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CLC number: Q75

On-line Access: 2016-12-05

Received: 2016-06-25

Revision Accepted: 2016-08-26

Crosschecked: 2016-11-10

Cited: 1

Clicked: 1807

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Qing-Yao Shu

http://orcid.org/0000-0002-9201-0593

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Journal of Zhejiang University SCIENCE B 2016 Vol.17 No.12 P.992-996

10.1631/jzus.B1600301


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


Author(s):  Wen-xu Li, San-ling Wu, Yan-hua Liu, Gu-lei Jin, Hai-jun Zhao, Long-jiang Fan, Qing-yao Shu

Affiliation(s):  State Key Laboratory of Rice Biology, Institute of Crop Sciences, Zhejiang University, Hangzhou 310058, China; more

Corresponding email(s):   qyshu@zju.edu.cn

Key Words:  Rice, Genetic variation, Genome-wide, Transfer-DNA, Transposon


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Wen-xu Li, San-ling Wu, Yan-hua Liu, Gu-lei Jin, Hai-jun Zhao, Long-jiang Fan, Qing-yao Shu. Genome-wide profiling of genetic variation in Agrobacterium-transformed rice plants[J]. Journal of Zhejiang University Science B, 2016, 17(12): 992-996.

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journal="Journal of Zhejiang University Science B",
volume="17",
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pages="992-996",
year="2016",
publisher="Zhejiang University Press & Springer",
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A1 - Long-jiang Fan
A1 - Qing-yao Shu
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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

目的:揭示转基因水稻全基因组遗传变异的特征与频率。
创新点: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: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: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。

关键词:水稻;遗传转化;全基因组;转移DNA;转座子

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

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[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

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