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Abstract: Agrobacterium-mediated transformation has been widely used in producing transgenic plants, and was recently used to generate “transgene-clean” targeted genomic modifications coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Although tremendous variation in morphological and agronomic traits, such as plant height, seed fertility, and grain size, was observed in transgenic plants, the underlying mechanisms are not yet well understood, and the types and frequency of genetic variation in transformed plants have not been fully disclosed. To reveal the genome-wide variation in transformed plants, we sequenced the genomes of five independent T₀ rice plants using next-generation sequencing (NGS) techniques. Bioinformatics analyses followed by experimental validation revealed the following: (1) in addition to transfer-DNA (T-DNA) insertions, three transformed plants carried heritable plasmid backbone DNA of variable sizes (855–5216 bp) and in different configurations with the T-DNA insertions (linked or apart); (2) each transgenic plant contained an estimated 338–1774 independent genetic variations (single nucleotide variations (SNVs) or small insertion/deletions); and (3) 2–6 new Tos17 insertions were detected in each transformed plant, but no other transposable elements or bacterial genomic DNA.

农杆菌介导的水稻转化植株遗传变异特征的全基因组分析

[1]Chen, K., Wallis, J.W., McLellan, M.D., et al., 2009. BreakDancer: an algorithm for high-resolution mapping of genomic structural variation. Nat. Methods, 6(9):677-681.

[2]Endo, M., Kumagai, M., Motoyama, R., et al., 2014. Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting. Plant Cell Physiol., 56(1):116-125.

[3]Hiei, Y., Komari, T., 2008. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat. Protoc., 3(5):824-834.

[4]Kawakatsu, T., Kawahara, Y., Itoh, T., et al., 2013. A whole-genome analysis of a transgenic rice seed-based edible vaccine against cedar pollen allergy. DNA Res., 20(6):623-631.

[5]Kovalic, D., Garnaat, C., Guo, L., et al., 2012. The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology. Plant Gen., 5:149-163.

[6]Ledford, H., 2015. CRISPR, the disruptor. Nature, 522(7554):20-24.

[7]Li, H., Handsaker, B., Wysoker, A., et al., 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics, 25(16):2078-2079.

[8]Li, R., Zhu, H., Ruan, J., et al., 2010. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res., 20(2):265-272.

[9]Sabot, F., Picault, N., El-Baidouri, M., et al., 2011. Transpositional landscape of the rice genome revealed by paired-end mapping of high-throughput re-sequencing data. Plant J., 66(2):241-246.

[10]Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, NY.

[11]Wei, F.J., Kuang, L.Y., Oung, H.M., 2016. Somaclonal variation does not preclude the use of rice transformants for genetic screening. Plant J., 85(5):648-659.

[12]Yang, L., Wang, C., Holst-Jensen, A., 2013. Characterization of GM events by insert knowledge adapted re-sequencing approaches. Sci. Rep., 3:2839.

[13]List of electronic supplementary materials

[14]Fig. S1 PCR validation of plasmid DNA in transgenic rice lines

[15]Fig. S2 PCR validation of newly inserted Tos17 with site specific primers

[16]Fig. S3 Chromosome structural variations detected uniquely in transgenic lines

[17]Fig. S4 Schematic diagram of artificial microRNA of OsMRP5 expression plasmid p1301-amiMRP5-OleN

[18]Fig. S5 Procedure used to produce transgenic plants from seed derived calli, and the particulars of 5 individual transgenic T₀ lines used for genome sequencing

[19]Table S1 Summary of the genome sequencing of five transgenic rice lines

[20]Table S2 Primers for validation of plasmid transfer and backbone DNA insertions

[21]Table S3 Primers used for validation of newly inserted Tos17 sequences

[22]Table S4 Information about new Tos17 insertions in transgenic rice

[23]Table S5 Primers for PCR validation of structural variation in transgenic line T182