Development and characterization of an oat TILLING-population and identification of mutations in lignin and β-glucan biosynthesis genes

Abstract

BackgroundOat, Avena sativa is the sixth most important cereal in the world. Presently oat is mostly used as feed for animals. However, oat also has special properties that make it beneficial for human consumption and has seen a growing importance as a food crop in recent decades. Increased demand for novel oat products has also put pressure on oat breeders to produce new oat varieties with specific properties such as increased or improved β-glucan-, antioxidant- and omega-3 fatty acid levels, as well as modified starch and protein content. To facilitate this development we have produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the spring oat cultivar SW Belinda.ResultsHere a population of 2600 mutagenised M2 lines, producing 2550 M3 seed lots were obtained. The M2 population was initially evaluated by visual inspection and a number of different phenotypes were seen ranging from dwarfs to giants, early flowering to late flowering, leaf morphology and chlorosis. Phloroglucinol/HCl staining of M3 seeds, obtained from 1824 different M2 lines, revealed a number of potential lignin mutants. These were later confirmed by quantitative analysis. Genomic DNA was prepared from the M2 population and the mutation frequency was determined. The estimated mutation frequency was one mutation per 20 kb by RAPD-PCR fingerprinting, one mutation per 38 kb by MALDI-TOF analysis and one mutation per 22.4 kb by DNA sequencing. Thus, the overall mutation frequency in the population is estimated to be one mutation per 20-40 kb, depending on if the method used addressed the whole genome or specific genes. During the investigation, 6 different mutations in the phenylalanine ammonia-lyase (AsPAL1) gene and 10 different mutations in the cellulose synthase-like (AsCslF6) β-glucan biosynthesis gene were identified.ConclusionThe oat TILLING population produced in this work carries, on average, hundreds of mutations in every individual gene in the genome. It will therefore be an important resource in the development of oat with specific characters. The population (M5) will be available for academic research via Nordgen http://www.nordgen.org as soon as enough seeds are obtained.[Genbank accession number for the cloned AsPAL1 is GQ373155 and GQ379900 for AsCslF6]

Creation of a TILLING Population in Barley After Chemical Mutagenesis with Sodium Azide and MNU

“…There are many successful examples of using TILLING for crop improvement, for example, in Triticum aestivum and Triticum turgidum subsp. durum for obtaining a better starch quality (Slade and Knauf 2005); in Hordeum vulgare for the manipulation of spike morphology (Gottwald et al 2009), starch content (Sparla et al 2014), and waterlogging tolerance (Mendiondo et al 2016); in Cucumis melo (Dahmani-Mardas et al 2010) and Solanum lycopersicum (Okabe et al 2011) for enhanced shelf life; and in Sorghum bicolor (Xin et al 2008) and Avena sativa (Chawade et al 2010) for increased digestibility.…”

Mutation Detection by Analysis of DNA Heteroduplexes in TILLING Populations of Diploid Species

“…Among them are techniques such as single-strand conformation polymorphism (SSCP) (Hestekin and Barron 2006), denaturing high-performance liquid chromatography (DHPLC) (McCallum et al 2000), temperature/denaturing gradient gel electrophoresis (TGGE/DGGE) (Hestekin and Barron 2006), conformation-sensitive gel/capillary electrophoresis (CSGE/CSCE) (Gady et al 2009), high-resolution melting (HRM) (Gundry et al 2003), mismatch cleavage (Colbert et al 2001;Till et al 2006a) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) (Chawade et al 2010). The improvement of new molecular techniques and increased knowledge of gene sequences has led to the invention of Targeting Induced Local Lesions IN Genomes (TILLING)-a reverse genetics method that combines the creation of a mutated population through classical mutagenesis with rapid and high-throughput screening for individuals that carry mutations in the desired genes (McCallum et al 2000).…”

Trends in breeding oat for nutritional grain quality - An overview

“…In a case study, oat population of 2600 mutagenised M2 lines, producing 2550 M3 seed lots was developed and the mutation frequency determined. Chawade et al, 2010 demonstrated the potential of the oat TILLING population by identifying ten different mutations in the cellulose synthase-like (AsCslF6) β-glucan biosynthes gene -key gene in the β-glucan biosynthetic pathway. Since β-glucans are becoming very important functional food ingredients, an oat variety with increased or modified β-glucan content in the seed will significantly increase the value of the crop.…”