The first sex determination study on Pistacia with molecular markers was done by Hormaza . One thousand Random Amplified Polymorphic DNA (RAPD) markers had used for discrimination in that study. Bulk P. vera L. male and female samples had amplified with 700 decamer primers and as a result OPO08945 (OPO-08) had defined as a sex marker. 945 bp size band was observed on female bulk sample and this band was absent on male samples. Yakubov et al. reported to verify the primer and develop OPO-08 RAPD primer with touchdown PCR and they determined PVF1-2 Sequence Characterized Amplified Region (SCAR) primers based on OPO-08. In that study 4-Point Base Deletion (PBD) for female samples and 1 PBD for males were defined as a mutation (again with P. vera) . In other completed gender discrimination study was associated RAPD markers in wild Pistacia which conducted by Kafkas et al. . BC156 (1300bp) & BC360 (500bp) markers for P. eurocarpa and OPAK-09 (850bp) & BC346 (700bp) markers for P. atlantica was indicated as a marker. In the articles published by Esfendiyari et al. [46, 47], the first twenty 10-mer RAPD primers than thirty different 10-mer RAPD primer were tried on 3 different species; P. atlantica, Desf subsp. mutica, P. khinjuk and P. vera. As a result, they reported that BC1200 primer is a discriminator marker and designed the primers PVF1 (forward) and PVF2 (reverse) from the PCR amplified region sequence (convert RAPD to SCAR). The SCAR primers which has 300 bp amplifying region, while generate a band in all female samples, there wasn't any band in male samples.
When studies are examined, it is seen that many of them are based on P. vera genus [48, 49, 43, 50, 44] due to the world wide trade income. On this basis, in 2008, P. vera L. cultivars were generated with Inter Simple Sequence Repeat (ISSR) primers and 2 of them were able to distinguish genders. These markers are (AC) 8GC and (AC) 8TA 10-mer repeats and band sizes are 2400 . Following year the same research group was experienced 32 arbitrary primers for RAPD analysis and mentioned FPK 106 and FPK 105 but there was no data related to primer sequence. Vedramin et al. carried out the SSR study covering 4 species . Their analysis contains 50 P. vera, 4 P. integerrima, 12 P. mutica and 16 P. terebinthus for a total of 82 accessions but the tested markers were insufficient for gender base discrimination.
Up to the present, the most reliable study about sex linked marker on Pistacia genus was published by Kafkas et al. . In that study 38 putative sex-associated SNP markers were identified as heterozygous in female individuals and homozygous in male individuals using RAD sEq. suggests a ZW/ZZ sex determination system in P. vera.
As a result, some of the 8 species in the genus (P. vera, P atlantica, P integerrima, P. terebinthus, P. mutica, P. khinjuk) were studied separately before, while about the remaining part of the genus (P. palaestina, P. lentiscus) there is no gender discrimination study. However, it has not been previously collected in a single study, which indicates the comprehensiveness of our study.
There are sex discrimination marker studies already done for the Pistacia species and different molecular marker methods have been used in these studies like RAPD  or ISSR . When the previous studies were compared with the results of KASP, there are extra steps in previous techniques like preparing gel, loading samples or screening the gel. These extra steps can cause disadvantageous in terms of time, effort and cost. While each extra step increases the risk of making mistakes, KASP is very suitable for scale-up as it does not contain any of these steps.
It’s obviously known that, this new and popular technique KASP assay has a wide range of usage on different species from crop improvements; maize , wheat , tomato  to detection of disease in human . Nonetheless, sex discrimination studies using KASP technique is not seen except for a few fish like salmon  or halibut  and one insect species . These results show that, this study is a not just first for Pistacia in terms of gender discrimination but also the precursor study for sex discrimination by KASP in plants.
In our study, 12 SNP regions were screened for 8 species and 5 SNP (SNP-PIS-133396, SNP-PIS-167992, P-ATL-91951-565, P-INT-91951-256, P-KHI-91951-115) showed clear allelic discrimination between sexes in species. As a result of this allelic distinction, 5 KASP primers identified as a sex-linked markers for 7 of 8 species. In the sex discrimination marker study conducted by Kafkas et al.  for P. vera species, 8 of 13 SNP had differentiated between P. vera genders. When these markers were tested with some other species (P. atlantica, P. terebinthus, P. eurycarpa, P. integerrima) in the genus, a distinction could not be observed in these wild types.
In terms of allelic frequency, the best marker assays were SNP-PIS-167992 and P-ATL-91951-565 (Fig. 4a, c). In allelic graphs of these markers, frequency differences are apparent for all individuals whose distinction was observed, and the distinction is exact in terms of homozygosis and heterozygosis (Fig. 4). At the same time, they can be considered more comprehensive because it differentiates several species at once for whole genus (Fig. 4a, b, c). Each SNP markers show discrimination for 3 Pistacia species; SNP-PIS-167992 for P. atlantica, P. terebinthus, P. vera (Fig. 4a); P-KHIN-91951-115 for P. atlantica, P. khinjuck, P. lentiscus (Fig. 4b); P-ATL-91951-565 for P. atlantica, P. khinjuck, P. mutica (Fig. 4c).
When all results are evaluated, the less discriminative result of allelic signals between genders showed itself in separation of P. lentiscus species with P-KHIN-91951-115 assay. While allele 2 (HEX) radiations were similar in this result and indicative dots seems close to each other, allele 1 (FAM) had more than 3 times stronger signaling difference in the female individuals. Some of the assays were irradiated after reaction but they did not display a difference on allelic discrimination plot. In one marker (P-VERA-10733-132), no signal was received, which is actually in fact an indication of the KASP system accuracy and the specificity of primer sequences.
The assay P-INT-91951-120 were discriminated only P. integerrima species apart from genus and actually it was not surprising, because the assay was designed special and in the direction of P. integerrima sequence. When we assess the SNP-PIS-167992 assay result, while the Kafkas et al.  research discriminate only P. vera, our assay discriminated 2 more species which are P. atlantica and P. terebinthus in addition.