Genome-wide profiling of genetic variation in Agrobacterium-transformed rice plants#

Genome-wide profiling of genetic variation in Agrobacterium -transformed rice plants # # 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 4 first-author 2 1 5 1 3 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:qyshu@zju.edu.cn †E-mail:qyshu@zju.edu.cn qyshu@zju.edu.cn State Key Laboratory of Rice Biology, Institute of Crop Sciences, Zhejiang University, Hangzhou 310058, China State Key Laboratory of Rice Biology, Institute of Crop Sciences, Zhejiang University, Hangzhou 310058, China Analysis Center of Agrobiology and Environmental Sciences, Faculty of Agriculture, Life and Environment Sciences, Zhejiang University, Hangzhou 310058, China Analysis Center of Agrobiology and Environmental Sciences, Faculty of Agriculture, Life and Environment Sciences, Zhejiang University, Hangzhou 310058, China IBM Biocomputational Laboratory, Institute of Crop Sciences, Zhejiang University, Hangzhou 310058, China IBM Biocomputational Laboratory, Institute of Crop Sciences, Zhejiang University, Hangzhou 310058, China Institute for Wheat Research, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China Institute for Wheat Research, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China Hangzhou Guhe Information and Technology Co., Ltd., Hangzhou 310058, China Hangzhou Guhe Information and Technology Co., Ltd., Hangzhou 310058, China 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 T 0 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. 目的 2 揭示转基因水稻全基因组遗传变异的特征与频率。 创新点 2 通过单核苷酸分辨率揭示了农杆菌介导法转化水稻植株全基因组水平遗传变异的类型和频率以及外源DNA的整合模式。 方法 2 应用Illumina Hiseq2000高通量测序技术测定了 5个T0代转基因水稻株系的基因组序列。结合生物信息学分析和聚合酶链反应（PCR）扩增，以及Sanger测序，我们检测和验证单核苷酸多态性（SNP）和Indel变化类型和数量，转移DNA（T-DNA）及其载体骨架序列和转座子整合位点及特征，大片段的结构变异等遗传变异。 结论 2 结果表明，农杆菌介导的水稻遗传转化，除T-DNA整合到水稻基因组外，还存在载体骨架整合现象；每个转基因水稻基因组的变异（SNP和小片段的缺失插入）数目为338-1774，与报道的组培过程中产生的变异类似；转基因水稻基因组仅存在Tos17转座子数量的变化，未检测到其他转座子数目和位置的变化。 Rice Genetic variation Genome-wide Transfer-DNA Transposon 水稻 遗传转化 全基因组 转移DNA 转座子 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 T 0 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. During the past three decades, Agrobacterium -mediated transformations have been widely applied to a broad range of species, resulting in a number of transgenic (or genetically modified (GM)) plants being commercialized globally ( http://www.isaaa.org http://www.isaaa.org ). The importance of this technology may further increase with the widespread application of the CRISPR/Cas9 system to produce targeted genomic modifications of agricultural plants (Ledford, 2015 2015 ). For assessment of both their safety and practical usefulness, a genome-wide analysis of genetic changes in transformed plants would provide the most comprehensive and fundamental information. NGS technologies, because of their high throughput and cost effectiveness, are appropriate choices for holistically examining genetic variation in transgenic plants. NGS-based methods have been used for characterizing complex cases of T-DNA insertions in soybean (Kovalic et al., 2012 2012 ) and rice (Yang et al., 2013 2013 ), and for examining other types of genetic variation in transgenic rice plants (Kawakatsu et al., 2013 2013 ; Endo et al., 2014 2014 ; Wei et al., 2016 2016 ). Despite the vast amount of transgenic plants produced and applied worldwide, the number of such studies and the representation of transgenic materials, are limited, our understanding of genome-wide variation in transformed plants is far from complete. Hence, more studies involving representative transgenic plants are needed. In the present study, we produced transgenic rice plants via Agrobacterium -mediated transformation and used NGS to sequence five independent T 0 plants to assess the frequencies, spectra, and nature of variations introduced into the rice genome. A total of about 11.5 Gb of clean data were generated for each of the five transgenic lines (T027, T182, T204, T273, and T400), of which over 98% mapped to the reference genome (Table S1 S1 ). A bioinformatics analysis of NGS data showed that there was one T-DNA sequence in each transgenic line (Fig. 1 1 ). The left and right borders of the T-DNA insertions were complete and clear in T027, T204, and T400, but the right borders of T273 and T182 had short backbone DNA segments (Fig. 1 1 ). Furthermore, we detected separate backbone DNA insertions in three transgenic lines, i.e. T027, T182, and T204 (Fig. 1 1 ). In T027, the backbone (3.5 kb) and T-DNA fragments were inserted into two different chromosomes (chromosomes 1 and 7), while, in T182, both the backbone (5.2 kb) and T-DNA fragments were integrated into chromosome 1, but separated by a about 3-kb rice genomic fragment (Fig. 1 1 ). While only one end of the T-DNA insertion was precisely located on chromosome 1 in T182, neither end of the small backbone DNA fragment (855 bp) in T204 could be located to any chromosome (Fig. 1 1 , Fig. S1 S1 , Table S2 S2 ). Rice genomic DNA is shown in empty boxes, and plasmid transfer and backbone DNA in black and gray filled boxes, respectively. The bold numbers around the insertions refer to the nucleotide number in respective rice chromosomes, and non-bold numbers refer to nucleotides in the expression vector. The insertions with defined positions in rice chromosomes are marked with lines and nucleotide numbers, while those that could not be located in rice chromosomes are marked with a question mark () To assess whether the transgenic plant production process generated other genetic variations, SNVs and small indels were profiled genome-wide. A total of 8204 SNVs and small indels were identified in the five transgenic lines compared with the reference genome. Further examination indicated that 2593 of these were common across the five transgenic lines. The remaining 5611 genomic variations (4566 SNVs and 1045 indels) were considered to be derived from transformation and in vitro culture. Among the five transgenic lines, four lines with a short in vitro culture period had similar numbers of variations (502–766). Line T204, generated from a callus with a prolonged in vitro culture period, had many more variations than the other lines, particular for the number of SNVs (Table 1 1 ). A genomic position analysis indicated that on average 26.36% of indels and 36.14% of SNVs were located in the coding regions (Table 1 1 ). Validation of randomly selected variations ( n =200) by Sanger sequencing confirmed 57% of them as true mutations. Hence, the actual number of mutations in the transgenic plants was less than 5611. The validation was performed by the Sanger sequencing of fragments encompassing SNV/indel sites (20 SNV and indel mutations were tested from each line) Transgenic line Type Total number Validation 1 1 Coding region 2 T027 Indel 183 13/20 41 SNV 319 14/20 119 2 T182 Indel 204 15/20 63 SNV 309 13/20 124 2 T204 Indel 165 11/20 51 SNV 3060 8/20 1085 2 T273 Indel 217 13/20 53 SNV 388 12/20 139 2 T400 Indel 301 11/20 74 SNV 465 14/20 147 Total 5611 114/200 1896 The number of transposable elements (TEs) in the transgenic plants was also determined. Compared with Nipponbare, 18 new Tos17 sequences, from 2 to 6 in each line, were identified across nine chromosomes (Fig. 2 2 , Table S3 S3 ). The new Tos17 insertions were validated in all lines except T400, where the amplification of both ends was observed for only two of its five events, while one-ended amplification was observed for the other three insertions (Fig. S2 S2 ). Interestingly, many of the Tos17 insertions were located in the vicinity of chromosome ends (Fig. 2 2 , Table S4 S4 ). Other TEs, including mPing, RN_363, and Tami2, were also examined. While a few differences were common to all the lines, none was unique to any single transgenic plant. For example, among over 40 mPings identified, three were present in all five transgenic lines, in chromosomes 1 (positions 40, 490, 011 bp), 3 (26, 791, 701 bp), and 12 (3, 328, 968 bp), respectively, but were not reported in the reference genome of Nipponbare. Conversely, one mPing (chromosome 1: 17, 514, 263 bp) reported in the reference genome of Nipponbare was not identified in any of the transgenic lines. In addition to SNVs and small indels, 793 structural variations (SVs), such as deletions of chromosomal fragments, inversions, interchromosomal exchanges, and complex variations, were identified in the five transgenic lines through bioinformatic analysis. About one third (267/793) of the SVs were common to the five transgenic lines. Therefore, there were differences between the “Nipponbare” used for genetic transformation and the “Nipponbare” used as a reference. The numbers and profiles of SVs were quite similar among the five transgenic lines (Fig. S3 S3 ). However, we were not able to validate any of the 10 randomly selected SVs in T182 by polymerase chain reaction (PCR) amplification (Table S5 S5 ). No Agrobacterium genomic sequence was identified in the transgenic plants. The present study revealed the complicated plasmid DNA integration scenarios and the types and frequency of genome-wide variations in transformed rice plants, greatly enriching our knowledge of the genetics and genomics of Agrobacterium- transformed plants. Materials and methods 1 Production of transgenic rice 2 Transgenic rice plants were developed via Agrobacterium -mediated transformation of the japonica cultivar “Nipponbare” following the method of Hiei and Komari ( 2008 2008 ). Rice calli induced from mature seeds were co-cultured with A. tumefaciens strain EHA105 containing plasmid p1301-amiMRP5-OleN (Fig. S4 S4 ). Because the in vitro culture process is known to cause somaclonal variation (Sabot et al., 2011 2011 ), we analyzed one transgenic T 0 line (T204) having an extended in vitro culture period (about 5 months) and four T 0 lines (T027, T182, T273, and T400) with a short culture period (about 3 months) (Fig. S5 S5 ). Genome sequencing and bioinformatics analysis 2 Genomic DNA was extracted from the leaf tissues of five independent transgene positive T 0 lines using the method of Sambrook and Russell ( 2001 2001 ). For NGS, DNA sequencing libraries were constructed according to the manufacturer’s instructions. Short paired-end (PE) reads (90 bp) were generated using the Illumina Hiseq2000 sequencing platform of BGI (Shenzhen, China). Raw reads were pre-processed to remove adaptors and reads of low quality (Li et al., 2010 2010 ). All sequences generated by this study have been deposited in GenBank (BioProject ID: PRJNA290293). For read mapping and SNV calling and detection of small insertion/deletions (indels < 10 bp), clean reads of each line were aligned to the reference genome using Burrows-Wheeler Aligner (BWA) v0.5.9 and SAMtools v0.1.18 (Li et al., 2009 2009 ). The SNVs and indels were further annotated with the Michigan State University (MSU) Rice Genome Annotation Project Database using SnpEff v2.0.5d, and structural variations were analyzed by Break Dancer v1.1 (Chen et al., 2009 2009 ). The identification and prediction of inserted DNAs (plasmid DNA, TE, and Agrobacterium DNA) were enabled using PE read pairs. BWA v0.5.9 was used for read mapping (Li et al., 2009 2009 ), and a custom in-house Perl script was written for locating insertion sites. To detect the presence of Agrobacterium DNA sequences in transgenic rice, the Agrobacterium C58 genome sequence was used as a reference (Li et al., 2009 2009 ). PCR and DNA Sanger sequencing 2 The presence of plasmid DNAs, T-DNA, and Tos17 insertion sequences revealed by NGS was further examined by PCR using site-specific primers (Tables S1 S1 and S2 S2 ). For indel or SNV validation, fragments encompassing the variation sites were amplified using event-specific primers. The variation was determined by clone sequencing of the amplified fragments (five clones for each SNV/indel). References K Chen , JW Wallis , MD McLellan , 等 . BreakDancer: an algorithm for high-resolution mapping of genomic structural variation . Nat Methods , 2009 . 6 ( 9 ): 677 - 681 . (Available from: http://dx.doi.org/10.1038/nmeth.1363 http://dx.doi.org/10.1038/nmeth.1363 ) . BreakDancer: an algorithm for high-resolution mapping of genomic structural variation M Endo , M Kumagai , R Motoyama , 等 . Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium -mediated gene targeting . Plant Cell Physiol , 2014 . 56 ( 1 ): 116 - 125 . (Available from: http://dx.doi.org/10.1093/pcp/pcu153 http://dx.doi.org/10.1093/pcp/pcu153 ) . Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting Y Hiei , T Komari . Agrobacterium -mediated transformation of rice using immature embryos or calli induced from mature seed . Nat Protoc , 2008 . 3 ( 5 ): 824 - 834 . (Available from: http://dx.doi.org/10.1038/nprot.2008.46 http://dx.doi.org/10.1038/nprot.2008.46 ) . Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed T Kawakatsu , Y Kawahara , T Itoh , 等 . A whole-genome analysis of a transgenic rice seed-based edible vaccine against cedar pollen allergy . DNA Res , 2013 . 20 ( 6 ): 623 - 631 . (Available from: http://dx.doi.org/10.1093/dnares/dst036 http://dx.doi.org/10.1093/dnares/dst036 ) . A whole-genome analysis of a transgenic rice seed-based edible vaccine against cedar pollen allergy D Kovalic , C Garnaat , L Guo , 等 . The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology . Plant Gen , 2012 . 5 149 - 163 . (Available from: http://dx.doi.org/10.3835/plantgenome2012.10.0026 http://dx.doi.org/10.3835/plantgenome2012.10.0026 ) . The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology H Ledford . CRISPR, the disruptor . Nature , 2015 . 522 ( 7554 ): 20 - 24 . (Available from: http://dx.doi.org/10.1038/522020a http://dx.doi.org/10.1038/522020a ) . CRISPR, the disruptor H Li , B Handsaker , A Wysoker , 等 . The Sequence Alignment/Map format and SAMtools . Bioinformatics , 2009 . 25 ( 16 ): 2078 - 2079 . (Available from: http://dx.doi.org/10.1093/bioinformatics/btp352 http://dx.doi.org/10.1093/bioinformatics/btp352 ) . The Sequence Alignment/Map format and SAMtools R Li , H Zhu , J Ruan , 等 . De novo assembly of human genomes with massively parallel short read sequencing . Genome Res , 2010 . 20 ( 2 ): 265 - 272 . (Available from: http://dx.doi.org/10.1101/gr.097261.109 http://dx.doi.org/10.1101/gr.097261.109 ) . De novo assembly of human genomes with massively parallel short read sequencing F Sabot , N Picault , M El-Baidouri , 等 . Transpositional landscape of the rice genome revealed by paired-end mapping of high-throughput re-sequencing data . Plant J , 2011 . 66 ( 2 ): 241 - 246 . (Available from: http://dx.doi.org/10.1111/j.1365-313X.2011.04492.x http://dx.doi.org/10.1111/j.1365-313X.2011.04492.x ) . Transpositional landscape of the rice genome revealed by paired-end mapping of high-throughput re-sequencing data J Sambrook , DW Russell . Molecular Cloning: A Laboratory Manual, 3rd Ed , 2001 . : NY Cold Spring Harbor Laboratory Press , . Molecular Cloning: A Laboratory Manual, 3rd Ed FJ Wei , LY Kuang , HM Oung . Somaclonal variation does not preclude the use of rice transformants for genetic screening . Plant J , 2016 . 85 ( 5 ): 648 - 659 . (Available from: http://dx.doi.org/10.1111/tpj.13132 http://dx.doi.org/10.1111/tpj.13132 ) . Somaclonal variation does not preclude the use of rice transformants for genetic screening L Yang , C Wang , A Holst-Jensen . Characterization of GM events by insert knowledge adapted re-sequencing approaches . Sci Rep , 2013 . 3 2839 (Available from: http://dx.doi.org/10.1038/srep02839 http://dx.doi.org/10.1038/srep02839 ) . Characterization of GM events by insert knowledge adapted re-sequencing approaches Compliance with ethics guidelines Wen-xu LI, San-ling WU, Yan-hua LIU, Gu-lei JIN, Hai-jun ZHAO, Long-jiang FAN, and Qing-yao SHU declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1600301 Project supported by the Ministry of Science and Technology, China (No. SQ2015IM3600010) and the Fundamental Research Funds for the Central Universities (No. 2016XZZX001-09), China 2016-06-25 2016-11-10 2016-08-26 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 17