Development and characterization of an oat TILLING-population and identification of mutations in lignin and beta-glucan biosynthesis genes.
BACKGROUND
Oat, 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 beta-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.


RESULTS
Here 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) beta-glucan biosynthesis gene were identified.


CONCLUSIONS
The 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].
Chawade
Aakash
A
Department of Plant and Environmental Sciences, Göteborg University, S-40530, Göteborg, Sweden. olof.olsson@gu.se.
Sikora
Per
P
Bräutigam
Marcus
M
Larsson
Mikael
M
Vivekanand
Vivekanand
V
Nakash
Montedar All
MA
Chen
Tingsu
T
Olsson
Olof
O
D018554:Avena / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D020090:Chromosome Segregation / Q000187:drug effects / Q000235:genetics; D003001:Cloning, Molecular; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D017343:Genes, Plant / Q000235:genetics*; D005820:Genetic Testing; D005828:Genetics, Population; D008031:Lignin / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D010696:Phloroglucinol / Q000378:metabolism; D016133:Polymerase Chain Reaction; D019105:Random Amplified Polymorphic DNA Technique; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D047071:beta-Glucans / Q000378:metabolism*
D047071:beta-Glucans; D008031:Lignin; D005020:Ethyl Methanesulfonate; D010696:Phloroglucinol
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