S rDNA were found near the centromeres of another pair of metacentric chromosomes, with one strong and one weak signal intensity. Conclusion: By using the male inflorescence of J. mandshurica when the tip of the anther turns red, a high-quality chromosome preparation and FISH technique system for J. mandshurica in the mitosis metaphase has been successfully established. This study provides a foundation for the molecular cytogenetics research of J. mandshurica and also serves as a reference for obtaining high-quality chromosome preparations in plants with large anthers.

Key words: Juglans mandshurica, male inflorescence, chromosome preparation, fluorescence in situ hybridization (FISH)