Development Of Molecular Markers To Authenticate The Hybrid Seed Of Shuza No.6, Brassica Napus

The objectives of this study are to develop RAPD markers in Shuza No. 6, Brassica napus, and convert them into SCAR markers for evaluating hybridity of FI hybrid. DNA samples extracted from the individuals of two parents of hybrid rapeseed Shuza No. 6 were mixed equally to form the parent DNA pools, which were used to screen the polymorphism with 100 arbitrary 10-mer primers. 5 primers, with which polymorphism bands were amplified in both parents, were selected to check their polymorphism in the DNA pool of F[ hybrid. Two primers, GE204 and GE222, could display the well distinctive band patterns among the parents and their F, hybrid and were chosen for further study. The GE204 could amplify two maternal-specific markers, GE204500 and GE204340, in the DNA pools corresponding to both maternal parent and Fl hybrid. The GE222, on the other hand, amplify two paternal-specific markers, GE222620 and GE222300, in the DNA pools of paternal parent and F1 hybrid, respectively. In order to improve the reproducibility and reliability of PCR assays, the RAPD markers were converted to SCAR markers. The four marker fragments, GE204500, GE204m, GE222620 and GE222300, were cloned and the identity of these clones with respect to the corresponding RAPD markers was examined by dots and restriction enzyme analysis. 23 clones were sequenced and the intact sequences of the four fragments were aligned with the sequences of the clones. According to the sequences of GE204500 and GE222300, three pairs of parent specific SCAR primers ranging from 19 to 25 bases in length were designed. The annealing temperature was optimized for each pair of primers and multiplexed primers. Based on these results, PCR conditions with mixed two pairs of parent specific SCAR primers were set up for authentication of the hybrid seed Shuza No. 6. The phenotypes of 300 plants of Shuza No. 6 were investigated in the field and their DNA samples were prepared individually for PCR reaction with multiplexed primers. The results show that the genotypes of individuals decided with SCAR markerscould match very well with the phenotypes examined in the field. Together with the protocol for rapid preparation of total rapeseed DNAs on a large scale, the use of SCAR markers provides a more efficient and rapid way to evaluate the hybridity of commercial rapeseed F, hybrid in the laboratory.