Identification Of RAPD And SCAR Markers Linked To Orange-red Color Of Internal Head Leaf Trait In Chinese Cabbage

Bulked segregant analysis was employed to identify molecular markers linked to orange-red color of internal head leaf(Or) trait based on RAPD in Chinese cabbage with doubled haploid (DH) population. By testing the RAPD marker with DH population and 78 F2 individual plants, we identified one RAPD marker linked to Or gene closely and transferred it into SCAR marker successfully. The technique of molecular marker-assisted-selection for breeding orange-red internal head Chinese cabbage cultivars was established. The chief results are as follows:Doubled haploid population derived from the microspore culture of F1 crossed between white internal head line 91-112 and orange-red internal head line T12-19 was obtained. According to the segregating results of leaf-head color in DH population, orange-red genotype proved to be controlled by a recessive allese and named Or gene.The method of DNA extraction was explored. DNA extracted in this method was very complete and can be digested completely by EcoRI. A stable and reliable PCR amplification can be obtained. Factors affecting RAPD reaction were analyzed. Optimizing the RAPD reaction conditions in Chinese cabbage, the optimal concentration of MgCl2, dNTP and the annealing temperature were investigated.By screening polymorphism between two parents with 500 Operon RAPD 10-mer-primers (25 sets), 462 primers produced clear bands, whichaccounted for 92.4%. A total of 2032 bands were obtained and the average bands produced by per primer were 4.4. 124 primers showed polymorphic bands between two parents and stable and clear polymorphic bands were obtained with 97 primers in two times replication. 8 lines were selected randomly from 59 orange-red head DH lines and 41 white-head DH lines respectively, their genomic DNA were mixed together to form orang-red pool and white pool. 13 primers showed polymorphic between two pools among 97 primers which showed polymorphic between the parents. However, the polymorphism of 12 primers was found to be false when every individual of each pool was checked. One RAPD marker OPB01-845 produced by primer OPB01(5'-GTTTCGCTCC-3')was observed in every plants of orange-head pool and disappeared in every plants of white-head pool. Identical results were obtained in three times replication, therefore OPB01-845 was thought to be linked to Or gene.The accuracy of 93% and 89.7% were identified in comparing the internal head leaf color and the OPB01 marker in 100 DH lines and 78 F2 plants respectively, the genetic distance between the OPB01 and Or gene was 3.8 cM and LOD value was 19.05. This can conform that the marker OPB01-845 is closely linked to Or gene.The specific DNA marker OPB01 amplified in orange-head pool was recovered, and then inserted into pGEM-T Easy Vector plasmid, and finally transported into E.coli.DH5α. After the specific band was cloned and sequenced, a pair of 20-base specific primers was designed. The SCAR analysis of DH population and 78 F2 plants displayed that the result of amplification with SCAR is identical with that of RAPD. The reliability of result showed that the marker was converted into SCAR marker successfully. The same accuracy of SCAR marker as RAPD 01-845 was obtained in the investigation of 100 DH population and 78 F2 plants. This SCAR marker can be used in marker-assisted selection in breeding program of head leaf color in Chinese cabbage.