Incidence and molecular markers of 2n pollen in Populus tomentosa Carr.

Microscopic examination, amplified fragment length polymorphism (AFLP) and SCAR (sequence-characterized amplified region) molecular markers were employed to determine the incidence of 2n pollen (unreduced pollen) in Chinese white poplar (Populus tomentosa Carr.) and to identify related molecular markers. The presence of a parallel and tripolar spindle at metaphase II and the absence of cytokinesis at telophase II were found to be determining factors in 2n pollen formation. A group of 298 clones that originated from their indigenous areas were investigated for the production of 2n pollen based on pollen size differences, both within a clone and between n and 2n pollen. Pollen grains were collected from 224 of the clones, six of which were subsequently determined to produce only normal pollen; the remainder produced 2n pollen at different frequencies (0.6-21.9%). The frequency at which 2n pollen was produced was significantly and highly significantly different among and within indigenous populations, respectively. Clones produced by the six normal and twenty-two 2n pollen clones were selected for AFLP analysis. Following an initial screening with 55 primer combinations, the E50-M38 (CAT/ACT) primer was identified: it generated a PCR fragment (246 bp) from the normal clones, but not from the 2n pollen producers. In addition, the E31-M50 (AAA/CAT)-amplified DNA fragment (204 bp) was present in 2n pollen producers, and absent in normal clones. These two discriminating AFLP markers were developed into easily detectable SCAR (sequence characterized amplified region) markers which can be used in combination with previously developed AFLP markers to distinguish between normal and 2n pollen clones.