Brachypodium distachyon Bd21-3 Tilling By Sequencing

TILLING (Targeting induced local lesions IN genomes) is an efficient reverse genetics technique to identify mutations in genes of interest through PCR-based screens of mutagenized populations.  To develop this platform for.  Brachypodium distachyon, an excellent model plant for the functional genomics of  temperate grasses, we have developed both genetics resources and an informatics pipeline to detect rare EMS-induced mutations in pools of individuals using high throughput sequencing technology.  We have established an EMS-mutagenized Bd21-3 population of approximately 4000 M2 families.  By integrating TILLING methods with Illumina sequencing of target gene amplicons, we have developed a protocol for efficiently identifying single basepair mutations. To date, we have screened for mutations in four genes of interest. A total of 47 SNPs were detected and 40 SNPs were confirmed through Sanger sequencing. Therefore, our Brachypodium distachyon mutant population and the TILLING by sequencing protocol are valuable and should greatly facilitate reverse genetic approaches for gene discovery in this model grass system.