See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/316428389 TILLING, high-resolution melting (HRM), and next-generation sequencing (NGS) techniques in plant mutation breeding Article in Molecular Breeding · February 2017 CITATION READS 1 199 5 authors, including: Sima Taheri Thohirah Lee Abdullah University of Malaya Universiti Putra Malaysia 49 PUBLICATIONS 190 CITATIONS 118 PUBLICATIONS 454 CITATIONS SEE PROFILE SEE PROFILE Shri Mohan Jain Mahbod Sahebi University of Helsinki Universiti Putra Malaysia 207 PUBLICATIONS 3,913 CITATIONS 73 PUBLICATIONS 402 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Oil Palm View project Salt stress effects on rice View project All content following this page was uploaded by Shri Mohan Jain on 01 August 2018. The user has requested enhancement of the downloaded file. Mol Breeding (2017) 37:40 DOI 10.1007/s11032-017-0643-7 REVIEW TILLING, high-resolution melting (HRM), and next-generation sequencing (NGS) techniques in plant mutation breeding Sima Taheri & Thohirah Lee Abdullah & Shri Mohan Jain & Mahbod Sahebi & Parisa Azizi Received: 15 June 2016 /Accepted: 19 February 2017 # Springer Science+Business Media Dordrecht 2017 Abstract Induced mutations have been used effectively identify single nucleotide mutations in a specific region for plant improvement. Physical and chemical mutagens of a gene of interest with a powerful detection method induce a high frequency of genome variation. Recently, resulted from chemical-induced mutagenesis. The main developed screening methods have allowed the detec- advantage of TILLING as a reverse genetics strategy is tion of single nucleotide polymorphisms (SNPs) and the that it can be applied to any species, regardless of identification of traits that are difficult to identify at the genome size and ploidy level. However, TILLING re- molecular level by conventional breeding. With the quires laborious and time-consuming steps, and a lack assistance of reverse genetic techniques, sequence vari- of complete genome sequence information for many ation information can be linked to traits to investigate crop species has slowed the development of suitable gene function. Targeting induced local lesions in ge- TILLING targets. Another method, high-resolution nomes (TILLING) is a high-throughput technique to melting (HRM), which has assisted TILLING in muta- tion detection, is faster, simpler and less expensive with non-enzymatic screening system. Currently, the se- S. Taheri (*) : T. L. Abdullah : P. Azizi quencing of crop genomes has completely changed our Department of Crop Science, Faculty of Agriculture, University vision and interpretation of genome organization and Putra Malaysia, 43400 Serdang, Selangor, Malaysia evolution. Impressive progress in next-generation se- e-mail: [email protected] quencing (NGS) technologies has paved the way for T. L. Abdullah the detection and exploitation of genetic variation in a e-mail: [email protected] given DNA or RNA molecule. This review discusses the applications of TILLING in combination with HRM P. Azizi e-mail: [email protected] and NGS technologies for screening of induced muta- tions and discovering SNPs in mutation breeding S. M. Jain programs. Department of Agricultural Sciences, University of Helsinki, PL-27, Latokartanonkaari 5, Helsinki, Finland e-mail: [email protected] Keywords Mutation breeding . Mutagenesis . EMS . M. Sahebi TILLING High resolution melting Next-generation Laboratory of Plantation Crops, Institute of Tropical Agriculture, sequencing . SNPs Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia e-mail: [email protected] Abbreviations M. Sahebi BSA Bulk segregant analysis Department of Land Management, Faculty of Agriculture, CEL I Celery endonuclease Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia CE Capillary electrophoresis 40 Page 2 of 23 Mol Breeding (2017) 37:40 CODDLE Codons optimized to detect deleterious therefore, physical and chemical mutagens are important lesions formutationinductionincropbreedingprograms.Muta- ComSeq Compressed sequencing approach tions theoretically encompass all changes that occur in the dCARE Deep candidate resequencing DNA sequence and result in changes in the genetic code DGGE Denaturing gradient gel electrophoresis (van Harten 1998). An induced mutation is an alternative dHPLC Denaturing high performance liquid and complementary technique in plant breeding for the chromatography introduction of genetic changes and the establishment of EMS Denaturing methanesulphonate new genetic resources. In the 1920s, Lewis Stadler at the HDA Heteroduplex analysis University of Missouri discovered the mutagenic effects HRM High-resolution melting of X-rays in barley by exposing barley seeds. Since the IAEA International Atomic Energy Agency; 1950s, it has been widely used, specifically in crops with indels: insertion/deletion low genetic variability and those that are not amenable to MAB Mutation-assisted breeding improvement through conventional breeding methods. MAF Mutant allele frequency The number of physical and chemical mutagens used in MAS Marker-assisted selection mutation breeding is large and continues to increase (Mba mRNA Messenger RNA et al. 2010;Mba2013). According to the International miRNA microRNA Atomic Energy Agency (IAEA) Mutant Varieties Data- NGM Next-generation mapping base (http://mvgs.iaea.org), 3233 mutant varieties of 214 NGS Next-generation sequencing different plant species have been officially released PCR Polymerase chain reaction worldwide (Joint 2015). Of these, 49.5, 21.9, and 15% SNP Single nucleotide polymorphism belongs to cereals, ornamental and decorative plants and SSCP Single-strand conformational legumes, respectively (Fig. 1). Gamma rays have been polymorphism most used successfully to develop new mutant varieties SSR Simple sequence repeat of ornamentals with novel leaf and flower colour/shape T-DNA Transfer DNA (Taheri et al. 2014). TGGE Temperature gradient gel electrophoresis In any plant breeding program, genetic variation is TGS Target genome sequencing necessary for crop improvement. Using mutagenesis to TILLING Targeting induced local lesions in genomes create novel genetic variation is extremely effective to TPSeq Targeted parallel sequencing promote natural genetic resources with the induction of WGS Whole genome sequencing random changes throughout the genome, resulting in a single plant with a large number of different mutations. Chemical and physical mutagenesis has successfully assisted the development of improved and new cultivars Introduction among both seed and vegetatively propagated crops (Parry et al. 2009). Seeds of seed-propagated crops are One of the keys to sustainable agriculture is the creation treated with the mutagens while in vegetatively propa- of genetic variation in plant crops (Griggs et al. 2013). gated plants stem cuttings, twigs, bulbs, buds, rhizomes Over the past 70 years, mutation breeding has generated and tubers are exposed to the mutagens. Chemical thousands of novel crop varieties in hundreds of crop mutagens most often produce point mutations (Kodym species and billions of dollars in additional revenue, and Afza 2003) and chromosome breaks that result in delivering higher yields, increased nutritional value and various chromosomal rearrangements that affect plant resilience to the effects of climate change, such as resis- fertility and cause lethality. Among the different chem- tance to diseases and tolerance to drought. Mutation ical mutagens, ethyl methane sulphonate (EMS) is the refers to sudden and heritable changes to the genetic most powerful, effective, reliable and frequently used material in any organism not caused by normal genetic chemical mutagens in plants. Treatment with the segregation or recombination including spontaneous mu- mutagen breaks the nuclear DNA, and during the tations and induced mutations (Suprasanna et al. 2015; process of DNA repair, new mutations are induced Mba 2013;Austinetal.2011; Oladosu et al. 2016). randomly and are heritable. The changes can also occur Spontaneous mutations occur in nature at a very low rate; in cytoplasmic organelles, again resulting in Mol Breeding (2017) 37:40 Page 3 of 23 40 Fig. 1 Percentage (%) of mutant varieties by crop type reported on the Joint FAO/IAEA Mutant Varieties Database (Joint FAO/ IAEA 2015) 15 % 49.5 % 21.9 % Cerals Flowers/Ornamentals Legumes Fruits/Nuts Vegetable crops Fiber crops Oil crops Others Forrage crops Root & Tuber crops Herbs Medicinal plants chromosomal or genomic mutations that enable plant and recombination of available desired genes from gene breeders to select useful mutants such as those with pools and the related plant species by sexual hybridiza- modified flower colour, flower shape, disease resistance tion, plant breeders could successfully develop new and early flowering phenotypes (Jain 2010). Breeders cultivars with high yield and resistance to abiotic and are most interested in point mutations because large- biotic stresses. One of the advantages of mutation in- scale changes can have negative effects on chromosome duction is the chance of obtaining unselected genetic structures. However, large-scale changes in chromo- variation when one or few characters of a prominent some structure increase the number of recombination cultivar are to be modified to improve plants (Jain events, and the breaking of undesirable