Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting.
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.
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan.
Masaki
M
Endo
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan Present address: Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan.
Masahiko
M
Kumagai
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan.
Ritsuko
R
Motoyama
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan.
Harumi
H
Sasaki-Yamagata
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan.
Satomi
S
Mori-Hosokawa
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan.
Masao
M
Hamada
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan.
Hiroyuki
H
Kanamori
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan.
Yoshiaki
Y
Nagamura
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan.
Yuichi
Y
Katayose
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan.
Takeshi
T
Itoh
Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa, Yokohama, 236-0027 Japan stoki@affrc.go.jp.
Seiichi
S
Toki
D000095:Acetolactate Synthase / Q000235:genetics; D060054:Agrobacterium / Q000235:genetics; D055028:Comparative Genomic Hybridization; D018556:Crops, Agricultural; D018390:Gene Targeting; D018745:Genome, Plant / Q000235:genetics*; D006540:Herbicides / Q000494:pharmacology*; D059014:High-Throughput Nucleotide Sequencing; D012275:Oryza / Q000187:drug effects / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D017354:Point Mutation; D017422:Sequence Analysis, DNA
D006540:Herbicides; D010940:Plant Proteins; D000095:Acetolactate Synthase
Acetolactate synthase; Gene targeting; Herbicide tolerance; Rice