基于45S rDNA-FISH的异源三倍体‘银中杨’小孢子母细胞减数分裂染色体行为追踪  

Chromosome Behaviors Tracking during Meiosis of Microsporocytes of Allotriploid Populus alba×P.berolinensis‘Yinzhong’Based on 45S rDNA-FISH

作　　者：王律几 张晓晓 王君[1] Wang Lüji;Zhang Xiaoxiao;Wang Jun(National Engineering Laboratory in Tree Breeding,Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education,College of Biological Sciences and Biotechnology,Beijing Forestry University,Beijing 100083)

机构地区：[1]北京林业大学林木育种国家工程实验室,林木花卉遗传育种教育部重点实验室,北京林业大学生物科学与技术学院,北京100083

出　　处：《林业科学》2022年第3期69-78,共10页Scientia Silvae Sinicae

基　　金：中央高校基本科研业务费专项资金资助(2021ZY60);国家自然科学基金项目(31470662)。

摘　　要：【目的】利用FISH技术追踪三倍体‘银中杨’小孢子母细胞减数分裂过程特定染色体的行为,明确其配对和分离规律,丰富对杨树异源三倍体减数分裂过程染色体行为的细胞遗传学认识。【方法】以三倍体‘银中杨’花药为材料,对不同酶液组合和酶解时间进行筛选,在此基础上均进行减数分裂染色体制片,以45S rDNA序列为探针,利用FISH技术,对‘银中杨’小孢子母细胞减数分裂过程染色体行为进行追踪和分析。【结果】1)筛选出适于‘银中杨’小孢子母细胞减数分裂染色体制片的酶解条件为3%纤维素酶+1%果胶酶混合液在37℃下酶解3 h,结合压片和冷冻脱盖片,可获得细胞分散度良好,细胞质薄,背景干净,染色体形态清晰的制片,适用于FISH分析。2)45S rDNA探针信号定位于‘银中杨’3条同源染色体上,它们在中期I呈现III、II+I、I+I+I 3种配对形式,其中三价体发生的频率最高,达69.28%,II+I和I+I+I的配对类型分别占28.10%和2.61%,表明信号所定位的染色体亲缘关系可能较近,但也存在联会松弛的现象。3)约80.37%~93.44%的细胞在后期I至中期II发育阶段呈现2/1分离模式,约63.00%~73.08%的细胞在后期II至末期II呈现2/2/1/1分离模式,表明探针信号所定位的染色体在大部分细胞中遵循较为正常的分离规律,但在部分细胞中也观察到探针信号所对应的1~2条染色体在后期I、中期II、后期II、末期II发生分离滞后或形成微核,进而可能在后续发育过程丢失的现象。4)末期II细胞存在一定比例的融合核,其中可观察到3~4个45S rDNA探针信号,可能形成具有2n甚至超数染色体的配子。【结论】利用FISH技术可对杨树减数分裂过程的特定染色体行为进行定位和精细分析,尽管三倍体‘银中杨’全染色体组的减数分裂过程染色体行为异常紊乱,但45S rDNA探针所定位的染色体行为相对稳定,丰富了杨树异源三倍体减数�【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

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 

分 类 号：S718.46[农业科学—林学]