For this study, additional primers from Operon Technologies (Alameda, CA) and the University of British Columbia (Vancouver, BC) were screened on gender-based bulked DNA from families 960, 961 and 962.
The initial marker, RAPD UBC354 clone 78-0-90) of families 960, 961 and 962 was amplified, electrophoresed in a 1.5% agarose/0.5X TBE gel, excised, and purified with Gene Clean II (Qbiogene 101, La Jolla, CA).
1998), although some level of hermaphroditic and female-biased sex ratios has been reported for both as a short-rotation energy crop (Åhman 1997; Alström-Rapaport et al. The development of molecular markers linked to sex has been attempted in a number of dioecious species through genetic mapping or breeding work (reviewed in Ainsworth 2000).
The GBA primers amplify a single 571 bp band in male samples but not in female samples, and DNA amplification using GBB primers could generate a 688 bp band only in the female individuals.
Finally, the SCAR primers were used to test 16 sex-unknown samples.
Then the sex-specific bands, S10-Band A and S10-Band B, were cloned and sequenced.
Based on the sequences two pairs of SCAR primers, GBA and GBB, were designed.
A custom primer was synthesized (Life Technologies, Rockville, MD) to append a described in Alström-Rapaport et al. The sequence of the custom primer was the following: 5′-TTTAAGCTTTCTAGAGGCCG-3′.