Molecular Markers Linked To Restorer Gene Of CMS In Chinese Cabbage

Bulked segregant analysis (BSA) was employed to screen molecular markers linked to restorer gene of pol CMS 02A1 in Chinese cabbage (Brassica campestris syn. rapa L . ssp. pekinensis (Lour) Olsson) based on RAPD, with 142 F2 population derived from a self-cross of fertile F1 A1-26×02S149-5 hybrid. By testing segregation of the special RAPD markers among the 142 individual plants of F2, one RAPD marker linked closely to the fertile gene was identified. The SRAP technology was also tried to use in Chinese cabbage. The main results are as follow:1. Observation of fertile segregation in F2 population derived from a self-cross of F1 hybrid A1-26×02S149-5 showed that the fertility restoration of the pol CMS 02A1 of Chinese cabbage is controlled by a single dominant gene.2. By screening polymorphism of RAPDs between fertile DNA pool and cytoplasmic male sterile DNA pool with 400 random 10-mer primers, the primer S1036 produced polymorphic between two DNA pools was selected. One RAPD marker named as S1036-657 produced by primer S1036 (AAGGCACGAG)was observed in all plants of fertile DNA pool and disappeared in all plants of cytoplasmic male sterile DNA pool.3. By analyzing the segregation of the specific DNA marker S1036-657 and fertility in 142 F2 population. It was found that the marker S1036-657 was closely linked to the restorer gene of the pol CMS 02A1 with the crossing-over value of 8.4% and genetic distance of 8.5cM.4. The marker S1036-657 was cloned and sequenced. The sequence analysis revealed that S1036-657 has an 87% homology with the MYB transcription factors in Arabidopsis thaliana L. and Brassica campestris L. in nucleic acid sequence.5. Using the genome DNA of Chinese Cabbage as template, the major components in SRAP, such as concentrations of Mg2+, dNTPs, Taq DNA polymerase, primers and template, were optimized by orthogonal design in five factors with four levels in this study. The results showed that the optimum SRAP reaction system includes Mg2+ 3.0mmol/L, dNTPs 0.2mmol/L, DNA template 73.2ng,Taq DNA polymerase 1.5U and primer 0.2μmol/L in the 25μl volume reaction. The most suitable protocol was initially denaturing at 94℃for 5 min, then pre-amplifying at 94℃1 min, 35℃1min and 72℃1 min for five cycles, finally amplifying for 35 cycles when the annealing temperature was adjusted to 50℃.6. SRAP technology was also used for finding molecular marker of male sterile in Chinese cabbage. From the primer combination of 8 F-primers and 11 R-primers 12 pairs of primers was found to produce the polymorphism between the two DNA pools. One pair of primers me6em3 was identified to produce one specific band (about 2000bps) only in fertile DNA pool.