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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.

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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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Examples for mapping of transcripts to the ASGR-carrier chromosome. a: amplification from PS26, N37 and apomictic BC8but not from IA4X or sexual BC8(PS26_c583: p1510/p1511). b: amplification from PS26 and apomictic BC8but not from IA4X, N37 or sexual BC8(PS26_c9369: p1514/p1515). c: amplification from PS26, IA4X, N37 and both apomictic and sexual BC8(no specificity; PS26_c4364: p1504/p1505). Specificity for PS26_c4364 subsequently was achieved by using a different primer pair (Table 2).
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Figure 4: Examples for mapping of transcripts to the ASGR-carrier chromosome. a: amplification from PS26, N37 and apomictic BC8but not from IA4X or sexual BC8(PS26_c583: p1510/p1511). b: amplification from PS26 and apomictic BC8but not from IA4X, N37 or sexual BC8(PS26_c9369: p1514/p1515). c: amplification from PS26, IA4X, N37 and both apomictic and sexual BC8(no specificity; PS26_c4364: p1504/p1505). Specificity for PS26_c4364 subsequently was achieved by using a different primer pair (Table 2).

Mentions: Up to four primer pairs per contig were used to test for linkage of the 61 contigs to the ASGR-carrier chromosome. Sequence characterized amplified region (SCAR) primer pairs were designed based on the PS26 contig sequence (Additional file 3, Table S1). After screening by PCR against PS26, IA4X (4 × P. glaucum), N37 (P. purpureum) and a small number of progeny from apomictic BC8 segregating for mode of reproduction, 45 contigs showed specific amplification from PS26 and apomictic BC8 but no amplification from IA4X or sexual BC8 individuals (Figure 4, Table 2) establishing linkage of 45 contigs to the ASGR-carrier chromosome. Single-strand conformation polymorphism analysis (SSCP) and a CAPS screen using two to four restriction enzymes was applied to the 14 primer pairs which amplified products in both PS26 and IA4X DNA. Four additional contigs could be linked to the ASGR-carrier chromosome using SSCP analysis (Table 2). The CAPS screen identified a HaeIII polymorphism for PS26_c2552, a transcript also mapped by SSCP.


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

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

Examples for mapping of transcripts to the ASGR-carrier chromosome. a: amplification from PS26, N37 and apomictic BC8but not from IA4X or sexual BC8(PS26_c583: p1510/p1511). b: amplification from PS26 and apomictic BC8but not from IA4X, N37 or sexual BC8(PS26_c9369: p1514/p1515). c: amplification from PS26, IA4X, N37 and both apomictic and sexual BC8(no specificity; PS26_c4364: p1504/p1505). Specificity for PS26_c4364 subsequently was achieved by using a different primer pair (Table 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3111391&req=5

Figure 4: Examples for mapping of transcripts to the ASGR-carrier chromosome. a: amplification from PS26, N37 and apomictic BC8but not from IA4X or sexual BC8(PS26_c583: p1510/p1511). b: amplification from PS26 and apomictic BC8but not from IA4X, N37 or sexual BC8(PS26_c9369: p1514/p1515). c: amplification from PS26, IA4X, N37 and both apomictic and sexual BC8(no specificity; PS26_c4364: p1504/p1505). Specificity for PS26_c4364 subsequently was achieved by using a different primer pair (Table 2).
Mentions: Up to four primer pairs per contig were used to test for linkage of the 61 contigs to the ASGR-carrier chromosome. Sequence characterized amplified region (SCAR) primer pairs were designed based on the PS26 contig sequence (Additional file 3, Table S1). After screening by PCR against PS26, IA4X (4 × P. glaucum), N37 (P. purpureum) and a small number of progeny from apomictic BC8 segregating for mode of reproduction, 45 contigs showed specific amplification from PS26 and apomictic BC8 but no amplification from IA4X or sexual BC8 individuals (Figure 4, Table 2) establishing linkage of 45 contigs to the ASGR-carrier chromosome. Single-strand conformation polymorphism analysis (SSCP) and a CAPS screen using two to four restriction enzymes was applied to the 14 primer pairs which amplified products in both PS26 and IA4X DNA. Four additional contigs could be linked to the ASGR-carrier chromosome using SSCP analysis (Table 2). The CAPS screen identified a HaeIII polymorphism for PS26_c2552, a transcript also mapped by SSCP.

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