Selecttion Of RAPD Marker And Specitic Fragment Clone For Cold Tolerance In Tomato(Lycopersicon Esculentum Mill.)

Tomato near isogenic lines of cold tolerance were used as experiment materials, 10 each of the cold tolerant and cold sensitive plants were selected for DNA extraction to develop tolerant and sensitive gene pools respectively. By the BSA,NILs and RAPD technique, 520 random decamer primers were used to amplify the two types of gene pools, 3 polymorphic specific fragments were detected and found from three primers (OPF-14,OPD-11,OPI-01).Using these primers to amplify the parents, NILs populations, the specific fragments were RAPD markers linked to cold tolerance of tomato. By the software Mapmaker/3.0, the OPF-14,OPD-11,OPI-01 markers were tested to be linked to the cold tolerance with genetic distance of 6.1 cM,9.8 cM ,12.3 cM respectively. Fragment OPF-14-800 was extracted from the agarose and mixed with pGEMR-T-Easy vector,then transferred into DH5-α. Through white-blue colonies screening ,white clones were picked out from LB/Amp/X-gal/IPTG plate,then the recombined vector was extracted and amplified with primers SP6 and T7. Finally,detected by electrophoring in agarose, the results confirmed that the OPF-14-800 has been cloned successfully. The clone was determined 792 bp after being sequenced. On internet,DNA Sequence of OPF-14 is homology to Pto gene.This research provides a base for transform to SCAR marker and further study on marker-assisted-selection for cold tolerance breeding in tomato.