Harbans Bariana

Transfer of leaf resistance genes into elite lines can assist in developing future wheat cultivars and mitigate economic losses caused by the leaf rust pathogen, Puccinia triticina Erikss. (Pt). Some previously reported leaf rust resistance QTL have been challenged by aggressive Pt pathotypes. This experiment aims to detect genetically diverse resistance QTL using a Tunisian landrace, Aus26670, which confers a high level of adult plant leaf rust resistance against Australian Pt pathotypes. One hundred nineteen F₇ recombinant inbred lines (RILs) were generated after crossing Aus26670 and a susceptible line Avocet ‘S’ (AvS). The Aus26670/AvS RIL population was evaluated against mixture of five Pt pathotypes under field conditions for two years. The same RIL population was also screened using three Pt pathotypes individually in the greenhouse under controlled conditions. Genetic analysis of the seedling leaf rust response against Pt104-2,3,6, (7) confirmed the presence of an all-stage resistance (ASR) gene, Lr13. The RIL population was genotyped using a targeted genotyping-by-sequencing (tGBS) assay, and Lr13 was mapped in the 153.9–182.1 Mb region in chromosome 2BS. QTL analysis suggested the involvement of three genomic regions/adult plant resistance genes (APR) namely QLr.sun-1BL/Lr46, QLr.sun-5DL and QLr.sun-7DS, in controlling leaf rust resistance. Data of seedling assays, known marker survey, and comparison of genomic regions detected in this study with previously reported APR indicated the uniqueness of QLr.sun-5DL (559.7 Mb) and QLr.sun-7DS (11 Mb). Both QTL represent new additions to the APR toolbox.

A seedling susceptible Australian common wheat cultivar Sentinel showed resistance to stripe rust under field conditions. A Sentinel/Nyabing3 (Nyb3)-derived recombinant inbred line (RIL) population was phenotyped. A DArTseq marker-based linkage map of the Sentinel/Nyb3 RIL population was used to determine the chromosomal location of the adult plant stripe rust resistance possessed by Sentinel. Three consistent quantitative trait loci (QTL); QYr.sun-1BL, QYr.sun-2AS and QYr.sun-3BS were detected, and they on an average explained 18%, 15.6% and 10.6% of the variation in stripe rust response, respectively. All three QTL were contributed by Sentinel. QYr.sun-1B corresponded to the previously characterized gene Yr29. Sentinel expressed resistance at the four-leaf stage at 21 ± 2 °C in the greenhouse. Monogenic segregation among the RIL population was observed when screened at the four-leaf stage at 21 ± 2 °C in the greenhouse, and the underlying resistance locus was temporarily named YrSen. QYr.sun-3BS peaked on YrSen. QYr.sun-2AS was mendelized by generating and phenotyping a mongenically sgregating F₆ RIL population, and it was temporarily designated YrSen2. RILs carrying Yr29, YrSen and YrSen2 in combination exhibited responses like the parent Sentinel. Based on a comparison of the genomic locations and resistance expression with stripe rust resistance genes previously located in their respective chromosomes, QYr.sun-2AS (YrSen2) and QYr.sun-3BS (YrSen) were concluded to represent new loci.

Key message Sr65 in chromosome 1A of Indian wheat landrace Hango-2 is a potentially useful all-stage resistance gene that currently protects wheat from stem rust in Australia, India, Africa and Europe. Abstract Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), threatened global wheat production with the appearance of widely virulent races that included TTKSK and TTRTF. Indian landrace Hango-2 showed resistance to Pgt races in India and Australia. Screening of a Hango-2/Avocet ‘S’ (AvS) recombinant inbred line population identified two stem rust resistance genes, a novel gene (temporarily named as SrH2) from Hango-2 and Sr26 from AvS. A mapping population segregating for SrH2 alone was developed from two recombinant lines. SrH2 was mapped on the short arm of chromosome 1A, where it was flanked by KASP markers KASP_7944 (proximal) and KASP_12147 (distal). SrH2 was delimited to an interval of 1.8–2.3 Mb on chromosome arm 1AS. The failure to detect candidate genes through MutRenSeq and comparative genomic analysis with the pan-genome dataset indicated the necessity to generate a Hango-2 specific assembly for detecting the gene sequence linked with SrH2 resistance. MutRenSeq however enabled identification of SrH2-linked KASP marker sunCS_265. Markers KASP_12147 and sunCS_265 showed 92% and 85% polymorphism among an Australian cereal cultivar diversity panel and can be used for marker-assisted selection of SrH2 in breeding programs. The effectiveness of SrH2 against Pgt races from Europe, Africa, India, and Australia makes it a valuable resource for breeding stem rust-resistant wheat cultivars. Since no wheat-derived gene was previously located in chromosome arm 1AS, SrH2 represents a new locus and named as SR65.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is among the major threats to global wheat production. The common wheat landraces AUS27506 and AUS27894 displayed stripe rust resistance against several commercially prevailing Pst pathotypes. These genotypes were crossed with a stripe-rust-susceptible landrace AUS27229 to understand the inheritance of resistance and to determine the genomic location(s) of underlying gene(s). F₃ generations of crosses AUS27506/AUS27229 and AUS27894/AUS27229 showed monogenic segregation for stripe rust resistance under greenhouse conditions. The absence of segregation for stripe rust response among the AUS27506/AUS27894-derived F₃ population suggested that both genotypes carry the same gene. The stripe rust resistance gene carried by AUS27506 and AUS27894 was tentatively named YrAW4. A bulked segregant analysis placed YrAW4 in the long arm of chromosome 2B. The AUS27506/AUS27229 F₃ population was enhanced to develop an F₆ recombinant inbred line (RIL) population for detailed mapping of chromosome 2BL. DArT-based SSR, STS and SNP markers were employed to enrich the 2BL map. DArT-based STS markers sun481 and SNP marker IWB12294 flanked YrAW4 proximally (1.8 cM) and distally (1.2 cM), respectively. Deletion mapping placed sun481 in the deletion bin 2BL-5. All stripe rust resistance genes, previously located on chromosome 2BL, neither express an infection type like YrAW4, nor are they mapped in the deletion bin 2BL-5. Hence, YrAW4 represented a new locus and was formally named Yr72. The usefulness of the markers IWB12294 and sun481 in marker-assisted selection was demonstrated by the amplification of alleles that are different to that linked with Yr72 in 19 common wheat and two durum wheat cultivars.

Breeding crops resilient to climate change is urgently needed to help ensure food security. A key challenge is to harness genetic diversity to optimise adaptation, yield, stress resilience and nutrition. We examined the genetic and phenotypic diversity of the A.E. Watkins landrace collection of bread wheat ( Triticum aestivum ), a major global cereal, through whole-genome re-sequencing (827 Watkins landraces and 208 modern cultivars) and in-depth field evaluation spanning a decade. We discovered that modern cultivars are derived from just two of the seven ancestral groups of wheat, leaving five groups as previously untapped sources for breeding. This provides access to landrace-specific functional variations using structured germplasm, genotyping and informatics resources. Employing complementary genetic populations and approaches, we identified thousands of high-resolution quantitative trait loci (QTL) and significant marker–trait associations for major traits, revealing many Watkins-unique loci that can confer superior traits in modern wheat. Furthermore, we identified and functionally verified causative genes for climate-change adaptation, nutritional enhancement and resistance to wheat blast. Finally, we assessed the phenotypic effects of 44,338 Watkins-unique haplotypes, introgressed from 143 prioritised QTL in the context of modern cultivars, bridging the gap between landrace diversity and current breeding. This study establishes a framework for systematically utilising genetic diversity in crop improvement to achieve sustainable food security.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a significant challenge to global wheat production. Common wheat landraces AUS27507 and AUS27894 displayed stripe rust resistance against several commercially prevailing Pst pathotypes. These genotypes were crossed with a stripe rust susceptible landrace AUS27229 to understand inheritance of resistance and to determine genomic location(s) of underlying gene(s). F3 generations of crosses AUS27507/AUS27229 and AUS27894/AUS27229 showed monogenic segregation for stripe rust resistance under greenhouse conditions. The absence of segregation for stripe rust response among the AUS27507/AUS27894-derived F3 population suggested that both genotypes carry the same gene. The stripe rust resistance gene carried by AUS27507 and AUS27894 was tentatively named YrAW4. Bulked segregant analysis placed YrAW4 in the long arm of chromosome 2B. The AUS27507/AUS27229 F3 population was enhanced to develop an F6 recombinant inbred line (RIL) population for detailed mapping of chromosome 2BL. DArT-based SSR, STS and KASP markers were employed to enrich the 2BL map. DArT-based STS markers sun481 and SNP marker IWB12294 flanked YrAW4 proximally (1.8 cM) and distally (1.2 cM), respectively. Deletion mapping placed sun481 in the deletion bin 2BL-5. All stripe rust resistance genes, previously located on chromosome 2BL, neither express infection type like YrAW4, nor are mapped in the deletion bin 2BL-5. Hence YrAW4 represented a new locus and was formally named Yr72. The robustness of IWB12294 marker in marker-assisted selection was demonstrated by amplification of and alternative allele to that linked Yr72 in 21 common wheat background.

The Ug99-effective stem rust resistance gene Sr48 was mapped to chromosome 2A based on its repulsion linkage with Yr1 in an Arina/Forno recombinant inbred line (RIL) population. Attempts to identify markers closely linked to Sr48 using available genomic resources were futile. This study used an Arina/Cezanne F_5:7 RIL population to identify markers closely linked with Sr48. Using the Arina/Cezanne DArTseq map, Sr48 was mapped on the short arm of chromosome 2D and it co-segregated with 12 markers. These DArTseq marker sequences were used for BlastN search to identify corresponding wheat chromosome survey sequence (CSS) contigs, and PCR-based markers were developed. Two simple sequence repeat (SSR) markers, sun590 and sun592, and two Kompetitive Allele-Specific PCR (KASP) markers were derived from the contig 2DS_5324961 that mapped distal to Sr48. Molecular cytogenetic analysis using sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) identified a terminal translocation of chromosome 2A in chromosome 2DL of Forno. This translocation would have led to the formation of a quadrivalent involving chromosomes 2A and 2D in the Arina/Forno population, which would have exhibited pseudo-linkage between Sr48 and Yr1 in chromosome 2AL. Polymorphism of the closet marker sunKASP_239 among a set of 178 wheat genotypes suggested that this marker can be used for marker-assisted selection of Sr48.

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.

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.