Identification of Rare Alleles in Soybean Population using TILLInG by Sequencing

Modified carbohydrate composition in soybean improves metabolizable energy (ME) for monogastric animals. Incompletely removed during standard processing of soybean meal, raffinose family oligosaccharides (RFOs) reduce ME due to fermentation in the gut of monogastric animals, while sucrose increases ME. The goal of this project is to identify genetic variation for carbohydrate composition. To achieve this we implemented chemical mutagenesis via N-nitroso-N-methylurea (NMU), which induces single-nucleotide polymorphism (SNP) mutations across the genome. Through the application of TILLING (Targeting Induced Local Lesions In Genomes), a reverse genetic technique, unique chemically induced mutations can be identified within target genes that have potential to alter gene function. The main challenge of TILLInG is the cost, compounded by the fact that in soybean multiple copies of homeologous genes are present. A strong mutation, or sometimes even a double mutant, is often required to observe a phenotype. Our method makes this more affordable by surveying the available diversity in the population using next-generation sequencing, then isolating mutant individuals by genotyping with a custom SNP marker. In this study we describe our method and report the successful identification of a mutant allele of RS3 (RAFFINOSE SYNTHASE3), which is expected to reduce levels of RFOs and impact carbohydrate composition.