Conversion of RAPD markers linked to cold tolerance to scar markers in common carp

RAPD-PCR on DNA samples were performed with 300 random primers in Heilongjiang wild common carp(Cyprinus carpio)(cold tolerance),cold-resistance strain of red purse carp(C.carpio var.)(cold tolerance),red purse carp(cold sensitive),Boshi carp(C.pellegini pellegini)(cold sensitive),and cross F2 of cold-resistance strain of red purse(♂) and Boshi carp(♀).Both AL04986 and AR02788 were identified to be molecular markers which were associated with cold tolerance traits of common carp.Also,specific fragments were extracted and purified from four primers above.The purified products were then inserted into pMD18-T vector which were transformed to competent Escherichia coli DH 5αfor sequencing.According to the sequences,three pairs of specific primers,SCAL04L/SCAL04R1,SCAL04L/SCAL04R2 and SCAR02L/SCAR02R,were designed,in which SCAR02L/SCAR02R was used for inversion of the difference of cold tolerance in cross F2 of cold-resistance strain of red purse and Boshi carp,and its optimal annealing temperature was 57 ℃.The PCR analysis showed that SCAR markers were associated with cold-resistance traits,which could be used for the identification of hybrid to cold tolerance traits of common carp.Unfortunately the conversed SCAR markers from AL04986 could not reproduce the polymorphic AL04986.The reasons for that were also discussed.