TILLING in Cereal Crops for Allele Expansion and Mutation Detection by Using Modern Sequencing Technologies

Abstract

A substantial increase in yield of food crops is crucial to feeding the burgeoning global population. There is a need to introduce new breeding strategies that will accelerate the average phenotypic values of crop plants. The use of induced mutations coupled with modern genomics tools is an effective strategy for identifying and manipulating genes for crop improvement. High-throughput TILLING (Targeting Induced local Lesions IN Genomes) methodology, detects mutations in mutagenized populations, and EcoTILLING identifies single nucleotide polymorphisms (SNPs) within a natural population and associates these variations with traits of breeding interest. The main advantage of these techniques as a “reverse genetics” strategy is that they can be applied to any species regardless of genome size and ploidy level. In cereals, several space-induced and EMS-induced mutant populations have been used to identify mutants with important traits including salinity tolerance, grain size, and recombinant crossovers via TILLING by sequencing (TbyS). Genes such as TaSSIV, which plays an important role in starch granule formation, and Pin a and Pin b, which have been associated with kernel hardness in wheat, have been exploited in cereals via the EcoTILLING approach. This review focused on the functions and challenges of TILLING and the relation of TILLING to next-generation sequencing (NGS) technologies which help to exploit the induced mutations and their potential applications in cereal crops.

Highly Efficient and Comprehensive Identification of Ethyl Methanesulfonate-Induced Mutations in Nicotiana tabacum L. by Whole-Genome and Whole-Exome Sequencing

“…As comprehensive reverse genetic resources, chemically or physically induced mutant libraries are extremely valuable tools for studying gene function as well as for increasing the number of useful alleles in plant breeding (Suprasanna et al, 2015). Mutant libraries have been produced in various crop plants to efficiently generate phenotypic variations, enabling functional analysis of genes and the ability to ameliorate the phenotype in molecular breeding (Gupta et al, 2017;Penna and Jain, 2017;Irshad et al, 2020). Ethyl methanesulfonate (EMS) has been used widely as an effective mutagen for chemical mutagenesis in diverse plant species.…”

Soluble Starch Synthase Enzymes in Cereals: An Updated Review

“…TaSSIV is expressed in leaves and seeds of wheat [75]. The identification of differences at the amino acid level in TaSSIVb and OsSSIVb in their glycosyltransferase domains might indicate different functional associations [76,77]. TaSSIV overexpression increases the accumulation of starch in both photosynthetic and sinks organs.…”

Accurate, Efficient and User-Friendly Mutation Calling and Sample Identification for TILLING Experiments

“…In brief, TILLING consists of mutagenesis, DNA extraction and pooling of several individuals of a population, PCR amplification of regions of interest, and high-throughput mutation discovery in target genes ( McCallum et al, 2000 ). Despite newer technologies being available for targeted modification of genes such as CRISPR-Cas, TILLING remains a useful and effective functional genomics tool for studying genes responsible for desired phenotypes because large populations can be screened for mutations before bringing plants to the field, thus reducing phenotyping costs, and it generates genome-wide mutations allowing to target in multiple genes at the same time ( Irshad et al, 2020 ). With the advance in high-throughput sequencing technologies and their current lower costs, TILLING by Sequencing proves to be the best choice for the identification of mutations and the corresponding mutant individuals in pooled samples, and for linking the identified base pair changes with their impact on specific traits ( Tsai et al, 2011 ).…”