Detection Of Nematode Resistant Gene In Sugarcane By Using PCR

The nematode resistance of forty-three sugarcane cultivars (without conventional identification of nematode resistance) was tested in the experiment. Firstly, the sugarcane genomic DNA was extracted. Secondly, according to the sequence of root-knot nematode resistant gene in tomato and the sequence of nematode resistant gene in sugar beet, two forward primers and two reverse primers were produced separately by synthetic, 4 pairs of primers that were in .each kind were composed, and a pair of primers in each was selected among them by PCR. Thirdly, conditions for PCR in sugarcane were optimized, the results showed that a special fragment of about 460bp and a special fragment of about 690bp were appeared, and PCR-Southern blotting about them was made further in order to affirm the homogeneous sequences of nematode resistant gene. Finally , 43 sugarcane cultivars were primarily detected by PCR technique. The results were summarized as following :1. An optimal reaction system for PCR in sugarcane was established : in a 20ul reaction solution, the optimal composition included 10 mmol/L Tris-HCl(pH8.3), 50 mmol/L KC1,1.5 mmol/L MgCl2,100umol/L dNTP ,0.2umol/L primers ,40ng of template DNA and 1.0 U Tag DNA Polymerase.The reaction program was devised in to 3 processes: The first one is denaturation at 94℃ for 5 min. The second one is 35 cycles of denaturation at 94 ℃ for 40s, annealing at 60℃ for 50s and extension at 72 ℃ for 90 s. The last one is extension at 72℃ for 10 min.2. Among 43 sugarcane cultivars, 37 cultivars appeared a special fragment of about 630bp, and 6 cultivars had nothing by using root-knot nematode resistant primers. 39 cultivars appeared a special fragment of about 630bp, and 4 cultivars had nothing by using cytocyst nematode resistant primers.3. The two kinds of special PCR products were hybridized by PCR-Southern blotting. The blotting signals appeared, which proved that there was a great homogeneous sequence between 460bp special fragment and root-knot nematode resistant gene in tomato and there was a great homogeneous sequence between 690bp special fragment and nematode resistant gene in sweet potato. They were the basis for cloning nematode resistant gene in sugarcane. 4.The results will be tested by field identification in the further.