Barcoding assessment of the Afghan citrus population

The identification and characterization of the local population of fruit tree could be of basic importance for developing countries. Usually, identification of the cultivated species relies on the use of phenotypic descriptors. A more reliable system to identify species is based on the genetic analysis and particularly on the barcoding procedure which consist in the comparison of the sequences of highly conserved fragments located in the mithocondrial or plastid DNA. Afghan citrus seeds have been collected in three areas where citrus is intensively cultivated. To verify that samples belong to a particular species, we have analyzed also samples of Citrus aurantium, C. sinensis and many other Citrus whose origin is certain as control. Total DNA has been extracted from the selected plants. The universal primers matK, Rbcl, ITS1, ITS2 and psbA have been used for PCR amplification; although they are expected to work with all higher plants, sometimes mutations arise in the sequence compromising the primer annealing during PCR; the primers have therefore been tested. The primers constructed on the sequence of the ITS1 and ITS2 resulted in a faint amplification, while primers designed on the sequence of matK and Rbcl genes have given very good amplification which have been sequenced. Unfortunately such sequences did not show any discriminatory ability being completely overlapping for most analyzed samples. Conversely, the primers of psbA gene showed either good amplification and sequencing results either a good discriminatory capacity. After amplification and sequencing PCR fragments of the psbA gene we proceed to perform a phylogenetic analysis.According to the results of our analysis we can state with reasonable certainty that the Afghan citrus samples belong to the C. aurantium species. This result, if confirmed can be helpful for devising a strategy for the improvement of the local citrus production and for a stepwise introduction of foreign citrus germplasm.