Development of microsatellite markers for Jatropha curcas L.

The objective of this research is to develop microsatellite DNA markers for classification and species specific identification of physic nut ( Jatropha curcas L.). DNAs from physic nut (SS20-Sbr3, USA, Nakhon Ratchasima) were digested with Mse I restriction enzyme, then ligated to adapters. The products were PCR amplified before hybridization to biotin-oligonucleotide probes of B-(GA) 15 , B-(CA) 15 , B-(ACC) 10 and B-(CCT) 10 containing magnetic beads. The DNA fragments were then PCR amplified again before ligation to plasmid vector. The recombinant plasmids were heat-shock transformed to supercompetent E. coli JM109 cells. A total of 996 white colonies were selected and 906 clones (90.96 %) were confirmed to carry DNA inserts by PCR amplification using primers specific to the plasmid. Dot blot hybridization was performed to reconfirm the microsatellite sequences in the inserts. Ninety-seven clones were selected and sequenced. A total of 51 clones (52.58 %) were found to contain microsatellite sequences. Most abundant iterated sequences were di-nucleotide repeat of (GA) n (31.37 %) and tri-nucleotide repeat of (GGA) n (23.53 %). Twenty six primer pairs were designed and four pairs could amplify the DNA, giving the expected PCR product with polymorphism among toxic and non-toxic physic nuts.