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

Salvemini M, D'Amato R, Petrella V, Ippolito D, Ventre G, Zhang Y, Saccone G - BMC Genet. (2014)

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

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.

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Analysis of SSH subtraction efficiency. Gel electrophoresis (upper rpP1, lower rpS1), showing expected PCR products after 18, 23, 28, and 33 cycles of PCR amplification. The quantity of transcripts from the constitutively expressed Ceratitis rpP1 and rpS21 gene was compared between subtracted (S) and unsubtracted (U) cDNA samples. As expected, there is a reduction in the subtracted cDNAs, which is more pronounced for rpS21.
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Figure 1: Analysis of SSH subtraction efficiency. Gel electrophoresis (upper rpP1, lower rpS1), showing expected PCR products after 18, 23, 28, and 33 cycles of PCR amplification. The quantity of transcripts from the constitutively expressed Ceratitis rpP1 and rpS21 gene was compared between subtracted (S) and unsubtracted (U) cDNA samples. As expected, there is a reduction in the subtracted cDNAs, which is more pronounced for rpS21.

Mentions: We applied Suppression Subtractive Hybridization (SSH), following the manufacturer's instructions, and produced a forward subtracted library potentially enriched in male-specific/male-biased mRNAs (tester minus driver: XX/XY mixed embryonic cDNAs subtracted with XX-only embryonic cDNAs). We also obtained as a control a reverse subtracted library (driver minus tester: XX-only cDNAs subtracted with the XY/XX cDNAs). We partially validated that the subtraction had indeed taken place by comparing the reduction of constitutive (common) transcripts from two C. capitata housekeeping genes, rpP1 [37] and rpS21 [38,39] by semi-quantitative PCR [40]. rpP1 cDNA fragments were detected in the non-subtracted cDNA after 18 PCR cycles of amplification and in the subtracted after 23 cycles (Figure 1). Similarly, rpS21 cDNA fragments were detected after 28 cycles in the non-subtracted cDNA while they were completely absent in the subtracted cDNAs sample (Figure 1). This marked reduction of both housekeeping mRNAs in the SSH subtracted cDNA suggested that the SSH has possibly reduced the relative quantity of specific group of transcripts and hopefully also the complexity of the cDNA population in the forward library.


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

Salvemini M, D'Amato R, Petrella V, Ippolito D, Ventre G, Zhang Y, Saccone G - BMC Genet. (2014)

Analysis of SSH subtraction efficiency. Gel electrophoresis (upper rpP1, lower rpS1), showing expected PCR products after 18, 23, 28, and 33 cycles of PCR amplification. The quantity of transcripts from the constitutively expressed Ceratitis rpP1 and rpS21 gene was compared between subtracted (S) and unsubtracted (U) cDNA samples. As expected, there is a reduction in the subtracted cDNAs, which is more pronounced for rpS21.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255797&req=5

Figure 1: Analysis of SSH subtraction efficiency. Gel electrophoresis (upper rpP1, lower rpS1), showing expected PCR products after 18, 23, 28, and 33 cycles of PCR amplification. The quantity of transcripts from the constitutively expressed Ceratitis rpP1 and rpS21 gene was compared between subtracted (S) and unsubtracted (U) cDNA samples. As expected, there is a reduction in the subtracted cDNAs, which is more pronounced for rpS21.
Mentions: We applied Suppression Subtractive Hybridization (SSH), following the manufacturer's instructions, and produced a forward subtracted library potentially enriched in male-specific/male-biased mRNAs (tester minus driver: XX/XY mixed embryonic cDNAs subtracted with XX-only embryonic cDNAs). We also obtained as a control a reverse subtracted library (driver minus tester: XX-only cDNAs subtracted with the XY/XX cDNAs). We partially validated that the subtraction had indeed taken place by comparing the reduction of constitutive (common) transcripts from two C. capitata housekeeping genes, rpP1 [37] and rpS21 [38,39] by semi-quantitative PCR [40]. rpP1 cDNA fragments were detected in the non-subtracted cDNA after 18 PCR cycles of amplification and in the subtracted after 23 cycles (Figure 1). Similarly, rpS21 cDNA fragments were detected after 28 cycles in the non-subtracted cDNA while they were completely absent in the subtracted cDNAs sample (Figure 1). This marked reduction of both housekeeping mRNAs in the SSH subtracted cDNA suggested that the SSH has possibly reduced the relative quantity of specific group of transcripts and hopefully also the complexity of the cDNA population in the forward library.

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

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.

Show MeSH
Related in: MedlinePlus