Highly efficient de novo mutant identification in a Sorghum bicolor TILLING population using the ComSeq approach

Abstract

SUMMARYScreening large populations for carriers of known or de novo rare single nucleotide polymorphisms (SNPs) is required both in Targeting induced local lesions in genomes (TILLING) experiments in plants and in screening of human populations. We previously suggested an approach that combines the mathematical field of compressed sensing with next-generation sequencing to allow such large-scale screening. Based on pooled measurements, this method identifies multiple carriers of heterozygous or homozygous rare alleles while using only a small fraction of resources. Its rigorous mathematical foundations allow scalable and robust detection, and provide error correction and resilience to experimental noise. Here we present a largescale experimental demonstration of our computational approach, in which we targeted a TILLING population of 1024 Sorghum bicolor lines to detect carriers of de novo SNPs whose frequency was less than 0.1%, using only 48 pools. Subsequent validation confirmed that all detected lines were indeed carriers of the predicted mutations. This novel approach provides a highly cost-effective and robust tool for biologists and breeders to allow identification of novel alleles and subsequent functional analysis.

Assessment of Phenotypic Variations and Correlation among Seed Composition Traits in Mutagenized Soybean Populations

“…Currently, more than 3200 mutant varieties have been released from 214 plant species throughout the world [23]. The efficient reverse genetics method to determine the function of gene interests in plants, TILLING (Targeting Induced Local Lesions IN Genomes), has been applied to identify the induced mutations from diverse mutagenized populations [24][25][26][27][28]. TILLING typically entails chemical mutagenesis and a high-throughput mutation screening method [29].…”

Characterization of the FAD2 Gene Family in Soybean Reveals the Limitations of Gel-Based TILLING in Genes with High Copy Number

“…TILLING has been commonly employed in many organisms (McCallum et al, 2000; Meksem et al, 2008; Moens et al, 2008; Dierking and Bilyeu, 2009; Colasuonno et al, 2016; Gauffier et al, 2016; Nida et al, 2016). To perform TILLING, a mutagenized population is developed using a chemical mutagen such as ethyl methanesulfonate (EMS).…”

RECAS9: Recombining wild species introgression via mitotic gene editing in barley

“…Nevertheless, the ddPCR method seems to overcome these difficulties although it does require extensive experimentation and DNA adjustments to make this a cost-effective tool, and for examining recombination beyond closely linked SNPs. Obviously, an efficient regeneration protocol from calli into plants would eventually allow screening and validating genetically modified plants by advanced allele mining approaches previously used in TILLING projects (Nida et al, 2016). Moreover, new Cas9 delivery systems, which have recently been developed like nanomaterials delivery(Demirer et al, 2019) and Haploid-Inducer Mediated Genome Editing (IMGE)(Wang et al, 2019)(Kelliher et al, 2019) can be utilized to enhance Cas9 delivery system and allow better penetration.…”