Combination of SCAR primers and Touchdown-PCR for sex identification in Pistacia vera L.

A combined method of sequence characterized amplified regions (SCAR) primers and Touchdown-PCR was used for the development of a female DNA marker in Pistacia vera L. The random decamer primer OPO-08 amplified a 905-bp fragment in all female trees, but also in several males. SCAR primers designed on the basis of the RAPD female molecular marker amplified a 905-bp female and a 909-bp male fragment. Sequencing these fragments revealed high homology, with several point mutations, four deletions in female and one in the male sequences. A second internal set of SCAR primers designed on the basis of a polymorphic locus, in combination with Touchdown-PCR technique, amplified a specific female 297-bp product. The diagnostic reliability of the new female specific marker was verified on 54 different genotypes. The method reported here offers a simple and reproducible way for early gender determination in P. vera.