Dataset Information

Efficiency to Discovery Transgenic Loci in GM Rice Using Next Generation Sequencing Whole Genome Re-sequencing.

ABSTRACT: Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS) methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF) in genetically modified rice cells. A total of 29.3 Gb (~72× coverage) was sequenced with a 2 × 150 bp paired end method by Illumina HiSeq2500, which was consecutively mapped to the rice genome and T-vector sequence. The compatible pairs of reads were successfully mapped to 10 loci on the rice chromosome and vector sequences were validated to the insertion location by polymerase chain reaction (PCR) amplification. The EGF transgenic site was confirmed only on chromosome 4 by PCR. Results of this study demonstrated the success of NGS data to characterize the rice genome. Bioinformatics analyses must be developed in association with NGS data to identify highly accurate transgenic sites.

SUBMITTER: Park D

PROVIDER: S-EPMC4623445 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Efficiency to Discovery Transgenic Loci in GM Rice Using Next Generation Sequencing Whole Genome Re-sequencing.

Park Doori D Kim Dongin D Jang Green G Lim Jongsung J Shin Yun-Ji YJ Kim Jina J Seo Mi-Seong MS Park Su-Hyun SH Kim Ju-Kon JK Kwon Tae-Ho TH Choi Ik-Young IY

Genomics & informatics 20150930 3

Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS) methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF) in g ...[more]

PMID: 26523132

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Project description:BackgroundHybridization between genetically diverged organisms is known as an important avenue that drives plant genome evolution. The possible outcomes of hybridization would be the occurrences of genetic instabilities in the resultant hybrids. It remained under-investigated however whether pollination by alien pollens of a closely related but sexually "incompatible" species could evoke genomic changes and to what extent it may result in phenotypic novelties in the derived progenies.Methodology/principal findingsIn this study, we have re-sequenced the genomes of Oryza sativa ssp. japonica cv. Matsumae and one of its derived introgressant RZ35 that was obtained from an introgressive hybridization between Matsumae and Zizanialatifolia Griseb. in general, 131 millions 90 base pair (bp) paired-end reads were generated which covered 13.2 and 21.9 folds of the Matsumae and RZ35 genomes, respectively. Relative to Matsumae, a total of 41,724 homozygous single nucleotide polymorphisms (SNPs) and 17,839 homozygous insertions/deletions (indels) were identified in RZ35, of which 3,797 SNPs were nonsynonymous mutations. Furthermore, rampant mobilization of transposable elements (TEs) was found in the RZ35 genome. The results of pathogen inoculation revealed that RZ35 exhibited enhanced resistance to blast relative to Matsumae. Notably, one nonsynonymous mutation was found in the known blast resistance gene Pid3/Pi25 and real-time quantitative (q) RT-PCR analysis revealed constitutive up-regulation of its expression, suggesting both altered function and expression of Pid3/Pi25 may be responsible for the enhanced resistance to rice blast by RZ35.Conclusions/significanceOur results demonstrate that introgressive hybridization by Zizania has provoked genomewide, extensive genomic changes in the rice genome, and some of which have resulted in important phenotypic novelties. These findings suggest that introgressive hybridization by alien pollens of even a sexually incompatible species may represent a potent means to generate novel genetic diversities, and which may have played relevant roles in plant evolution and can be manipulated for crop improvements.

Project description:BackgroundThe molecular characterization information of T-DNA integration is not only required by public risk assessors and regulators, but is also closely related to the expression of exogenous and endogenous genes. At present, with the development of sequencing technology, whole-genome resequencing has become an attractive approach to identify unknown genetically modified events and characterise T-DNA integration events.ResultsIn this study, we performed genome resequencing of Pb29, a transgenic high-resistance poplar 741 line that has been commercialized, using next-generation and Nanopore sequencing. The results revealed that there are two T-DNA insertion sites, located at 9,283,905-9,283,937 bp on chromosome 3 (Chr03) and 10,868,777-10,868,803 bp on Chr10. The accuracy of the T-DNA insertion locations and directions was verified using polymerase chain reaction amplification. Through sequence alignment, different degrees of base deletions were detected on the T-DNA left and right border sequences, and in the flanking sequences of the insertion sites. An unknown fragment was inserted between the Chr03 insertion site and the right flanking sequence, but the Pb29 genome did not undergo chromosomal rearrangement. It is worth noting that we did not detect the API gene in the Pb29 genome, indicating that Pb29 is a transgenic line containing only the BtCry1AC gene. On Chr03, the insertion of T-DNA disrupted a gene encoding TAF12 protein, but the transcriptional abundance of this gene did not change significantly in the leaves of Pb29. Additionally, except for the gene located closest to the T-DNA integration site, the expression levels of four other neighboring genes did not change significantly in the leaves of Pb29.ConclusionsThis study provides molecular characterization information of T-DNA integration in transgenic poplar 741 line Pb29, which contribute to safety supervision and further extensive commercial planting of transgenic poplar 741.

Dataset Information

Efficiency to Discovery Transgenic Loci in GM Rice Using Next Generation Sequencing Whole Genome Re-sequencing.

Publications

Efficiency to Discovery Transgenic Loci in GM Rice Using Next Generation Sequencing Whole Genome Re-sequencing.

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