Copy number variation of <i>TdDof</i> controls solid-stemmed architecture in wheat

Stem solidness is an important agronomic trait of durum (<i>Triticum turgidum</i> L. var. <i>durum</i>) and bread (<i>Triticum aestivum</i> L.) wheat that provides resistance to the wheat stem sawfly. This dominant trait is conferred by the <i>SSt1</i> locus on chromosome 3B. However, the molecular identity and mechanisms underpinning stem solidness have not been identified. Here, we demonstrate that copy number variation of <i>TdDof</i>, a gene encoding a putative DNA binding with one finger protein, controls the stem solidness trait in wheat. Using map-based cloning, we localized <i>TdDof</i> to within a physical interval of 2.1 Mb inside the <i>SSt1</i> locus. Molecular analysis revealed that hollow-stemmed wheat cultivars such as Kronos carry a single copy of <i>TdDof</i>, whereas solid-stemmed cultivars such as CDC Fortitude carry multiple identical copies of the gene. Deletion of all <i>TdDof</i> copies from CDC Fortitude resulted in the loss of stem solidness, whereas the transgenic overexpression of <i>TdDof</i> restored stem solidness in the <i>TdDof</i> deletion mutant <i>pithless1</i> and conferred stem solidness in Kronos. In solid-stemmed cultivars, increased <i>TdDof</i> expression was correlated with the down-regulation of genes whose orthologs have been implicated in programmed cell death (PCD) in other species. Anatomical and histochemical analyses revealed that hollow-stemmed lines had stronger PCD-associated signals in the pith cells compared to solid-stemmed lines, which suggests copy number-dependent expression of <i>TdDof</i> could be directly or indirectly involved in the negative regulation of PCD. These findings provide opportunities to manipulate stem development in wheat and other monocots for agricultural or industrial purposes.