关键词: 银中杨’, 三倍体, 减数分裂, 原位杂交, 45S rDNA, 染色体行为, 分离规律

Abstract:

Objective: In this study, the FISH technology was used to track the behavior of specific chromosomes during meiosis of microsporocytes of triploid Populus alba × P. berolinensis 'Yinzhong', to clarify the pattern of chromosome pairing and separation, which would enhance understanding of the cytogenetic knowledge of chromosome behavior during meiosis of allotriploid plants and promote the polyploid breeding of poplars. Method: The anthers of triploid Populus alba × P. berolinensis 'Yinzhong' were used as materials to screen suitable enzyme solution and hydrolysis time. On this basis, the slides of meiotic chromosomes were prepared. The 45S rDNA probe was used to trace the chromosomal behavior of pollen mother cells during meiosis by fluorescence in situ hybridization (FISH) analysis. Result: 1) The suitable enzymolysis conditions for meiotic chromosome preparations of pollen mother cells of 'Yinzhong' were selected as the mixture with 3% cellulase and 1% pectolyase, enzymolysis at 37 ℃ for 3 h, combined with squashing coverslip and frozen coverslip removing. The preparations were characterized by good cell dispersion, thin cytoplasm, clean background, and clear chromatid morphology, which were suitable for FISH analysis. 2) The 45S rDNA probe signals were located on three homologous chromosomes of 'Yinzhong', which showed three different chromosome pairing types at metaphase I: one trivalent (Ⅲ), one bivalent and one univalent (Ⅱ+Ⅰ), and three univalents (Ⅰ+Ⅰ+Ⅰ), among them the frequency of cells with one trivalent was the highest, reaching 69.28%. The pairing types of Ⅱ+Ⅰ and Ⅰ+Ⅰ+Ⅰ accounted for 28.10% and 2.61% respectively, indicating that the chromosomes located by the 45S rDNA signals might be closely related, but the synaptic relaxation also existed. 3) Approximately 80.37%-93.44% of the cells presented a 2/1 distribution pattern from the anaphase Ⅰ to the metaphase Ⅱ, and about 63.00%-73.08% of the cells presented a 2/2/1/1 distribution pattern from anaphase Ⅱ to telophase Ⅱ, indicating that the chromosomes tended to follow a relatively normal separation in most of cells during the second meiotic division. However, in some cells, 1-2 chromosomes corresponding to the probe signals were observed to lag or form micronucleus in anaphase Ⅰ, metaphase Ⅱ, anaphase Ⅱ and telophase Ⅱ, which might be lost in the subsequent development. 4) There were some proportions of fusion nuclei in telophase Ⅱ cells with 3-4 45S rDNA probe signals, which might develop into gametes with 2n or even supernumerary chromosomes. Conclusion: FISH technology can be used to locate and analyze the specific chromosome behavior during the meiosis of poplars. Although the triploid 'Yinzhong' has abnormal chromosome behavior during the meiosis, the chromosome behavior located by 45S rDNA probe is relatively stable, which enriches the understanding of the meiotic chromosome behavior of allotriploid poplars.

Key words: Populus alba × P. berolinensis 'Yinzhong', triploid, meiosis, fluorescence in situ hybridization, 45S rDNA, chromosome behavior, separation regulation