Study On Analysis And Identification Of Hybrid Rapeseed Seed Purity With Molecular Markers Technique

Rapeseed is one of main food oil plants in china. Using heterosis of rapeseed is a important approach to increase yield and improve quality.The sown area of hybrid rapeseed is about 50% of total sown rapeseed area.The hybrid purity play a very important role in the heterosis. The off-types in hybrid rapeseed are mostly sterile line plants and restorer plants. So the identification of hybrid rapeseed seeds purity is to identify parent seeds from hybrid seeds.CMS hybrid Qinyou No.7 in Brassica napus L. and its sterile line Shaan 3A and restorer K407 were used as materials in this experiment to study the technique of identification of hybrid rapeseed seeds purity with RAPD and SSR molecular markers. The main results are as follows:1.Two RAPD primers (S₉₃ and S₄₆₉) and one pair of SSR primers (S_a82) were screened out from 814 RAPD primers (10mer) and 100 pairs of SSR primers. And they can be used to distinguish hybrid rapeseed Qinyou No.7 and its parents at same time. The pattern of hybrid seed amplification products showed complemental pattern of its parents. The patterns of the hybrid were distinguished from its parents clearly and stably.2.A simple and rapid method of DNA extraction was established on rapeseed. The rapeseed sprouts gerorminated for 3-5 days were used as materials of DNA extraction. Chloroform was used before rubbing, this way made mostly protein and enzyme lose activity or passivation, and prevented DNA from degradation. The steps of extracting and washing were omitted to simplify extraction process,at the same time,the time of heating in warm water and colding in ice and centrifugating was shortened. So the speed and efficiency of DNA extraction were increased.3.RAPD and SSR reaction system that can be used to identify hybrid rapeseed seed purity was built by optimizing reaction system.The optimum RAPD and amplification reaction system was:20μL of PCR reaction mixture containing 2μL of 10 × PCR buffer(with (NH₄)₂SO₄), 2.0mmol/L of Mg²⁺,0.2mmol/L of dNTPs, 1U Taq polymerase, 30-40ng of template DNA, 0.25μmol/L of RAPD random primers or 2.5ng/μL of SSR primers.The descended anneal temperature was used in the SSR PCR program toincrease the stability of amplification.4. Comparing with method of esterase isoenzyme, RAPD and SSR technique can be used to distinguish the hybrid Qinyou No.7 and its restorer K407 obviously but method of esterase isoenzyme can not. Comparing with RAPD marker, SSR marker has the characteristics of better stability and simpler pattern and easier distinguish. So it has wide foreground of application in identification of hybrid rapeseed seed purity.5. RAPD and SSR markers technique,which were based on screening suitable primers, can be used practically in identification of hybrid rapeseed seed purity by simplifying the method of DNA extraction, standardizing the test system and using repeatly agarose gels to cut cost and increase efficiency.