Department of Horticulture, The University of Georgia Tifton Campus, Tifton, GA 31973, USA.
BMC Genomics. 2011 Apr 26;12:206. doi: 10.1186/1471-2164-12-206.
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.
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.
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.
无融合生殖是植物无性种子生产，作为杂种优势固定的一种手段，具有很大的农业潜力。无孢子生殖是一种无融合生殖形式，其中胚胎通过有丝分裂从源自体细胞珠心细胞的未减数卵发育而来，该细胞是无孢子原基。了解调节无孢子原基特化的分子机制将是阐明无融合生殖的关键步骤，也将为发育调控和导致无融合生殖的下游信号提供深入了解。为了在 P. squamulatum 中发现调节无孢子原基特化的候选转录本，我们比较了来自微分离无融合生殖供体亲本 P. squamulatum（PS26 号）和仅包含来自 P. squamulatum 的无孢子特异性基因组区域（ASGR）-载体染色体的无融合生殖回交 8（BC8）系的处于无孢子原基形成阶段的胚珠的两个转录组。为此，我们使用 454-FLX 技术对无融合生殖供体亲本及其无融合生殖回交衍生体的胚珠在无孢子起始阶段的两个转录组进行了测序。
使用 454-FLX 技术，我们为 PS26 胚珠转录组文库生成了 332,567 个reads，平均读长为 147 个碱基对（bp），为 BC8 胚珠转录组文库生成了 363,637 个reads，平均读长为 142 bp。使用多功能惯性参考组装程序对来自 PS26 胚珠转录组文库的 33,977 个 contigs 和来自 BC8 胚珠转录组文库的 26,576 个 contigs 进行了组装。使用严格的计算机参数，预测有 61 个转录本映射到 ASGR-载体染色体，其中 49 个转录本被验证为 ASGR-载体染色体特异性。通过筛选无融合生殖和有性 F1，可以将一个外来表达基因分配为与 ASGR 紧密连锁。只有一个转录本未映射到 ASGR，但主要在生殖组织中表达。
我们的结果表明，在包含感兴趣的外来染色体的供体亲本和回交系之间进行转录组比较测序的策略可能是识别染色体添加系中外来染色体衍生转录本的有效方法。
