Kirby T. Nilsen, Sean Walkowiak,  Daoquan Xiang,  Peng Gao,  Teagen D. Quilichini,  Ian R. Willick,  Brook Byrns,  Amidou N’Diaye,  Jennifer Ens,  Krystalee Wiebe,  Yuefeng Ruan,  Richard D. Cuthbert,  Melanie Craze,  Emma J. Wallington, James Simmonds, Cristobal Uauy,  Raju Datla, and Curtis J. Pozniak

PNAS November 17, 2020 117 (46) 28708-28718

Significance

Solid-stemmed wheat cultivars are resistant to the wheat stem sawfly, an important agricultural pest. Here, we identify TdDof as the causal gene that controls stem solidness in wheat. We show that copy number gain of TdDof correlates with its increased expression and the solid-stem phenotype. Our results suggest TdDof could function as a key regulator of genes involved in programmed cell death of the pith parenchyma cells. This research provides the framework to manipulate stem architecture in wheat and other monocots, which can be applied toward downstream agricultural and industrial applications. These include enhancing wheat stem sawfly resistance, modifying carbon partitioning and water-soluble carbohydrate remobilization in plants under drought and temperature stress, and bioenergy production.

Abstract

Stem solidness is an important agronomic trait of durum (Triticum turgidum L. var. durum) and bread (Triticum aestivum L.) wheat that provides resistance to the wheat stem sawfly. This dominant trait is conferred by the SSt1 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 TdDof, a gene encoding a putative DNA binding with one finger protein, controls the stem solidness trait in wheat. Using map-based cloning, we localized TdDof to within a physical interval of 2.1 Mb inside the SSt1 locus. Molecular analysis revealed that hollow-stemmed wheat cultivars such as Kronos carry a single copy of TdDof, whereas solid-stemmed cultivars such as CDC Fortitude carry multiple identical copies of the gene. Deletion of all TdDof copies from CDC Fortitude resulted in the loss of stem solidness, whereas the transgenic overexpression of TdDof restored stem solidness in the TdDof deletion mutant pithless1 and conferred stem solidness in Kronos. In solid-stemmed cultivars, increased TdDof 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 TdDof 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.

See https://www.pnas.org/content/117/46/28708

Figure 1: CNV of TdDof is associated with stem solidness in wheat. (A) Fine-map interval of SSt1 in the Kofa/W9262-260D3 F₂ population. The region between closest flanking markers usw306 and usw308 is shown in blue. (B) Chromium read coverage plots within the SSt1 interval on chromosome 3B of five durum lines. The y axis of each coverage plot denotes the minimum and maximum read coverage for each line within the interval. GemCode molecule associations spanning structural variants are indicated by the green bars (CDC Fortitude, W9262-260D3) and red bar (pithless1). Positions are in megabases. (C) Diagram showing the organization of the TdDof CNV region in CDC Fortitude (solid-stemmed) and Kofa (hollow-stemmed) with CNV breakpoints indicated by the black bars. Three copies of TdDof (TdDof1-3) are present in CDC Fortitude vs. a single copy in Kofa. At the CNV breakpoint between TdDof1-2 and TdDof2-3, a unique 25-bp insertion is present that is only found in solid-stemmed cultivars. (D) High-throughput KASP (kompetitive allele-specific PCR) marker usw275 accurately distinguishes stems that are hollow (red) and solid (blue) in both bread and durum wheat.