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Homoeologous gene silencing in tissue cultured wheat callus.

ABSTRACT: In contrast to diploids, most polyploid plant species, which include the hexaploid bread wheat, possess an additional layer of epigenetic complexity. Several studies have demonstrated that polyploids are affected by homoeologous gene silencing, a process in which sub-genomic genomic copies are selectively transcriptionally inactivated. This form of silencing can be tissue specific and may be linked to developmental or stress responses.Evidence was sought as to whether the frequency of homoeologous silencing in in vitro cultured wheat callus differ from that in differentiated organs, given that disorganized cells are associated with a globally lower level of DNA methylation. Using a reverse transcription PCR (RT-PCR) single strand conformation polymorphism (SSCP) platform to detect the pattern of expression of 20 homoeologous sets of single-copy genes known to be affected by this form of silencing in the root and/or leaf, we observed no silencing in any of the wheat callus tissue tested.Our results suggest that much of the homoeologous silencing observed in differentiated tissues is probably under epigenetic control, rather than being linked to genomic instability arising from allopolyploidization. This study reinforces the notion of plasticity in the wheat epi-genome.

SUBMITTER: Bottley A

PROVIDER: S-EPMC2576348 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Homoeologous gene silencing in tissue cultured wheat callus.

Bottley Andrew A Chapman Natalie H NH Koebner Robert M D RM

BMC genetics 20081017

<h4>Background</h4>In contrast to diploids, most polyploid plant species, which include the hexaploid bread wheat, possess an additional layer of epigenetic complexity. Several studies have demonstrated that polyploids are affected by homoeologous gene silencing, a process in which sub-genomic genomic copies are selectively transcriptionally inactivated. This form of silencing can be tissue specific and may be linked to developmental or stress responses.<h4>Results</h4>Evidence was sought as to ...[more]

PMID: 18928533

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Project description:BackgroundEST-PCR markers normally represent specific products from target genes, and are therefore effective tools for genetic analysis. However, because wheat is an allohexaploid plant, PCR products derived from homoeologous genes are often simultaneously amplified. Such products may be easier to differentiate if they include intron sequences, which are more polymorphic than exon sequences. However, genomic sequence data for wheat are limited; therefore it is difficult to predict the location of introns. By using the similarities in gene structures between rice and wheat, we developed a system called PLUG (PCR-based Landmark Unique Gene) to design primers so that PCR products include intron sequences. We then investigated whether products amplified using such primers could serve as markers able to distinguish multiple products derived from homoeologous genes.ResultsThe PLUG system consists of the following steps: (1) Single-copy rice genes (Landmark Unique Gene loci; LUGs) exhibiting high degrees of homology to wheat UniGene sequences are extracted; (2) Alignment analysis is carried out using the LUGs and wheat UniGene sequences to predict exon-exon junctions, and LUGs which can be used to design wheat primers flanking introns (TaEST-LUGs) are extracted; and (3) Primers are designed in an interactive manner. From a total of 4,312 TaEST-LUGs, 24 loci were randomly selected and used to design primers. With all of these primer sets, we obtained specific, intron-containing products from the target genes. These markers were assigned to chromosomes using wheat nullisomic-tetrasomic lines. By PCR-RFLP analysis using agarose gel electrophoresis, 19 of the 24 markers were located on at least one chromosome.ConclusionIn the development of wheat EST-PCR markers capable of efficiently sorting products derived from homoeologous genes, it is important to design primers able to amplify products that include intron sequences with insertion/deletion polymorphisms. Using the PLUG system, wheat EST sequences that can be used for marker development are selected based on comparative genomics with rice, and then primer sets flanking intron sequences are prepared in an interactive, semi-automatic manner. Hence, the PLUG system is an effective tool for large-scale marker development.

Project description:BackgroundEucalyptus is a highly diverse genus of the Myrtaceae family and widely planted in the world for timber and pulp production. Tissue culture induced callus has become a common tool for Eucalyptus breeding, however, our knowledge about the genes related to the callus maturation and shoot regeneration is still poor.ResultsWe set up an experiment to monitor the callus induction and callus development of two Eucalyptus species - E. camaldulensis (high embryogenic potential) and E. grandis x urophylla (low embryogenic potential). Then, we performed transcriptome sequencing for primary callus, mature callus, shoot regeneration stage callus and senescence callus. We identified 707 upregulated and 694 downregulated genes during the maturation process of the two Eucalyptus species and most of them were involved in the signaling pathways like plant hormone and MAPK. Next, we identified 135 and 142 genes that might play important roles during the callus development of E. camaldulensis and E. grandis x urophylla, respectively. Further, we found 15 DEGs shared by these two Eucalyptus species during the callus development, including Eucgr.D00640 (stem-specific protein TSJT1), Eucgr.B00171 (BTB/POZ and TAZ domain-containing protein 1), Eucgr.C00948 (zinc finger CCCH domain-containing protein 20), Eucgr.K01667 (stomatal closure-related actinbinding protein 3), Eucgr.C00663 (glutaredoxin-C10) and Eucgr.C00419 (UPF0481 protein At3g47200). Interestingly, the expression patterns of these genes displayed "N" shape in the samples. Further, we found 51 genes that were dysregulated during the callus development of E. camaldulensis but without changes in E. grandis x urophylla, such as Eucgr.B02127 (GRF1-interacting factor 1), Eucgr.C00947 (transcription factor MYB36), Eucgr.B02752 (laccase-7), Eucgr.B03985 (transcription factor MYB108), Eucgr.D00536 (GDSL esterase/lipase At5g45920) and Eucgr.B02347 (scarecrow-like protein 34). These 51 genes might be associated with the high propagation ability of Eucalyptus and 22 might be induced after the dedifferentiation. Last, we performed WGCNA to identify the co-expressed genes during the callus development of Eucalyptus and qRT-PCR experiment to validate the gene expression patterns.ConclusionsThis is the first time to globally study the gene profiles during the callus development of Eucalyptus. The results will improve our understanding of gene regulation and molecular mechanisms in the callus maturation and shoot regeneration.

Project description:Fracture healing requires controlled expression of thousands of genes. Only a small fraction of these genes have been isolated and fewer yet have been shown to play a direct role in fracture healing. The purpose of this study was threefold: (1) to develop a reproducible open femur model of fracture healing that produces consistent fracture calluses for subsequent RNA extraction, (2) to use this model to determine temporal expression patterns of known and unknown genes using DNA microarray expression profiling, and (3) to identify and validate novel gene expression in fracture healing. In the initial arm of the study, a total of 56 wild-type C57BL/6 mice were used. An open, stabilized diaphyseal femur fracture was created. Animals were killed at 1, 5, 7, 10, 14, 21, and 35 days after surgery and the femurs were harvested for analysis. At each time point, fractures were radiographed and sectioned for histologic analyses. Tissue from fracture callus at all stages following fracture yielded reproducibly large amounts of mRNA. Expression profiling revealed that genes cluster by function in a manner similar to the histologic stages of fracture healing. Based on the expression profiling of fracture tissue, temporal expression patterns of several genes known to be involved in fracture healing were verified. Novel expression of multiple genes in fracture callous tissue was also revealed including leptin and leptin receptor. In order to test whether leptin signaling is required for fracture repair, mice deficient in leptin or its receptor were fractured using the same model. Fracture calluses of mice deficient in both leptin or leptin receptor are larger than wild-type mice fractures, likely due to a delay in mineralization, revealing a previously unrecognized role of leptin signaling in fracture healing. This novel model of murine fracture repair is useful in examining both global changes in gene expression as well as individual signaling pathways, which can be used to identify specific molecular mechanisms of fracture healing.

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Homoeologous gene silencing in tissue cultured wheat callus.

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Homoeologous gene silencing in tissue cultured wheat callus.

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