title: Identification of sex-linked SNP markers using RAD sequencing suggests ZW/ZZ sex determination in Pistacia vera L.
abstract: BACKGROUND
  Pistachio (Pistacia vera L.) is a dioecious species that has a long juvenility period. Therefore, development of marker-assisted selection (MAS) techniques would greatly facilitate pistachio cultivar-breeding programs. The sex determination mechanism is presently unknown in pistachio. The generation of sex-linked markers is likely to reduce time, labor, and costs associated with breeding programs, and will help to clarify the sex determination system in pistachio.
  
  
  RESULTS
  Restriction site-associated DNA (RAD) markers were used to identify sex-linked markers and to elucidate the sex determination system in pistachio. Eight male and eight female F1 progenies from a Pistacia vera L. Siirt × Bağyolu cross, along with the parents, were subjected to RAD sequencing in two lanes of a Hi-Seq 2000 sequencing platform. This generated 449 million reads, comprising approximately 37.7 Gb of sequences. There were 33,757 polymorphic single nucleotide polymorphism (SNP) loci between the parents. Thirty-eight of these, from 28 RAD reads, were detected as putative sex-associated loci in pistachio. Validation was performed by SNaPshot analysis in 42 mature F1 progenies and in 124 cultivars and genotypes in a germplasm collection. Eight loci could distinguish sex with 100% accuracy in pistachio. To ascertain cost-effective application of markers in a breeding program, high-resolution melting (HRM) analysis was performed; four markers were found to perfectly separate sexes in pistachio. Because of the female heterogamety in all candidate SNP loci, we report for the first time that pistachio has a ZZ/ZW sex determination system. As the reported female-to-male segregation ratio is 1:1 in all known segregating populations and there is no previous report of super-female genotypes or female heteromorphic chromosomes in pistachio, it appears that the WW genotype is not viable.
  
  
  CONCLUSIONS
  Sex-linked SNP markers were identified and validated in a large germplasm and proved their suitability for MAS in pistachio. HRM analysis successfully validated the sex-linked markers for MAS. For the first time in dioecious pistachio, a female heterogamety ZW/ZZ sex determination system is suggested.
authors.affiliation: Department of Horticulture, Faculty of Agriculture, University of Çukurova, Adana, Turkey. skafkas@cu.edu.tr.
authors.forename: Salih
authors.initials: S
authors.lastname: Kafkas
authors.affiliation: Department of Horticulture, Faculty of Agriculture, University of Çukurova, Adana, Turkey. mortazakhodaei@yahoo.com.
authors.forename: Mortaza
authors.initials: M
authors.lastname: Khodaeiaminjan
authors.affiliation: Department of Horticulture, Faculty of Agriculture, University of Çukurova, Adana, Turkey. muratguney.dna@gmail.com.
authors.forename: Murat
authors.initials: M
authors.lastname: Güney
authors.affiliation: Department of Horticulture, Faculty of Agriculture, University of Çukurova, Adana, Turkey. ebru@cu.edu.tr.
authors.forename: Ebru
authors.initials: E
authors.lastname: Kafkas
mesh_terms: D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D017343:Genes, Plant; D056426:Genetic Loci; D005819:Genetic Markers; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D027927:Pistacia / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D019849:Sex Determination Processes / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D004247:DNA