Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting.

Masaki Endo,Masao Hamada,Satomi Mori-Hosokawa,Seiichi Toki,Masahiko Kumagai,Hiroyuki Kanamori,Harumi Sasaki-Yamagata,Yuichi Katayose,Ritsuko Motoyama,Takeshi Itoh,Yoshiaki Nagamura

doi:10.1093/pcp/pcu153

Abstract

Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20–100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing.

Highlights

Since the first report of gene targeting (GT) of an integrated antibiotic resistance gene in the tobacco genome (Paszkowski et al 1988), various approaches aimed at homologous recombination (HR)-dependent GT have been attempted, and several instances of successful GT of an endogenous gene have been reported in crops
We reported the successful production of novel herbicide-tolerant rice plants via introduction of two point mutations (W548L and S627I) into the rice acetolactate synthase (ALS) gene via HR-dependent GT using an Agrobacterium T-DNAmediated transformation system (Endo et al 2007)
A schematic representation of this GT system is shown in Fig. 1A: the GT vector contains a truncated ALS coding sequence with two point mutations; tryptophan to leucine (TGG!TTG) at amino acid 548 (W548L), and serine to isoleucine (AGT!ATT) at amino acid 627 (S627I)

Summary

Introduction

Since the first report of gene targeting (GT) of an integrated antibiotic resistance gene in the tobacco genome (Paszkowski et al 1988), various approaches aimed at homologous recombination (HR)-dependent GT have been attempted, and several instances of successful GT of an endogenous gene have been reported in crops (for a review, see Endo and Toki 2013). We have introduced targeted point mutations into the rice genome via GT; herbicide-tolerant rice (Endo et al 2007) and tryptophan-hyperaccumulating rice (Saika et al 2011) were generated successfully by inducing point mutations in the acetolactate synthase (ALS) gene and the gene encoding anthranilate synthase a-subunit 2 (ASA2), respectively. In both cases, truncated target genes containing desirable mutations were transformed to wild-type (WT) rice calli, and GT cells were identified by subsequent selection using an ALSinhibiting herbicide and an analog of tryptophan. Random integration of GT vectors by non-homologous endjoining (NHEJ) often occurred (Endo et al 2007)

Methods

Results

Conclusion