Occurrence And Molecular Markers Of Unreduced Pollen In Chinese White Poplar

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.