Chromosome Location Of HbMyb1 Gene And Linkage Marker Of OPV-10₃₉₀ From Hevea Brasiliensis By In Situ PCR

The HbMybl Gene Associated with Tapping Panel Dryness(TPD) and OPV-10₃₉₀ linked mildew-resistance gene were physically located onto chromosomes in Hevea brasiliensis by in situ PCR.The results were as following:The method of direction in situ PCR on chromosomes of Hevea brasiliensis was developed successfully,and its main Procedure was as following:The component of direction in situ PCR reaction were including 1×buffer,4.5 mM MgCl₂,0.5 mg/ml BSA, 200μM dATP,dGTP and dCTP,72μM dTTP,4μM DIG-11-dUTP,5U Taq DNA Polymerase,2.4μM Primer,the total volume was 50μl;The in situ PCR amplification procedure was liked this:Predenaturalization with 95℃,then 20 cycles including denaturalization of 94℃for 1 rain,anneling at 53℃(by Tm of primers) for 1 min, extension at 72℃for 70 sec,the last step was extension at 72℃for 7min;After cycles was completed,the slides were washed in 0.1×PBS for 4 to 5 rain,and in 0.1×SSC/ (Tween20) for 2×4 to 5 min.One or two signals were detected on the different phases of the cells of Reyan 7-33-97 and RRIM600 leavies used the developed in situ PCR method.The signal sites of the HbMybl gene distributed on the Long arm of the fifth chromosomes on Reyan 7-33-97,the percent distances from centromere to detection site was 15.21.The signal sites of the OPV-10₃₉₀ on RRIM600 mainly distributed on the Long arm of the eighth chromosomes and the short arm of the eleventh,the percent distances from centromere to detection site were 16.99 and 49.58.Karyotypes on Reyan7-33-97and RRIM600 were analysed.The karyotype formula of Reyan 7-33-97 was 2n=36=34m(4sat)+2sm.The fifth chromosome was sub-median kinetochores,others were median kinetochores,the sixth chromosome and seventh had satellites,the absolute length of chromosomes was 3.14～6.36μm,the relative length was 3.67%～7.44%.the average arm ratio was 1.44.The karyotype belonged to 2B type. As to RRIM600,The karyotype formula of RRIM60 was 2n=36=28m(4sat)+8sm.The eleventh chromosome,twelfth,fourteenth and seventeenth were sub-median kinetochores, and others were median kinetochores,the sixth chromosome and seventh had satellites, the absolute length of chromosome was 3.92×8.47μm,and the relative length was 3.45%～7.47%,the average arm ratio was 1.60,The karyotype RRIM600 was also belong to 2B type.