Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis

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Abstract
The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in the dioecious species, Pistacia vera. Progenies from two female parents pollinated by a common male parent were studied. Two bulks of DNA were made in each cross, one from males and one from females, by pooling an equal weight of fresh leaves from each individual contributing to the bulk prior to DNA extraction. DNA was extracted from each bulked sample and from each of the contributing individuals. DNA was also extracted from 14 cultivars of P. vera and from 94 open-pollinated, fewweeks-old P. vera seedlings of unknown sex. Seven hundred different decamer oligonucleotide primers were used to perform DNA amplification, with 1 of these (OPO08) producing a 945 bp amplification band that was present only in the bulked female samples and absent in the bulked male samples of the two crosses. The relationship between band presence and female sex expression was conserved in every individual obtained from the two crosses and in the 14 cultivars unrelated to the crosses. We propose that this band is tightly linked to the gene(s) that control sex determination in pistachio. The OPO08945 RAPD marker could be used in a breeding program to screen the gender of pistachio plants long before they reach reproductive maturity, resulting in considerable savings of time and economic resources. In order to verify that assumption we screened 94 additional seedlings with the OPO08 primer and obtained results consistent with a 1∶1 male:female ratio.