title: Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes.
abstract: The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal. We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) anddifferential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome. We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.
authors.forename: Marco
authors.initials: M
authors.lastname: Salvemini
authors.forename: Rocco
authors.initials: R
authors.lastname: D'Amato
authors.forename: Valeria
authors.initials: V
authors.lastname: Petrella
authors.forename: Domenica
authors.initials: D
authors.lastname: Ippolito
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ventre
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Saccone
mesh_terms: D000818:Animals; D033622:Ceratitis capitata / Q000235:genetics*; D004625:Embryo, Nonmammalian; D005260:Female; D005786:Gene Expression Regulation; D015723:Gene Library; D017344:Genes, Insect; D008297:Male; D009693:Nucleic Acid Hybridization; D012737:Sex Factors; D014158:Transcription, Genetic