A single nucleotide polymorphism in the Fra gene results in fractured starch granules in barley

We report here that the mutation causing fractured starch granules in the barley line Franubet results from a point mutation in the barley gene corresponding to the rice FLO6 gene. The fra mutation in barley, which was originally isolated and characterized over 30 years ago, results in fractured starch granules and an opaque phenotype. This mutation has been used in breeding programs, since it appears to be useful in the production of pearled barley for human consumption. However, selection for this phenotype is difficult, since wild-type and heterozygous kernels cannot be distinguished phenotypically, and until now, the gene involved in this mutation has not been determined. Here, we used a map-based cloning approach using nanopore sequencing to obtain long reads from a BAC clone carrying markers on either side of the fra locus. By fine mapping followed by aligning RNA-seq reads to four genes within the mapped region, we were able to determine that the fra mutation is caused by the introduction of a stop codon in the barley homologue of the rice FLOURY ENDOSPERM 6 (FLO6) gene. This gene has a CBM48 domain that binds to starch, and may act through interactions with isoamylase1 (ISA1), assisting in the binding of ISA1 to starch granules. Perfect markers able to distinguish all genotypes were designed and tested in several large populations; in all cases, the markers were able to distinguish wild-type, heterozygous, and mutant genotypes.