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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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Molecular analysis of the BA11 clone. A) Schematic representation of alignments between the BA11 clone and the three corresponding Medfly ESTs. Percentages of nucleotide identities are reported. Red arrows indicate the position of the primers utilized. B) Genomic PCR and RT-PCR product analyses on adult male and female samples with Cc114+/BA11- primers pair. C) Genomic PCR and RT-PCR analyses on adult male and female samples with CcBA11+/CcBA11- primers pair. D) Genomic PCR and RT-PCR analyses on adult male and female samples with Cc483+/483-1- primer pair. E) Schematic representation of alignments between the PCR fragments amplified with Cc483+/CcBA11- primer pair. In light grey alignments between CcGen1 and CcGen2 clones and in dark grey alignments between CcGen2 and CcDmale clones. Percentages of nucleotide identities are also reported. Red arrows indicate the position of the primers utilized. F) Genomic PCR and RT-PCR analyses on adult male and female samples with Cc483+/CcBA11- primer pair, which led to identify a Y-linked paralog.
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Figure 4: Molecular analysis of the BA11 clone. A) Schematic representation of alignments between the BA11 clone and the three corresponding Medfly ESTs. Percentages of nucleotide identities are reported. Red arrows indicate the position of the primers utilized. B) Genomic PCR and RT-PCR product analyses on adult male and female samples with Cc114+/BA11- primers pair. C) Genomic PCR and RT-PCR analyses on adult male and female samples with CcBA11+/CcBA11- primers pair. D) Genomic PCR and RT-PCR analyses on adult male and female samples with Cc483+/483-1- primer pair. E) Schematic representation of alignments between the PCR fragments amplified with Cc483+/CcBA11- primer pair. In light grey alignments between CcGen1 and CcGen2 clones and in dark grey alignments between CcGen2 and CcDmale clones. Percentages of nucleotide identities are also reported. Red arrows indicate the position of the primers utilized. F) Genomic PCR and RT-PCR analyses on adult male and female samples with Cc483+/CcBA11- primer pair, which led to identify a Y-linked paralog.

Mentions: We used BA11 as an in silico probe for BLASTN analyses at NCBI of the C. capitata Expressed Sequence Tags (ESTs) database and identified three partially overlapping ESTs (named EST114, EST796 and EST483, ranging from 800 to 700 bp long) which showed respectively identities of 99% over 461 nt, 99% over 378 nt and 95% over 72 bp (see Figure 4A). EST114 and EST796 are derived respectively from embryos and from adult head [41]; they have only four nt differences over 752 bp and correspond most likely to transcripts derived from the same gene (with either SNPs or few DNA sequencing errors).


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)

Molecular analysis of the BA11 clone. A) Schematic representation of alignments between the BA11 clone and the three corresponding Medfly ESTs. Percentages of nucleotide identities are reported. Red arrows indicate the position of the primers utilized. B) Genomic PCR and RT-PCR product analyses on adult male and female samples with Cc114+/BA11- primers pair. C) Genomic PCR and RT-PCR analyses on adult male and female samples with CcBA11+/CcBA11- primers pair. D) Genomic PCR and RT-PCR analyses on adult male and female samples with Cc483+/483-1- primer pair. E) Schematic representation of alignments between the PCR fragments amplified with Cc483+/CcBA11- primer pair. In light grey alignments between CcGen1 and CcGen2 clones and in dark grey alignments between CcGen2 and CcDmale clones. Percentages of nucleotide identities are also reported. Red arrows indicate the position of the primers utilized. F) Genomic PCR and RT-PCR analyses on adult male and female samples with Cc483+/CcBA11- primer pair, which led to identify a Y-linked paralog.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Molecular analysis of the BA11 clone. A) Schematic representation of alignments between the BA11 clone and the three corresponding Medfly ESTs. Percentages of nucleotide identities are reported. Red arrows indicate the position of the primers utilized. B) Genomic PCR and RT-PCR product analyses on adult male and female samples with Cc114+/BA11- primers pair. C) Genomic PCR and RT-PCR analyses on adult male and female samples with CcBA11+/CcBA11- primers pair. D) Genomic PCR and RT-PCR analyses on adult male and female samples with Cc483+/483-1- primer pair. E) Schematic representation of alignments between the PCR fragments amplified with Cc483+/CcBA11- primer pair. In light grey alignments between CcGen1 and CcGen2 clones and in dark grey alignments between CcGen2 and CcDmale clones. Percentages of nucleotide identities are also reported. Red arrows indicate the position of the primers utilized. F) Genomic PCR and RT-PCR analyses on adult male and female samples with Cc483+/CcBA11- primer pair, which led to identify a Y-linked paralog.
Mentions: We used BA11 as an in silico probe for BLASTN analyses at NCBI of the C. capitata Expressed Sequence Tags (ESTs) database and identified three partially overlapping ESTs (named EST114, EST796 and EST483, ranging from 800 to 700 bp long) which showed respectively identities of 99% over 461 nt, 99% over 378 nt and 95% over 72 bp (see Figure 4A). EST114 and EST796 are derived respectively from embryos and from adult head [41]; they have only four nt differences over 752 bp and correspond most likely to transcripts derived from the same gene (with either SNPs or few DNA sequencing errors).

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