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Comprehensive transcriptome analysis of early male and female Bactrocera jarvisi embryos.

Morrow JL, Riegler M, Gilchrist AS, Shearman DC, Frommer M - BMC Genet. (2014)

Bottom Line: No strong candidates for transcripts derived solely from the Y chromosome were recovered from the poly(A+) fraction.Bactrocera jarvisi provides an excellent model for embryonic studies due to available Y-chromosome markers and the compact time frame for zygotic transcription and the sex-determined state.Our data contribute fundamental information to sex-determination research, and provide candidates for the sourcing of gene promoters for transgenic pest-management strategies of tephritid fruit flies.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Developing embryos are provided with maternal RNA transcripts and proteins, but transcription from the zygotic nuclei must be activated to control continuing embryonic development. Transcripts are generated at different stages of early development, and those involved in sex determination and cellularisation are some of the earliest to be activated. The male sex in tephritid fruit flies is determined by the presence of a Y chromosome, and it is believed that a transcript from the Y-chromosome sets in motion a cascade that determines male development, as part of the greater maternal to zygotic transition (MTZ). Here we investigate the poly(A+) transcriptome in early male and female embryos of the horticultural pest Bactrocera jarvisi (Diptera: Tephritidae).

Results: Bactrocera jarvisi embryos were collected over two pre-blastoderm time periods, 2-3h and 3-5h after egg laying. Embryos were individually sexed using a Y-chromosome marker, allowing the sex-specific poly(A+) transcriptome of single-sex embryo pools to be deep-sequenced and assembled de novo. Transcripts for sixteen sex-determination and two cellularisation gene homologues of Drosophila melanogaster (Diptera: Drosophilidae) were identified in early embryos of B. jarvisi, including transcripts highly upregulated prior to cellularisation. No strong candidates for transcripts derived solely from the Y chromosome were recovered from the poly(A+) fraction.

Conclusions: Bactrocera jarvisi provides an excellent model for embryonic studies due to available Y-chromosome markers and the compact time frame for zygotic transcription and the sex-determined state. Our data contribute fundamental information to sex-determination research, and provide candidates for the sourcing of gene promoters for transgenic pest-management strategies of tephritid fruit flies.

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Principal co-ordinates analysis of the eight transcriptome samples mapped to the CLC assembly. Samples are male 3-5h (blue), female 3-5h (green), male 2-3h (red) and female 2-3h (yellow).
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Figure 2: Principal co-ordinates analysis of the eight transcriptome samples mapped to the CLC assembly. Samples are male 3-5h (blue), female 3-5h (green), male 2-3h (red) and female 2-3h (yellow).

Mentions: Within the RNA-Seq module of CLC Genomics, each trimmed library was mapped to the CLC assembly and levels of expression were recorded as normalised RPKM values. Validation of the assembly and mapping was performed. Principal components analysis of the eight samples showed samples clustering according to age, except for two samples, BJ1[male 3-5h] and BJ5[male 2-3h], which clustered together and not with their replicates (Figure 2). To investigate this discrepancy, the comparative expression levels over the two time points for the genes Sxl, tra, tra-2, dsx and slam [24] served as controls to validate the RPKM values. As there is no difference between male and female embryos in expression pattern for Sxl, tra-2 and female-specific tra and dsx over the first 5h, differential expression across the two developmental periods, irrespective of sex, was examined. However, the results for Sxl (maternal and zygotic expression) and slam (zygotic only) were most useful because both exhibit an increase in transcript abundance over this time course [24]. Sxl RPKM values indicated that BJ1[male 3-5h] had low expression levels equivalent to the 2-3h samples, rather than the higher levels of the other 3-5h samples. The RPKM values for slam, a zygotically transcribed gene validated by RT-PCR, were low in BJ1[male 3-5h] (36 RPKM) compared to the other three 3-5h samples (299-461 RPKM), and were very low in three 2-3h samples (0.25-2.02 RPKM) with the fourth 2-3h sample, BJ8[female 2-3h] showing a somewhat higher level (13 RPKM). The difference in RPKM values over time periods was not significant for slam, unless BJ1[male 3-5h] alone or BJ1[male 3-5h] and BJ5[male 2-3h] were excluded (FDR < 0.001). A similar phenomenon was also observed for sisA, run, gro, female lethal d (fl(2)d), emc and dpn (see sex-determination gene analysis, below), where each gene exhibited increased transcription in the other three 3-5h embryos (and significant up-regulation following exclusion of BJ1[male 3-5h] and BJ5[male 2-3h]; FDR<0.001).


Comprehensive transcriptome analysis of early male and female Bactrocera jarvisi embryos.

Morrow JL, Riegler M, Gilchrist AS, Shearman DC, Frommer M - BMC Genet. (2014)

Principal co-ordinates analysis of the eight transcriptome samples mapped to the CLC assembly. Samples are male 3-5h (blue), female 3-5h (green), male 2-3h (red) and female 2-3h (yellow).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255828&req=5

Figure 2: Principal co-ordinates analysis of the eight transcriptome samples mapped to the CLC assembly. Samples are male 3-5h (blue), female 3-5h (green), male 2-3h (red) and female 2-3h (yellow).
Mentions: Within the RNA-Seq module of CLC Genomics, each trimmed library was mapped to the CLC assembly and levels of expression were recorded as normalised RPKM values. Validation of the assembly and mapping was performed. Principal components analysis of the eight samples showed samples clustering according to age, except for two samples, BJ1[male 3-5h] and BJ5[male 2-3h], which clustered together and not with their replicates (Figure 2). To investigate this discrepancy, the comparative expression levels over the two time points for the genes Sxl, tra, tra-2, dsx and slam [24] served as controls to validate the RPKM values. As there is no difference between male and female embryos in expression pattern for Sxl, tra-2 and female-specific tra and dsx over the first 5h, differential expression across the two developmental periods, irrespective of sex, was examined. However, the results for Sxl (maternal and zygotic expression) and slam (zygotic only) were most useful because both exhibit an increase in transcript abundance over this time course [24]. Sxl RPKM values indicated that BJ1[male 3-5h] had low expression levels equivalent to the 2-3h samples, rather than the higher levels of the other 3-5h samples. The RPKM values for slam, a zygotically transcribed gene validated by RT-PCR, were low in BJ1[male 3-5h] (36 RPKM) compared to the other three 3-5h samples (299-461 RPKM), and were very low in three 2-3h samples (0.25-2.02 RPKM) with the fourth 2-3h sample, BJ8[female 2-3h] showing a somewhat higher level (13 RPKM). The difference in RPKM values over time periods was not significant for slam, unless BJ1[male 3-5h] alone or BJ1[male 3-5h] and BJ5[male 2-3h] were excluded (FDR < 0.001). A similar phenomenon was also observed for sisA, run, gro, female lethal d (fl(2)d), emc and dpn (see sex-determination gene analysis, below), where each gene exhibited increased transcription in the other three 3-5h embryos (and significant up-regulation following exclusion of BJ1[male 3-5h] and BJ5[male 2-3h]; FDR<0.001).

Bottom Line: No strong candidates for transcripts derived solely from the Y chromosome were recovered from the poly(A+) fraction.Bactrocera jarvisi provides an excellent model for embryonic studies due to available Y-chromosome markers and the compact time frame for zygotic transcription and the sex-determined state.Our data contribute fundamental information to sex-determination research, and provide candidates for the sourcing of gene promoters for transgenic pest-management strategies of tephritid fruit flies.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Developing embryos are provided with maternal RNA transcripts and proteins, but transcription from the zygotic nuclei must be activated to control continuing embryonic development. Transcripts are generated at different stages of early development, and those involved in sex determination and cellularisation are some of the earliest to be activated. The male sex in tephritid fruit flies is determined by the presence of a Y chromosome, and it is believed that a transcript from the Y-chromosome sets in motion a cascade that determines male development, as part of the greater maternal to zygotic transition (MTZ). Here we investigate the poly(A+) transcriptome in early male and female embryos of the horticultural pest Bactrocera jarvisi (Diptera: Tephritidae).

Results: Bactrocera jarvisi embryos were collected over two pre-blastoderm time periods, 2-3h and 3-5h after egg laying. Embryos were individually sexed using a Y-chromosome marker, allowing the sex-specific poly(A+) transcriptome of single-sex embryo pools to be deep-sequenced and assembled de novo. Transcripts for sixteen sex-determination and two cellularisation gene homologues of Drosophila melanogaster (Diptera: Drosophilidae) were identified in early embryos of B. jarvisi, including transcripts highly upregulated prior to cellularisation. No strong candidates for transcripts derived solely from the Y chromosome were recovered from the poly(A+) fraction.

Conclusions: Bactrocera jarvisi provides an excellent model for embryonic studies due to available Y-chromosome markers and the compact time frame for zygotic transcription and the sex-determined state. Our data contribute fundamental information to sex-determination research, and provide candidates for the sourcing of gene promoters for transgenic pest-management strategies of tephritid fruit flies.

Show MeSH
Related in: MedlinePlus