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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.

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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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Differential hybridization screening. Differentially expressed clones in a 8-10 h AEL embryonic suppression subtraction hybridization (SSH) / mirror orientation selection (MOS) library. 410 randomly selected clones were spotted onto 5 nylon membranes in four replicates (A to E) and hybridized with labelled probe from: subtracted XX/XY vs XX-only SSH/MOS cDNAs (forward); subtracted XX-only vs XX/XY SSH/MOS cDNAs (reverse); unsubtracted XX/XY cDNAs (unsub test); unsubtracted XX-only cDNAs (unsub driv). Hybridization signals were scored visually. Red circles indicate the selected 26 clones.
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Figure 3: Differential hybridization screening. Differentially expressed clones in a 8-10 h AEL embryonic suppression subtraction hybridization (SSH) / mirror orientation selection (MOS) library. 410 randomly selected clones were spotted onto 5 nylon membranes in four replicates (A to E) and hybridized with labelled probe from: subtracted XX/XY vs XX-only SSH/MOS cDNAs (forward); subtracted XX-only vs XX/XY SSH/MOS cDNAs (reverse); unsubtracted XX/XY cDNAs (unsub test); unsubtracted XX-only cDNAs (unsub driv). Hybridization signals were scored visually. Red circles indicate the selected 26 clones.

Mentions: The clones that hybridize with the forward-subtracted SSH-MOS (XX/XY) and unsubtracted tester probes (XX/XY), but not with the reverse-subtracted SSH (XX minus XX/XY) or unsubtracted driver probes (XX), usually correspond to differentially expressed genes, namely real positive clones (see also the manual of the manufacturer; Methods). Furthermore, those clones from SSH-MOS having no detectable hybridization signals with any probe could still represent differentially expressed transcripts as well as false positives but having a very low abundance. Finally, those clones hybridizing equally with both subtracted probes and unsubtracted probes, are usually the most highly expressed clones, including either real positive or false positive clones. The results of the differential screening are shown in Figure 3 and hybridization signals were scored visually. In our experiment, from 410 clones, 25 of them were identified as hybridizing with the forward-subtracted SSH-MOS tester probe (XX/XY minus XX), but not or only weakly hybridizing with the reverse-subtracted SSH probe. An additional positive clone was identified as hybridizing with unsubtracted tester probe (XX/XY). The 26 clones either failed to hybridize or weakly hybridized with the reverse-subtracted (XX minus XX/XY) and driver (XX) probes. The 26 cDNA sequences when aligned via Macaw software to search for identity/similarity revealed that AE5 and BE6 clones are identical in sequence and length respectively to DB4 and BE8 clones. The 24 different cDNA clones (ranging approximately from 300 to 900 bp) were analyzed by BLASTX revealing that most of them (18 cDNAs) encode for putative ORFs with significant homology with known dipteran proteins (Additional file 3 - Table S1). 6 cDNA clones seem to encode either unknown or weakly conserved proteins or they could correspond to untranslated regions of transcripts.


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)

Differential hybridization screening. Differentially expressed clones in a 8-10 h AEL embryonic suppression subtraction hybridization (SSH) / mirror orientation selection (MOS) library. 410 randomly selected clones were spotted onto 5 nylon membranes in four replicates (A to E) and hybridized with labelled probe from: subtracted XX/XY vs XX-only SSH/MOS cDNAs (forward); subtracted XX-only vs XX/XY SSH/MOS cDNAs (reverse); unsubtracted XX/XY cDNAs (unsub test); unsubtracted XX-only cDNAs (unsub driv). Hybridization signals were scored visually. Red circles indicate the selected 26 clones.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Differential hybridization screening. Differentially expressed clones in a 8-10 h AEL embryonic suppression subtraction hybridization (SSH) / mirror orientation selection (MOS) library. 410 randomly selected clones were spotted onto 5 nylon membranes in four replicates (A to E) and hybridized with labelled probe from: subtracted XX/XY vs XX-only SSH/MOS cDNAs (forward); subtracted XX-only vs XX/XY SSH/MOS cDNAs (reverse); unsubtracted XX/XY cDNAs (unsub test); unsubtracted XX-only cDNAs (unsub driv). Hybridization signals were scored visually. Red circles indicate the selected 26 clones.
Mentions: The clones that hybridize with the forward-subtracted SSH-MOS (XX/XY) and unsubtracted tester probes (XX/XY), but not with the reverse-subtracted SSH (XX minus XX/XY) or unsubtracted driver probes (XX), usually correspond to differentially expressed genes, namely real positive clones (see also the manual of the manufacturer; Methods). Furthermore, those clones from SSH-MOS having no detectable hybridization signals with any probe could still represent differentially expressed transcripts as well as false positives but having a very low abundance. Finally, those clones hybridizing equally with both subtracted probes and unsubtracted probes, are usually the most highly expressed clones, including either real positive or false positive clones. The results of the differential screening are shown in Figure 3 and hybridization signals were scored visually. In our experiment, from 410 clones, 25 of them were identified as hybridizing with the forward-subtracted SSH-MOS tester probe (XX/XY minus XX), but not or only weakly hybridizing with the reverse-subtracted SSH probe. An additional positive clone was identified as hybridizing with unsubtracted tester probe (XX/XY). The 26 clones either failed to hybridize or weakly hybridized with the reverse-subtracted (XX minus XX/XY) and driver (XX) probes. The 26 cDNA sequences when aligned via Macaw software to search for identity/similarity revealed that AE5 and BE6 clones are identical in sequence and length respectively to DB4 and BE8 clones. The 24 different cDNA clones (ranging approximately from 300 to 900 bp) were analyzed by BLASTX revealing that most of them (18 cDNAs) encode for putative ORFs with significant homology with known dipteran proteins (Additional file 3 - Table S1). 6 cDNA clones seem to encode either unknown or weakly conserved proteins or they could correspond to untranslated regions of transcripts.

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