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Mycroarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion

ABSTRACT: Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring during rice improvement through transgenesis versus mutation breeding. We used oligonucleotide microarrays to analyze gene expression in four different pools of four types of rice plants and respective controls: (i) a gamma-irradiated stable mutant, (ii) the M1 generation of a 100-Gy gamma-irradiated plant, (iii) a stable transgenic plant obtained for production of an anticancer antibody, and (iv) the T1 generation of a transgenic plant produced aiming for abiotic stress improvement, and all of the unmodified original genotypes as controls. We found that the improvement of a plant variety through the acquisition of a new desired trait, using either mutagenesis or transgenesis, may cause stress and thus lead to an altered expression of untargeted genes. In all of the cases studied, the observed alteration was more extensive in mutagenized than in transgenic plants. We propose that the safety assessment of improved plant varieties should be carried out on a case-by-case basis and not simply restricted to foods obtained through genetic engineering.

ORGANISM(S): Oryza sativa

PROVIDER: GSE12069 | GEO | 2008/07/31

SECONDARY ACCESSION(S): PRJNA113319

REPOSITORIES: GEO

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Project description:Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance; of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring during rice improvement through transgenesis versus mutation breeding. We used oligonucleotide microarrays to analyze gene expression in four different pools of four types of rice plants and respective controls: (i) a; gamma-irradiated stable mutant, (ii) the M1 generation of a 100-Gy gamma-irradiated plant, (iii) a stable transgenic plant obtained for production of an anticancer antibody, and (iv) the T1 generation of a transgenic plant produced aiming for abiotic stress improvement, and all of the unmodified original genotypes as controls. We found that the improvement of a plant variety through the acquisition of a new desired trait, using either mutagenesis or transgenesis, may cause stress and thus lead to an altered expression of untargeted genes. In all of the cases studied, the observed alteration was more extensive in mutagenized than in transgenic plants. We propose that the safety assessment of improved plant varieties should be carried out on a case-by-case basis and not simply restricted to foods obtained through genetic engineering. Experiment Overall Design: Plant Materials Experiment Overall Design: Two genetically stable Oryza sativa L. ssp. japonica lines: a gamma-irradiated ricemutant(cv. EstrelaA)and a well characterized transgenic rice line (cv. Bengal) were used as well as controls. The stable mutant was obtained in 1988 by gamma-irradiation, had already gone over 10 generations of self-pollination, and had a mature average height about 45 cm lower than the wild type. The stable transgenic line, which was already in the third generation of self-pollination after transformation, expresses a ScFV antibody (ScFvT84.66) against carcinoembryonic antigen, a well characterized tumor-associated marker antigen. Experiment Overall Design: We have also used two genetically unstable rice lines: the M1 generation of a 100-Gy gamma-irradiated line (98% survival after mutagenesis) and the T1 generation of an Agrobacterium-transformed transgenic line (both cv. Nipponbare) containing one copy of the BCBF1 gene driven by the AtRD29A promoter from Arabidopsis and one copy of the hpt II gene. We used seeds from the same self-pollinated panicle for control and irradiation/transgenesis. The unstable mutant line chosen for this experiment was the one showing a phenotype more similar to that of the nonirradiated control. Experiment Overall Design: In the case of the transgenic lines, stability was based on the stable Experiment Overall Design: inheritance of the introduced transgenes in the homozygous progeny. Regarding the mutagenized plants we have defined as genetically stable plants those that, after mutagenesis, had already gone through several cycles of self-pollination while maintaining the desirable traits. Experiment Overall Design: RNA Extraction and Microarrays Experiment Overall Design: Two pools of six whole seedlings were prepared for each condition under test, and RNA was isolated with the RNeasy Plant Mini Kit (Qiagen) following the manufacturerâ??s instructions. Total RNA was kept at -80Â°C and sent to the Affymetrix core facility (Instituto Gulbenkian de CiÃªncia, Experiment Overall Design: Oeiras, Portugal), where quality-control analysis was carried out before cDNA synthesis from themRNA[with appropriate oligo(dT) primers], labeling (through synthesis of cRNA with incorporation of biotinylated ribonucleotide analogs), and hybridization to the GeneChip Rice Genome Array (Affymetrix). This array contains probes to query 51,279 transcripts representing two rice subspecies (48,564 japonica transcripts and 1,260 transcripts of indica subspecies). Experiment Overall Design: Data Analysis Experiment Overall Design: Microarrays data analysis was performed with Partek Genomics Suite software. Affymetrix CEL files were imported by using the Robust Multichip Average method, which involves four steps: background correction of the perfect match values, quintile normalization across all of the chips in the experiment, Log2 transformation, and median polish summarization. The logged data were used for hierarchical cluster analysis and statistical analysis. Experiment Overall Design: Hierarchical cluster analysis was performed by using Pearsonâ??s dissimilarity product moment correlation coefficient and Wardâ??s algorithm. Experiment Overall Design: For the identification of differentially expressed genes we used ANOVA Experiment Overall Design: and a false discovery rate with a 0.05 threshold.

Project description:Somatic embryogenesis in nucellar tissues is widely recognized to induce polyembryony in major citrus varieties such as sweet oranges, satsuma mandarins and lemons. This capability for apomixis is attractive in agricultural production systems using hybrid seeds, and many studies have been performed to elucidate the molecular mechanisms of various types of apomixis. To identify the gene responsible for somatic embryogenesis in citrus, a custom oligo-DNA microarray including predicted genes in the citrus polyembryonic locus was used to compare the expression profiles in reproductive tissues between monoembryonic and polyembryonic varieties. The full length of CitRKD1, which was identified as a candidate gene responsible for citrus somatic embryogenesis, was isolated from satsuma mandarin and its molecular function was investigated using transgenic ‘Hamlin’ sweet orange by antisense-overexpression. The candidate gene CitRKD1, predominantly transcribed in reproductive tissues of polyembryonic varieties, is a member of the plant RWP-RK domain proteins. CitRKD1 of satsuma mandarin comprised two alleles (CitRKD1-mg1 and CitRKD1-mg2) at the polyembryonic locus controlling embryony type (mono/polyembryony) that were structurally divided into two types with or without a miniature inverted-repeat transposable element (MITE)-like insertion in the upstream region. CitRKD1-mg2 with the MITE insertion was the predominant transcript in flowers and young fruits where somatic embryogenesis of nucellar cells occurred. Loss of CitRKD1 function by antisense-overexpression abolished somatic embryogenesis in transgenic sweet orange and the transgenic T1 plants were confirmed to derive from zygotic embryos produced by self-pollination by DNA diagnosis. Genotyping PCR analysis of 95 citrus traditional and breeding varieties revealed that the CitRKD1 allele with the MITE insertion (polyembryonic allele) was dominant and major citrus varieties with the polyembryonic allele produced polyembryonic seeds.

Project description:Plant architecture is a critical trait in fruit crops that can significantly influence yield, pruning, planting density and harvesting. Most of the existing varieties of olive are traditional and their architecture is poorly suited for modern growing and harvesting systems. This study focuses on the identification of candidate genes involved in determining plant architecture in olive that could help in selecting phenotypes adapted to modern cultivation practices. We previously developed the first microarray for olive, as a means to discover candidate genes involved in relevant agronomical traits. The microarray has already been applied to identify candidates genes involved in regulating juvenile to adult transition in the shoot apex of seedlings. In the present study, made in the framework of an olive breeding program, varieties displaying differences in architecture and pooled seedlings grouped by their architecture-related phenotypes, were analysed using microarray analysis of meristematic tissue. We identify 2,258 differentially expressed genes potentially involved in determining plant architecture. Varieties with opposite architecture phenotypes and individuals from segregating progenies displaying extreme architecture features, constitute our key to linking phenotype to expression. We analyze some of the genes with potentially interesting functional annotation using quantitative RT-PCR assays, in the reference varieties and individual seedlings. Arabidopsis mutants in putative orthologs of some of the candidate genes show altered architecture, indicating functional conservation between the two species and supporting both, the biological relevance of the results, and the potential of the identified genes as markers for assisted breeding for olive varieties suited for high density orchards. Active or dormant meristems to be used for expression analysis were collected from individual olive trees from four varieties Picual, Arbequina, Arbosana and Chiquitita and from seedlings of a Picual x Arbequina progeny, all of the showing variability for growth habit. Plant material was provided by the olive breeding program of Cordoba. Harvesting was carried out between 8:00 to 11:00 a.m., at the end of Spring. Samples were immediately frozen in liquid nitrogen, and maintained afterwards at -80 ÂºC. Samples of the Picual x Arbequina seedlings used to generate pools were harvested individually and 0.2 g of tissue per individual mixed and processed together to obtain RNA to be used for microarray analysis.

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Mycroarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion

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