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. 2011 Apr 26;12:206.
doi: 10.1186/1471-2164-12-206.

Identification of Ovule Transcripts From the Apospory-Specific Genomic Region (ASGR)-carrier Chromosome

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Free PMC article

Identification of Ovule Transcripts From the Apospory-Specific Genomic Region (ASGR)-carrier Chromosome

Yajuan Zeng et al. BMC Genomics. .
Free PMC article

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.

Figures

Figure 1
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.
Figure 2
Figure 2
Agilent Bioanalyzer 2100 analysis result of the ds-cDNA samples.
Figure 3
Figure 3
Blast2GO Level 3 biological processes for PS26 and BC8.
Figure 4
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).
Figure 5
Figure 5
Examples for mapping of transcripts to the ASGR. a: amplification of apomictic F1s but not sexual F1s (PS26_c9369: p1514/p1515). b: amplification of both apomictic F1s and sexual F1s (PS26_c5080: p1506/p1507).
Figure 6
Figure 6
Examples of expression patterns for ASGR-carrier chromosome linked sequences. a: most genes showed expression in all four organs tested (Ps26_c194: p1604/p1605). b: one gene was expressed in only ovary and anther (PS26_ c33813: p1565/p1566). RT(+): RT with reverse transcriptase; RT(-): RT without reverse transcriptase as DNA contamination control.

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