Australian wheat cultivar Axe produced resistant to moderately resistant stripe rust responses under eld conditions and was exhibiting seedling response varying from 33C to 3+ under greenhouse conditions. Experiments covering tests at different growth stages (2 nd , 3 rd and 4 th leaf stages) demonstrated the clear expression of resistance at the 4 th leaf stage under controlled-environment greenhouse conditions. A recombinant inbred line (RIL) population was developed from the Axe/Nyabing-3 (Nyb) cross. Genetic analysis of Axe/Nyb RIL population in the greenhouse at the 4 th leaf stage showed monogenic inheritance of stripe rust resistance. Selective genotyping using the iSelect 90K In nium SNP genotyping array was performed and the resistance locus was mapped to long arm of chromosome 7A and named Yr75. The Axe/Nyb RIL population was genotyped using a targeted genotype-by-sequencing (tGBS) assay and the resistance-linked SNPs were converted into kompetitive allele speci c PCR (KASP) markers. These markers were tested on the entire Axe/Nyb RIL population and markers sunKASP_430 and sunKASP_427 showed close association with Yr75 in Axe/Nyabing-3 RIL population. A high-resolution mapping family of 1032 F 2 plants from the Axe/Nyb cross was developed and genotyped with sunKASP_430 and sunKASP_427 and these markers anked Yr75 at 0.3 cM and 0.4 cM, respectively. These markers covers 1.24Mb of the physical map of Chinese Spring and can be used for future map-based cloning of this gene.

The utility of CRISPR in plants has remained limited by the dual difficulties of delivering the molecular machinery to target cells and the use of somatic cell techniques that require tissue culture-based de novo organogenesis. We developed 5-10 nm isodiametric polyplex nanoassemblies, comprising poly [2-(dimethylamino)ethylmethacrylate] PDMAEMA (PD) polycationic linear homopolymers and CRISPR/Cas9 ribonucleoproteins (RNPs), that enable endocytosis-driven RNP uptake into pollen grains. Pollen from wheat plants (genotype Gladius+Sr50), homozygous for monogenic Sr50-mediated resistance to stem rust (Puccinia graminis f. sp. tritici -Pgt), were incubated with RNP/PD nanoassemblies targeting the dominant, Sr50 rust resistance gene. The treated pollen grains were then used to fertilize Gladius+Sr50 florets and the resulting M1 plants were tested for loss of Sr50 function via rust resistance screens. The identification of fully susceptible M1 seedlings indicated that the Sr50 RNPs acted on both alleles, indicating they were transferred via the treated pollen to the zygote. The ability to readily deliver CRISPR RNPs to reproductive cells via biodegradable, polymeric nanocomplexes has significant implications for the efficiency of gene editing in plants.

The use of biotechnology for the genetic improvement of wheat (Triticum aestivum L.) has been hampered by its recalcitrance to standard transformation and regeneration protocols. Male gametes present a potentially useful option for introducing gene edits via clustered regularly interspaced short palindromic repeats (CRISPR). However, the utility of male gametes for introducing genetic improvements would be dependent on the retention of viability after treatment to introduce the CRISPR components. We have studied wheat pollen morphology and its viability in a range of germination media to identify conditions that optimize the viability of in vitro hydrated pollen. The size, shape, and aperture from seven different wheat genotypes were compared using scanning electron microscope (SEM). The SEM results revealed that the pollen of all of the wheat genotypes examined in this study were monoporate; however, a significant variation in the size of the mature pollen grains was observed. The hydrated pollen of the wheat genotypes remained viable for up to five hours at 20 ± 2 °C. Of all of the germination media tested, the medium containing 5% sucrose, 10% PEG4000, 100 mg/L boric acid, 200 mg/L calcium nitrate, 100 mg/L potassium nitrate, and 100 mg/L magnesium sulphate at pH 6.5 achieved the highest percentage of pollen germination after 5 h of hydration. Impedance Flow Cytometry (IFC) provided similar results to the in vitro germination study. This work elucidates important factors that can form the basis for a pollen-based non-genetically modified system for gene editing in wheat.

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Australian wheat cultivar Axe produced resistant to moderately resistant stripe rust responses under field conditions and was exhibiting seedling response varying from 33C to 3+ under greenhouse conditions. Experiments covering tests at different growth stages (2nd, 3rd and 4th leaf stages) demonstrated the clear expression of resistance at the 4th leaf stage under controlled-environment greenhouse conditions. A recombinant inbred line (RIL) population was developed from the Axe/Nyabing-3 (Nyb) cross. Genetic analysis of Axe/Nyb RIL population in the greenhouse at the 4th leaf stage showed monogenic inheritance of stripe rust resistance. Selective genotyping using the iSelect 90K Infinium SNP genotyping array was performed and the resistance locus was mapped to long arm of chromosome 7A and named Yr75. The Axe/Nyb RIL population was genotyped using a targeted genotype-by-sequencing (tGBS) assay and the resistance-linked SNPs were converted into kompetitive allele specific PCR (KASP) markers. These markers were tested on the entire Axe/Nyb RIL population and markers sunKASP_430 and sunKASP_427 showed close association with Yr75 in Axe/Nyabing-3 RIL population. A high-resolution mapping family of 1032 F2 plants from the Axe/Nyb cross was developed and genotyped with sunKASP_430 and sunKASP_427 and these markers flanked Yr75 at 0.3 cM and 0.4 cM, respectively. These markers covers 1.24Mb of the physical map of Chinese Spring and can be used for future map-based cloning of this gene.