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Identification of ovule transcripts from the Apospory-Specific Genomic Region (ASGR)-carrier chromosome.

Zeng Y, Conner J, Ozias-Akins P - BMC Genomics (2011)

Bottom Line: Apospory is a form of apomixis where the embryo develops from an unreduced egg that is derived from a somatic nucellar cell, the aposporous initial, via mitosis.Only one transcript, which did not map to the ASGR, showed expression primarily in reproductive tissue.Our results suggest that a strategy of comparative sequencing of transcriptomes between donor parent and backcross lines containing an alien chromosome of interest can be an efficient method of identifying transcripts derived from an alien chromosome in a chromosome addition line.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticulture, The University of Georgia Tifton Campus, Tifton, GA 31973, USA.

ABSTRACT

Background: Apomixis, asexual seed production in plants, holds great potential for agriculture as a means to fix hybrid vigor. Apospory is a form of apomixis where the embryo develops from an unreduced egg that is derived from a somatic nucellar cell, the aposporous initial, via mitosis. Understanding the molecular mechanism regulating aposporous initial specification will be a critical step toward elucidation of apomixis and also provide insight into developmental regulation and downstream signaling that results in apomixis. To discover candidate transcripts for regulating aposporous initial specification in P. squamulatum, we compared two transcriptomes derived from microdissected ovules at the stage of aposporous initial formation between the apomictic donor parent, P. squamulatum (accession PS26), and an apomictic derived backcross 8 (BC8) line containing only the Apospory-Specific Genomic Region (ASGR)-carrier chromosome from P. squamulatum. Toward this end, two transcriptomes derived from ovules of an apomictic donor parent and its apomictic backcross derivative at the stage of apospory initiation, were sequenced using 454-FLX technology.

Results: Using 454-FLX technology, we generated 332,567 reads with an average read length of 147 base pairs (bp) for the PS26 ovule transcriptome library and 363,637 reads with an average read length of 142 bp for the BC8 ovule transcriptome library. A total of 33,977 contigs from the PS26 ovule transcriptome library and 26,576 contigs from the BC8 ovule transcriptome library were assembled using the Multifunctional Inertial Reference Assembly program. Using stringent in silico parameters, 61 transcripts were predicted to map to the ASGR-carrier chromosome, of which 49 transcripts were verified as ASGR-carrier chromosome specific. One of the alien expressed genes could be assigned as tightly linked to the ASGR by screening of apomictic and sexual F1s. Only one transcript, which did not map to the ASGR, showed expression primarily in reproductive tissue.

Conclusions: Our results suggest that a strategy of comparative sequencing of transcriptomes between donor parent and backcross lines containing an alien chromosome of interest can be an efficient method of identifying transcripts derived from an alien chromosome in a chromosome addition line.

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Microdissection and ovary clearing. a: cleared ovary showing no aposporous initials and prior to megasporogenesis. b: cleared ovary showing two aposporous initials, indicated by solid arrows.
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Figure 1: Microdissection and ovary clearing. a: cleared ovary showing no aposporous initials and prior to megasporogenesis. b: cleared ovary showing two aposporous initials, indicated by solid arrows.

Mentions: Ovules from PS26 and BC8 around the stage of aposporous initial formation were manually dissected from pistils (Figure 1). Three biological replicates of 40 ovules each were collected for both PS26 and BC8. The yield of total RNA from each replicate was approximately 20 ng from which 15 ng was used for one-round of T7 RNA polymerase-based RNA amplification. The average yield from one round of amplification was 90 μg. For each library, equal amounts of amplified RNA from each replicate were combined and 15 μg amplified RNA was used for ds-cDNA synthesis. The majority of the ds-cDNA synthesized from amplified RNA was distributed in a size range from 200 bp to 1000 bp (Figure 2).


Identification of ovule transcripts from the Apospory-Specific Genomic Region (ASGR)-carrier chromosome.

Zeng Y, Conner J, Ozias-Akins P - BMC Genomics (2011)

Microdissection and ovary clearing. a: cleared ovary showing no aposporous initials and prior to megasporogenesis. b: cleared ovary showing two aposporous initials, indicated by solid arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3111391&req=5

Figure 1: Microdissection and ovary clearing. a: cleared ovary showing no aposporous initials and prior to megasporogenesis. b: cleared ovary showing two aposporous initials, indicated by solid arrows.
Mentions: Ovules from PS26 and BC8 around the stage of aposporous initial formation were manually dissected from pistils (Figure 1). Three biological replicates of 40 ovules each were collected for both PS26 and BC8. The yield of total RNA from each replicate was approximately 20 ng from which 15 ng was used for one-round of T7 RNA polymerase-based RNA amplification. The average yield from one round of amplification was 90 μg. For each library, equal amounts of amplified RNA from each replicate were combined and 15 μg amplified RNA was used for ds-cDNA synthesis. The majority of the ds-cDNA synthesized from amplified RNA was distributed in a size range from 200 bp to 1000 bp (Figure 2).

Bottom Line: Apospory is a form of apomixis where the embryo develops from an unreduced egg that is derived from a somatic nucellar cell, the aposporous initial, via mitosis.Only one transcript, which did not map to the ASGR, showed expression primarily in reproductive tissue.Our results suggest that a strategy of comparative sequencing of transcriptomes between donor parent and backcross lines containing an alien chromosome of interest can be an efficient method of identifying transcripts derived from an alien chromosome in a chromosome addition line.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticulture, The University of Georgia Tifton Campus, Tifton, GA 31973, USA.

ABSTRACT

Background: Apomixis, asexual seed production in plants, holds great potential for agriculture as a means to fix hybrid vigor. Apospory is a form of apomixis where the embryo develops from an unreduced egg that is derived from a somatic nucellar cell, the aposporous initial, via mitosis. Understanding the molecular mechanism regulating aposporous initial specification will be a critical step toward elucidation of apomixis and also provide insight into developmental regulation and downstream signaling that results in apomixis. To discover candidate transcripts for regulating aposporous initial specification in P. squamulatum, we compared two transcriptomes derived from microdissected ovules at the stage of aposporous initial formation between the apomictic donor parent, P. squamulatum (accession PS26), and an apomictic derived backcross 8 (BC8) line containing only the Apospory-Specific Genomic Region (ASGR)-carrier chromosome from P. squamulatum. Toward this end, two transcriptomes derived from ovules of an apomictic donor parent and its apomictic backcross derivative at the stage of apospory initiation, were sequenced using 454-FLX technology.

Results: Using 454-FLX technology, we generated 332,567 reads with an average read length of 147 base pairs (bp) for the PS26 ovule transcriptome library and 363,637 reads with an average read length of 142 bp for the BC8 ovule transcriptome library. A total of 33,977 contigs from the PS26 ovule transcriptome library and 26,576 contigs from the BC8 ovule transcriptome library were assembled using the Multifunctional Inertial Reference Assembly program. Using stringent in silico parameters, 61 transcripts were predicted to map to the ASGR-carrier chromosome, of which 49 transcripts were verified as ASGR-carrier chromosome specific. One of the alien expressed genes could be assigned as tightly linked to the ASGR by screening of apomictic and sexual F1s. Only one transcript, which did not map to the ASGR, showed expression primarily in reproductive tissue.

Conclusions: Our results suggest that a strategy of comparative sequencing of transcriptomes between donor parent and backcross lines containing an alien chromosome of interest can be an efficient method of identifying transcripts derived from an alien chromosome in a chromosome addition line.

Show MeSH