Development of genome-specific primers for homoeologous genes in allopolyploid species: the waxy and starch synthase II genes in allohexaploid wheat (Triticum aestivum L.) as examples

<p>Abstract</p> <p>Background</p> <p>In allopolypoid crops, homoeologous genes in different genomes exhibit a very high sequence similarity, especially in the coding regions of genes. This makes it difficult to design genome-specific primers to amplify individual genes from different genomes. Development of genome-specific primers for agronomically important genes in allopolypoid crops is very important and useful not only for the study of sequence diversity and association mapping of genes in natural populations, but also for the development of gene-based functional markers for marker-assisted breeding. Here we report on a useful approach for the development of genome-specific primers in allohexaploid wheat.</p> <p>Findings</p> <p>In the present study, three genome-specific primer sets for the <it>waxy </it>(<it>Wx</it>) genes and four genome-specific primer sets for the <it>starch synthase II </it>(<it>SSII</it>) genes were developed mainly from single nucleotide polymorphisms (SNPs) and/or insertions or deletions (Indels) in introns and intron-exon junctions. The size of a single PCR product ranged from 750 bp to 1657 bp. The total length of amplified PCR products by these genome-specific primer sets accounted for 72.6%-87.0% of the <it>Wx </it>genes and 59.5%-61.6% of the <it>SSII </it>genes. Five genome-specific primer sets for the <it>Wx </it>genes (one for Wx-7A, three for Wx-4A and one for Wx-7D) could distinguish the wild type wheat and partial waxy wheat lines. These genome-specific primer sets for the <it>Wx </it>and <it>SSII </it>genes produced amplifications in hexaploid wheat, cultivated durum wheat, and <it>Aegilops tauschii </it>accessions, but failed to generate amplification in the majority of wild diploid and tetraploid accessions.</p> <p>Conclusions</p> <p>For the first time, we report on the development of genome-specific primers from three homoeologous <it>Wx </it>and <it>SSII </it>genes covering the majority of the genes in allohexaploid wheat. These genome-specific primers are being used for the study of sequence diversity and association mapping of the three homoeologous <it>Wx </it>and <it>SSII </it>genes in natural populations of both hexaploid wheat and cultivated tetraploid wheat. The strategies used in this paper can be used to develop genome-specific primers for homoeologous genes in any allopolypoid species. They may be also suitable for (i) the development of gene-specific primers for duplicated paralogous genes in any diploid species, and (ii) the development of allele-specific primers at the same gene locus.</p

Identification of genes preferentially expressed in wheat egg cells and zygotes

Wheat genes differentially expressed in the egg cell before and after fertilization were identified. The data support zygotic gene activation before the first cell division in wheat. To have an insight into fertilization-induced gene expression, cDNA libraries have been prepared from isolated wheat egg cells and one-celled zygotes. Two-hundred and twenty-six egg cell and 253 zygote-expressed EST sequences were determined. Most of the represented transcripts were detected in the wheat egg cell or zygote transcriptome at the first time. Expression analysis of fourteen of the identified genes and three controls was carried out by real-time quantitative PCR. The preferential expression of all investigated genes in the female gametophyte-derived samples (egg cells, zygotes, two-celled proembryos, and basal ovule parts with synergids) in comparison to the anthers, and the leaves were verified. Three genes with putative signaling/regulatory functions were expressed at a low level in the egg cell but exhibited increased (2-to-33-fold) relative expression in the zygote and the proembryo. Genes with high EST abundance in cDNA libraries exhibited strong expression in the egg cell and the zygote, while the ones coding for unknown or hypothetical proteins exhibited differential expression patterns with preferential transcript accumulation in egg cells and/or zygotes. The obtained data support the activation of the zygotic genome before the first cell division in wheat

Diversification of Genes Encoding Granule-Bound Starch Synthase in Monocots and Dicots Is Marked by Multiple Genome-Wide Duplication Events

Starch is one of the major components of cereals, tubers, and fruits. Genes encoding granule-bound starch synthase (GBSS), which is responsible for amylose synthesis, have been extensively studied in cereals but little is known about them in fruits. Due to their low copy gene number, GBSS genes have been used to study plant phylogenetic and evolutionary relationships. In this study, GBSS genes have been isolated and characterized in three fruit trees, including apple, peach, and orange. Moreover, a comprehensive evolutionary study of GBSS genes has also been conducted between both monocots and eudicots. Results have revealed that genomic structures of GBSS genes in plants are conserved, suggesting they all have evolved from a common ancestor. In addition, the GBSS gene in an ancestral angiosperm must have undergone genome duplication ∼251 million years ago (MYA) to generate two families, GBSSI and GBSSII. Both GBSSI and GBSSII are found in monocots; however, GBSSI is absent in eudicots. The ancestral GBSSII must have undergone further divergence when monocots and eudicots split ∼165 MYA. This is consistent with expression profiles of GBSS genes, wherein these profiles are more similar to those of GBSSII in eudicots than to those of GBSSI genes in monocots. In dicots, GBSSII must have undergone further divergence when rosids and asterids split from each other ∼126 MYA. Taken together, these findings suggest that it is GBSSII rather than GBSSI of monocots that have orthologous relationships with GBSS genes of eudicots. Moreover, diversification of GBSS genes is mainly associated with genome-wide duplication events throughout the evolutionary course of history of monocots and eudicots

Microspore embryogenesis in wheat: New marker genes for early, middle and late stages of embryo development

10 Págs., 3 figs. The definitive version is available at: http://link.springer.com/journal/497Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a 'star-like' morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation. © 2013 Springer-Verlag Berlin Heidelberg.RA Sánchez-Díaz was recipient of a predoctoral fellowship, from Junta Ampliación de Estudios, Consejo Superior de Investigaciones Científicas (JAE-CSIC) of Spain. This work was supported by Project AGL2010-17509, from ‘Plan Nacional de Recursos y Tecnologías Agroalimentarias’ of Spain and by COST Action FAO0903 ‘Harnessing of Reproduction for Plant Improvement’ (HAPRECI)Peer reviewe