Using Bulk Segregation Analysis for Mutant Identification in Wheat

Next generation sequencing has paved the way for simultaneous mapping and identification of phenotype inducing mutations in living organisms. Such approaches are extremely useful in well-characterized genomes such as Arabidopsis but can be more difficult to adapt to crop species where genome resources are often poor and the genome sizes are large.

Here we identify a mapping interval containing a deletion that is responsible for early flowering in the domesticated diploid wheat T. monococcum with use of an alternative strategy, based on the SHOREmapping¹ approach. The large size of the bread wheat allohexaploid genome (~17GB) makes sequencing to a sufficient depth for mutant identification prohibitively expensive so this project was undertaken with use of our NimbleGen wheat gene enrichment array.

RIL25 (early flowering) and RIL71 (wild type) parental lines were developed and two F2 bulk segregant populations corresponding to wild type phenotype (Bulk A) and Bulk B (mutant) were created from extracted DNA, encompassing over 250 plants. The two bulk segregant samples along with DNA from the 2 RIL parental lines were enriched using the wheat gene capture array and sequenced using the Illumina HiSeq technology. Analysing the frequency of parental homozygotes in the Bulk A/Bulk B samples allowed identification of a 600Kb homozygote rich region at the end of chromosome 3 containing the early flowering phenotype inducing deletion. We also demonstrate through simulated data that this method could be implemented successfully on tetraploid or hexaploid wheat.

1.         Schneeberger, K. et al. SHOREmap: simultaneous mapping and mutation identification by deep sequencing. Nat Methods 6, 550–551 (2009).