Cloning and Sequencing of psbA Gene from Phalaris minor Biotypes for Isoproturon Resistance

Phalaris minor is one of the most predominant and troublesome weed of wheat crop. Excessive use of isoproturon, an urea substituted herbicide in wheat fields has caused resistance in weed, which have become detrimental for the wheat crop. Metabolic detoxification and modification of herbicide binding site (D1 protein of chloroplast) have been predicted to be the major mechanism conferring resistance in P. minor against isoproturon. D1 protein is encoded by chloroplast genome (psbA gene) and Cytochrome P450 mediated detoxification of isoproturon is encoded by nuclear genes. In the present experiment, the psbA gene from 4 biotypes (resistant R, susceptible S, reciprocal crosses RxS, SxR) of P. minor was amplified using PCR and cloned in pGEM T easy vector. Reciprocal crosses were also made to see the inheritance pattern of the R/S biotypes. Sequence analysis of the fragment from R and S biotypes of P. minor exhibited a substitution from serine to glycine at position 136 of the D1 protein encoded by the psbA gene. However, the reciprocal crosses showed similar sequence to that of susceptible biotype. This mutation is not the same as reported earlier in the herbicide binding region of Photosystem II for other substituted ureas. The existence of isoproturon resistance biotypes of P. minor lacking mutation in the herbicide-binding region clearly indicates that psbA gene alone do not account for the resistant biotype but resistance to this herbicide can be attributed to some other mechanism.