Identification of Rare Alleles in Soybean using TILLInG by Sequencing

One of the significant problems with establishing a TILLING resource for soybean is the paleopolyploid origin of soybean, which means that multiple highly similar sequences are present for each gene.  This creates both technical complications for amplification and sequence analysis, as well as a practical problem that both highly similar copies may have to be mutated in order to obtain the desired effect of a gene knock-out. These factors make TILLING in soybean cost prohibitive. In this project we have pilot tested a method to survey the available diversity and select the most damaging mutations and identify the individuals that carry them by genotyping pools and subsequently individual plants. We have used this approach to identify mutations in genes that affect seed carbohydrate biosynthesis. The reverse-genetics approach will be compared to forward genetics approaches in soybean in which we have also utilized next generation sequencing to identify causative alleles.