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Coffee Leaf Rust(CLR)is a fungal disease caused by Hemileia vastatrix and is one the major disease of coffee.Resistant to CLR is conferred by SH3 a major dominant gene that has been introgressed from a wild coffee species Coffea liberica.The two DNA markers,BA-124-12K-f and Sat244,reported to be closely linked to SH3 gene of C.liberica,were introduced in this experiment for validated by the real-time PCR.The amplification protocol was defined ‘Tm calling’ application in realtime PCR was used for analysis.The amplified product of C.liberica by BA-124-12K-f revealed a sharp and specific peak with a melting temperature at 80.94°C while no product were amplified in C.canephora (Robusta)and C.arabica var.typica.Melting temperature of C.liberica by Sat244 that reported to indicate zygosity status revealed two peaks at 82.28 and 86.63°C while those of C.canephora (Robusta) were at different temperatures.Thus,this method provided a simple,fast,cost-effective and high-throughput protocol for screening of SH3 related Coffee Leaf Rust (CLR) resistant population.