Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes

BMC Genetics

Research

Valeria Petrella¹  Marco Salvemini¹  Ying Zhang¹  Giuseppe Saccone¹  Rocco D'Amato¹  Domenica Ippolito¹  Giuseppe Ventre² 

[1] Department of Biology, University of Naples Federico II, 80134, Naples, Italy;Department of Biology, University of Naples Federico II, 80134, Naples, Italy;Laboratorio Analisi Cliniche "La Salute", 84086, Roccapiemonte, Salerno, Italy;

关键词: male-specific;    Y chromosome;    sexing;    embryonic;    Suppression Subtractive Hybrydization;    Mirror Orientation Selection;    Differential Screening Hybridization;    SIT;   

DOI  :  10.1186/1471-2156-15-S2-S5

来源: Springer

【 摘 要 】

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) and differential 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.

【 授权许可】

CC BY   
© Salvemini et al.; licensee BioMed Central Ltd. 2014