High-Throughput Detection of Induced Mutations and Natural Variation Using KeyPoint™ Technology

Abstract

Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variants among mutant or natural populations. Current (pre-) screening methods such as TILLING and EcoTILLING are based on the detection of single base mismatches in heteroduplexes using endonucleases such as CEL 1. However, there are drawbacks in the use of endonucleases due to their relatively poor cleavage efficiency and exonuclease activity. Moreover, pre-screening methods do not reveal information about the nature of sequence changes and their possible impact on gene function. We present KeyPoint™ technology, a high-throughput mutation/polymorphism discovery technique based on massive parallel sequencing of target genes amplified from mutant or natural populations. KeyPoint combines multi-dimensional pooling of large numbers of individual DNA samples and the use of sample identification tags (“sample barcoding”) with next-generation sequencing technology. We show the power of KeyPoint by identifying two mutants in the tomato eIF4E gene based on screening more than 3000 M2 families in a single GS FLX sequencing run, and discovery of six haplotypes of tomato eIF4E gene by re-sequencing three amplicons in a subset of 92 tomato lines from the EU-SOL core collection. We propose KeyPoint technology as a broadly applicable amplicon sequencing approach to screen mutant populations or germplasm collections for identification of (novel) allelic variation in a high-throughput fashion.

Genetic Diversity in Tomato (Solanum lycopersicum) and Its Wild Relatives

“…TILLING allows generating variants in cultivated genetic background (Piron, Nicolaï et al 2010) and thus transfer rapidly interesting mutations into cultivars (Gady, Hermans et al 2009). Application of TILLING technique to screen for natural variation within tomato germplasm collection is now performed (Rigola, van Oeveren et al 2009). …”

Micro-Tom mutants for functional analysis of target genes and discovery of new alleles in tomato

“…To date, identification of mutations in tomato by TILLING using various technologies has been reported for several EMS mutant collections generated in either the determinate tomato cultivars M82, Red Setter, and TPAADASU used for processing tomatoes (Gady et al 2009;Menda et al 2004;Minoia et al 2010;Rigola et al 2009) or in the semi-determinate cultivar Arka Vikas used for fresh market (Sreelakshmi et al 2010). Recently published results highlight how powerful this approach can be in providing new alleles for deciphering the function of tomato genes involved in virus resistance Rigola et al 2009), plant growth and architecture (Busch et al 2011;MacAlister et al 2012;Martín-Trillo et al 2011) and fruit nutritional quality (Di Matteo et al 2013;Gady et al 2012;Jones et al 2012).…”

“…Recently published results highlight how powerful this approach can be in providing new alleles for deciphering the function of tomato genes involved in virus resistance Rigola et al 2009), plant growth and architecture (Busch et al 2011;MacAlister et al 2012;Martín-Trillo et al 2011) and fruit nutritional quality (Di Matteo et al 2013;Gady et al 2012;Jones et al 2012). In Micro-Tom, screening 3,052 EMS mutants from the Japan Micro-Tom collection for allelic series of 10 genes involved in ethylene signaling (the SlETR1, SlETR2, SlETR3, SlETR4, SlETR5 and SlETR6 genes), γ-aminobutyric acid (GABA) metabolism (the SlSSADH and SlGABAT1 and SlGABAT3 genes) and fruit softening (the SlPL gene) allowed the identification of up to 35 allelic mutants .…”

Next-Generation Sequencing for Targeted Discovery of Rare Mutations in Rice

“…Instead, mutation discovery using massive parallel sequencing approaches have been focused on the gene space, where mutations are most likely to affect function. This can be accomplished by 3D DNA multiplexing followed by pooling of amplicons derived from genes of interest (Tsai et al 2011(Tsai et al , 2013Rigola et al 2009) or via the use of sequence capture methods (Gnirke et al 2009;Fisher et al 2011) to select for gene coding sequences (Porreca et al 2007;Ng et al 2009;Nijman et al 2010). The latter of the two strategies represents a global or genome-wide approach to mutation discovery by focusing on all or most of the gene coding regions within a genome of interest (i.e., the exome).…”

“…Sequencing of PCR amplicons represents a natural extension of the traditional TILLING method (Tsai et al 2011;Rigola et al 2009). An advantage of mutation detection using next-generation sequencing platforms is increased sensitivity, which enables discovery in more complex DNA pools (64Â deep) than traditional TILLING (8Â deep).…”