Screening And Identification On TuMV Resistance Of Chinese Cabbage Germplasms And Their Research And Application On SSR Marker Technique

Chinese cabbage (Brassica campestris L. ssp. Pekinensis) is native to China and has other names, such as heading cabbage, cabbage and huangyacai. It is rich in Chinese resources. In this study, 667 Chinese cabbage accessions were identified on the resistance to turnip mosaic virus and research on technique of simple sequence repeats for Chinese cabbage. The result of study has offered the important data materials for establishing core databases, for selecting and mating parents in the resist disease breed engineering and for rational utilization and preservation potentializing gene resources of Chinese cabbage germplasms. The results were shown as following:The result showed that 26 accessions were highly resistant (HR) to TuMV, making up 3.9% of the total accessions identified, 78 were resistant (R) to TuMV 11.7%, 205 were medium resistant (T) to TuMV 30.7%, 222 were susceptible (S) to TuMV 3.3%, 136 were highly susceptible (HS) to TuMV 20.3%. And the investigation of morphological characters indicated that some resistant accessions had the same characters, such as, deep color in leaf, wide leaf vein and straight type. It is difficult differentiate the type of resistant only based their mainly morphological characters. Some accessions had the same on the morphological characters but they had the different on the type of resistance.A simple rapid and useful method was developed and a procedure of detecting turnip mosaic virus was set up by RT-PCR. According to the published, the primer pair of coat protein gene was synthesized. The reverse transcription step was performed with total RNA extracted from infected Chinese cabbage tissue. The 980 bp nucleotide fragments were amplified, which were the same as what we had expected. And it will give us an important preparation virus diagnosis and quarantine on Chinese cabbage in the field.A standard SSR system fitting for identification of Chinese cabbage cultivars was established finally. In 10μL reaction solution, contained 1×buffer, 2.50mmol/L MgCl2, 0.10mmol/L dNTPs, 0.35μmol/L SSR primers, 1.00 ng/μL DNA template and 0.40U Taq polymerase. Amplification was carried out in two steps. After one hot starting step of 3 minutes at 72℃and one denaturing step of 2 minutes at 94℃, DNA was denatured for 60 seconds at 94℃, annealing temperature for 60 seconds at 68℃for 2 cycles and subsequently was dropped 10℃every 2 cycles until a final temperature of 58℃was reached. For the last 20 cycles of the amplification, an annealing temperature of 58℃was employed. After all the cycles, amplification was done for 10 minutes at 72℃. The SDS- polyacrylamide gel electrophoresis and silver staining was applied as the new and effective way.In this study, laborious work was conducted in primer selection, 52 SSR primer pairs with over one bands were screened from Brassica database and cabbage-EST database. After study, EST-SSR marker is most effective, convenient and low-priced then other markers. Based on this experience, 27 pairs of Chinese cabbage SSR primers were used for building various multiplex-PCR-sets according to their amplified bands'size range. Under the same conditions as single PCR reaction, 45 two-PCR-sets appeared six different amplification behaviors, of which 36 were amplified normally. 18 two-PCR-sets, 8 three-PCR-sets and 1 four-PCR-sets were amplified normally. These screened normally amplified multiplex-PCR-sets could be applied in Chinese cabbage DNA fingerprinting database.The polymorphisms of SSR marker and the potentiality of SSR fingerprint were analyzed through the screened SSR markers in 5 Chinese cabbage cultivars. Primer Ra3-D04 and Ra2-G09 were more effective and they can be identified 5 varieties separately. Primers Ra3-H10 and Bn-38A can be identified 2 and 3 varieties separately.Genetic similarity and cluster analysis based on morphological marker and SSR molecular all indicated that genetic similarities of paired varieties ranged from 0.08 to 0.85 and from 0.42 to 0.87, respectively. And genetic basis of most varieties was dissimilar. Some varieties were similar by SSR molecular, but their different on the morphological. Most of varieties were similar and has the same type to TuMV. But some resistance and susceptible varieties have highly genetic similarities, and all could cluster together based two markers. There was no remarkable relationship between genetic difference and their showed resistance or susceptible to TuMV.