【作者】 张正海；

【导师】 康向阳；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2008， 博士

【摘要】 毛白杨(Populus tomentosa Carr.)种群中有天然未减数花粉存在,开展毛白杨天然未减数花粉形成机制研究,对未减数花粉高产植株选择和利用具有重要的理论和实际应用价值。本研究主要通过花粉形态和小孢子母细胞减数分裂观察,以及AFLP和cDNA-AFLP分子标记分析,围绕毛白杨未减数花粉发生频率、细胞学机制、相关分子标记等进行了初步研究,取得了一定的进展。1.根据未减数花粉相对于正常减数花粉表现为巨大性而易于判别的特点,利用醋酸洋红染色法对224个毛白杨雄性无性系的花粉进行了显微观察和统计,结果显示,除了河南和陕西的6个无性系无未减数花粉发生外,其它无性系均可产生未减数花粉,发生频率介于0.6-21.9%。方差分析表明,毛白杨未减数花粉的发生频率在不同地理种源间呈现显著性差异,而在各无性系之间存在极显著性差异。2.利用醋酸洋红压片观察,发现在毛白杨小孢子母细胞减数分裂中期Ⅱ,纺锤体表现为平行,或互为一定的角度;在减数分裂的四分体阶段,还存在一定比率的三分体和二分体。进一步通过间接免疫荧光法观察小孢子母细胞减数分裂,发现在一些细胞中,两个纺锤体在一个方向或两个方向上的微管系统存在共极现象,即存在融合(两极)纺锤体和三极纺锤体。由此可以推断,由于异常纺锤体的存在,纺锤体微管系统定位紊乱,部分细胞的纺锤体极点融合,导致二分体或三分体的形成,最终形成未减数花粉。此外,减数第二次分裂存在胞质分裂提前现象,也可能会导致二分体的形成,从而产生未减数花粉。其中,来源于平行纺锤体的未减数花粉为FDR型,而因胞质分裂提前而形成的未减数花粉则属于SDR型。3.利用AFLP和SCAR标记进行了毛白杨未减数花粉发生相关分子标记的研究。从52对AFLP引物组合中筛选出2对引物组合及其与毛白杨产生未减数花粉无性系和正常无性系相关的AFLP标记,其中采用引物组合E31-M50(AAA/CAT)可以从产生未减数花粉的毛白杨无性系中扩增出1条大小为204bp的多态性条带,而采用引物组合E50-M38(CAT/ACT)能够从正常不产生未减数花粉的毛白杨无性系中扩增出1条大小为246bp的多态性条带。并进一步回收由AFLP标记筛选出的2条多态性条带,成功转化为操作简单、方便检测的SCAR标记。4.利用EcoRⅠ和MseⅠ双酶切引物组合对毛白杨小孢子母细胞减数分裂各阶段的花芽进行cDNA-AFLP转录分析,用筛选出的12对多态性突出的引物组合共扩增到减数分裂阶段特异性转录来源片段52个,其中只有引物E33-M40(AAG/AGC)扩增得到了未减数花粉无性系在减数分裂Ⅱ差异表达的转录来源序列标签(TDF24#);进一步对TDF24#进行BLASTN和BLASTX分析,发现与已知EST序列、基因或蛋白没有同源匹配,特异TDF24#与未减数花粉发生是否相关有待进一步研究。更多还原

【Abstract】 The production of unreduced pollen in Chinese white poplar (Populus tomentosa Carr.) has been reported. Studying the mechanisms of unreduced pollen was important for selection and utilization of unreduced pollen in triploid breeding. The objective of this research was to determine the mechanism of unreduced pollen formation in indigenous populations of Chinese white poplar. Occurrence frequency, cytological mechanisms and molecular markers involved in unreduced pollen production were investigated by morphology of pollen grains, cytology of meiosis, AFLP and cDNA-AFLP markers.1. Pollen grains of 224 clones were investigated for the production of unreduced pollen based on pollen size difference. It was observed that six clones which from Henan and Shannxi were subsequently determined to only produce normal pollen and the remainder produce unreduced pollen at different frequency range from 0.6% to 21.9%. The analysis of variance on the frequency of unreduced pollen in Chinese white poplar revealed significant differences between indigenous populations and highly significant differences among clones within indigenous populations.2. Microscopic examination was used to determine the second meiosis of pollen mother cells in Chinese white poplar. Two types of spindles (parallel spindles and tripolar spindles) were observed as indicated by the orientation of the chromosomes. So did dyads and triads at the tetrad stage. Furthermore, indirect immunofluorescence analysis of meiosis revealed that some of the spindles fused in one or two poles, i.e. fused (bipolar) spindle and tripolar spindle. The results revealed that the abnormal spindles caused irregular operation of microtubules leading to spindle poles fusion. Dyads and triads were induced by the fusion of spindles and induced unreduced pollen formation. In addition, the premature cytokinesis during the second meiotic division also produced dyads. The abnormal spindles produce unreduced pollen is genetically equivalent to first-division restitution (FDR) mechanism, while that from premature cytokinesis is genetically equivalent to second division restitution (SDR).3. Molecular markers of amplified fragment length polymorphism (AFLP) and SCAR (sequence-characterized amplified region) were employed to identify related molecular markers of unreduced pollen in Chinese white poplar. Following an initial screening with 55 primer combinations, the E31-M50 (AAA/CAT) primer was identified: it generated a PCR fragment (204 bp) from the unreduced pollen producers, but not from the normal clones. In addition, the E50-M38 (CAT/ACT)-amplified DNA fragment (246 bp) was present in normal clones, and absent in unreduced pollen producers. These two discriminating AFLP markers were developed into easily detectable SCAR markers which can be used in combination with the previously developed AFLP markers to distinguish between normal and unreduced pollen clones.4. Differential gene expression profiles of meiosis during unreduced pollen formation were analyzed by cDNA-AFLP technique. From 12 primer combinations, 52 regulated differentially transcription-derived fragments (TDFs) were observed, of which TDF24# was a differential expression profile in unreduced pollen clone. However, the TDF24# that expressed at the second division of unreduced pollen clone was not found similarity to any ESTs, genes or proteins by the BLAST program at the GenBank database.更多还原