BioSample

This sequence was obtained by cDNA-AFLP where cDNAs were synthesized from reverse transcription of mRNA isolated from chickpea roots at different days after infection with Fusarium oxysporum f. sp. ciceri Race 1. Three cDNA libraries were made using Zap-cDNA synthesis kit Gigapack III cloning Kit from Stratagene, 11011 North Torrey Pines Road, La Jolla, CA 92037; 1) infected ressitant chickpea cultivar WR 315; 2) un-infected control of WR 315, 3) infected susceptable chickpea cultivar JG 62. cDNAs from the libraries were rescued by PCR amplification using flanking T3 and T7 promoter primers. Amplified cDNA was digested with EcoRI and MseI restriction endonucleases simultaneously. Subsequently,the cDNA fragments were ligated to EcoRI and MseI adapters to generate template DNA for amplification. These common adapter sequences flanking variable cDNA sequences serve as the primer binding sites on these restriction fragments. Small DNA fragments were generated, then amplified by PCR using different primer combinations including two selective bases in the presence of 33P for radioactive labeling of the amplified fragments. The PCR products from all the three cDNA libraries were run on a denaturing polyacrylamide gel. The up-regulated bands from resistant infected library were isolated from the gel, reamplified using the same primers and cloned in pGEMT easy vector (Promega) and sequenced in one direction using the T7 or SP6 primer.