Whole-Genome Analysis of Herbicide-Tolerant Mutant Rice Generated by Agrobacterium-Mediated Gene Targeting

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.

Genomic Variation between PRSV Resistant Transgenic SunUp and Its Progenitor Cultivar Sunset

“…S2). This result support previous reports on the pattern of nucleotide substitutions, regardless of whether SNPs were caused by spontaneous and somaclonal mutations [28,39], chemical and physical mutagens [45], or by Agrobacterium-mediated gene targeting and transformation [29,46]. Overabundance of G/C to A/T fit the earlier theory that G:C sites in CpG contexts are more likely to be methylated [47], and spontaneous deamination of methylated cytosine would lead to thymine substitution [48,49].…”

“…Whole-genome sequencing (7×) and CGH arrays were performed to evaluate the molecular composition of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting (GT). In inspected GT rice plants, more than 1,000 SNPs and InDels were identified and over 300 somaclonal mutations were predicted to be induced between generations, although no integration of Agrobacterium-derived DNA fragments had been detected [29]. The availability of the 'SunUp' draft genome, the rapid evolution of deepsequencing technology along with increasingly robust bioinformatics tools make it possible to decipher genomewide structural perturbations at the single-base resolution level in the transgenic papaya genome after subjection to bombardment, tissue culture, and other spontaneous mutations during 20-year's separation.…”

Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing

“…For example, NGS-based analysis has been used for examining genetic variations in transgenic rice plants ( Kawakatsu et al, 2013;Endo et al, 2015) and for investigating the molecular spectrum of somaclonal variation in regenerated rice (Miyao et al, 2012). Recently, NGS techniques were also used to study the frequency and type of spontaneous (Ossowski et al, 2010) and induced mutations, i.e.…”

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

“…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) and rice (Yang et al, 2013), and for examining other types of genetic variation in transgenic rice plants (Kawakatsu et al, 2013;Endo et al, 2014;Wei et al, 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.…”