Cloning And Sequence Measuring Of The Marker Linked To Seed Trait In Soft-seeded Pomegranate

Baiyushizi was a sport cultivar found in natural mutant of Sanbai. The result of former study on Baiyushizi and its stock plant showed that there were no significant differences at maturity date, dehiscent fruit pre-harvest fruit dehiscence tolerance to storage and transportation and resistant to adverse circumstance. The result of fuzzy synthetic valuation showed that the synthetic economic trait of Baiyushizi was better than Sanbai markedly. The outstanding advantages of varied trait of Baiyushizi were the weight of per hundred arils, which increased by 84.3%, and the seed hardness of which was softer and appeared as double-peak curve. It was postulated that main gene probably existed in seed hardness controlling.Taking Baiyushizi and Sanbai as materials the differences between the two cultivars analysed by PCR to find the gene marker related to trait of soft seed in pomegranate. The specific markers were cloned and their sequences were measured, and then transformed to SCAR marker to provide molecule biological evidence for pomegranate breeding.The genomic DNAs of pomegranate leaves were extracted by the improved CTAB method. The parameters of RAPD-PCR including the concentration of Taq polymerase, template DNA, magnesium ion, dNTPs and random primer were optimised gradually to establish the optimal reaction system of RAPD-PCR. The total volume of the reaction was 25μL, containing Taq polymerase 1.0 unit, random primer 0.3μmol/L, dNTPs 0.2mmol/L, MgCl2 2.0mmol/L, and template DNA 40 ng /μl.There were 11 specific bands amplified by 9 random primers, which were screened out from 113 random primers accounting for a wide range of variation of the mutant in genomic DNA occurred in Baiyushizi, the three clear and stable specific bands were isolated and purified by repeated amplification with the 9 random primers. The purified target DNA fragment was linked to the PMT18-T vector, then transforming them into to multiply. The result of transformation was checked by clone PCR. The sequences of the target fragment were measured by using of liquid culture of E.coli.According to the measured sequence the specific primer of 18~22 bp was designed with the software Primer Premier 5.0, then taking the genomic DNA of Baiyushizi and Sanbai as template SCAR-PCR amplification was carried out with the specific primers, and the result of which showed that two of the there target fragments existed in Baiyushizi and Sanbai actually, their molecular sizes were 562 bp and 804 bp respectively. Only one of the specific bands, which molecular size was 537 bp was true SCAR marker in Baiyushizi not presented in Sanbai. It proved there was a varied locus existed in Baiyushizi and Sanbai.The RAPD maker of genetic variation in Baiyushizi had been transformed to be SCAR maker successfully in this experiment. Also the results supplied a technical system for transforming the RAPD marker to the SCAR marker, and provided a DNA evidence for using RAPD marker to site the gene of soft seeded trait and to map and clone the gene related to fruit trait in pomegranate. It provided a basis for early selection of soft-seeded or big-aril trait of offspring in pomegranate breeding.