Harnessing landrace diversity empowers wheat breeding.

Harnessing genetic diversity in major staple crops through the development of new breeding capabilities is essential to ensure food security1. Here we examined the genetic and phenotypic diversity of the A.E. Watkins landrace collection2 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 and maintain very long-range haplotype integrity. The remaining five groups represent untapped genetic sources, providing access to landrace-specific alleles and haplotypes for breeding. Linkage disequilibrium (LD) based haplotypes and association genetics analyses link Watkins genomes to the thousands of high-resolution quantitative trait loci (QTL), and significant marker-trait associations identified. Using these structured germplasm, genotyping and informatics resources, we revealed many Watkins-unique beneficial haplotypes that can confer superior traits in modern wheat. Furthermore, 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.

Discovery of the New Leaf Rust Resistance Gene Lr82 in Wheat: Molecular Mapping and Marker Development.

Breeding for leaf rust resistance has been successful worldwide and is underpinned by the discovery and characterisation of genetically diverse sources of resistance. An English scientist, Arthur Watkins, collected pre-Green Revolution wheat genotypes from 33 locations worldwide in the early part of the 20th Century and this collection is now referred to as the 'Watkins Collection'. A common wheat genotype, Aus27352 from Yugoslavia, showed resistance to currently predominating Australian pathotypes of the wheat leaf rust pathogen. We crossed Aus27352 with a leaf rust susceptible wheat selection Avocet S and a recombinant inbred line (RIL) F6 population of 200 lines was generated. Initial screening at F3 generation showed monogenic segregation for seedling response to leaf rust in Aus27352. These results were confirmed by screening the Aus27352/Avocet S RIL population. The underlying locus was temporarily named LrAW2. Bulked segregant analysis using the 90K Infinium SNP array located LrAW2 in the long arm of chromosome 2B. Tests with molecular markers linked to two leaf rust resistance genes, Lr50 and Lr58, previously located in chromosome 2B, indicated the uniqueness of LrAW2 and it was formally designated Lr82. Kompetitive allele-specific polymerase chain reaction assays were developed for Lr82-linked SNPs. KASP_22131 mapped 0.8 cM proximal to Lr82 and KASP_11333 was placed 1.2 cM distal to this locus. KASP_22131 showed 91% polymorphism among a set of 89 Australian wheat cultivars. We recommend the use of KASP_22131 for marker assisted pyramiding of Lr82 in breeding programs following polymorphism check on parents.

Lr80: A new and widely effective source of leaf rust resistance of wheat for enhancing diversity of resistance among modern cultivars.

KEY MESSAGE: A new leaf rust resistance gene Lr80 was identified and closely linked markers were developed for its successful pyramiding with other marker-tagged genes to achieve durable control of leaf rust. Common wheat landrace Hango-2, collected in 2006 from the Himalayan area of Hango, District Kinnaur, in Himachal Pradesh, exhibited a very low infection type (IT;) at the seedling stage to all Indian Puccinia triticina (Pt) pathotypes, except the pathotype 5R9-7 which produced IT 3+. Genetic analysis based on Agra Local/Hango-2-derived F3 families indicated monogenic control of leaf rust resistance, and the underlying locus was temporarily named LrH2. Bulked segregant analysis using 303 simple sequence repeat (SSR) markers located LrH2 in the short arm of chromosome 2D. An additional set of 10 chromosome 2DS-specific markers showed polymorphism between the parents and these were mapped on the entire Agra Local/Hango-2 F3 population. LrH2 was flanked by markers cau96 (distally) and barc124 (proximally). The 90 K Infinium SNP array was used to identify SNP markers linked with LrH2. Markers KASP_17425 and KASP_17148 showed association with LrH2. Comparison of seedling leaf rust response data and marker locations across different maps demonstrated the uniqueness of LrH2 and it was formally named Lr80. The Lr80-linked markers KASP_17425, KASP_17148 and barc124 amplified alleles/products different to Hango-2 in 82 Australian cultivars indicating their robustness for marker-assisted selection of this gene in wheat breeding programs.

Fine Mapping of Lr49 Using 90K SNP Chip Array and Flow-Sorted Chromosome Sequencing in Wheat.

Leaf rust, caused by Puccinia triticina, threatens global wheat production due to the constant evolution of virulent pathotypes that defeat commercially deployed all stage-resistance (ASR) genes in modern cultivars. Hence, the deployment of combinations of adult plant resistance (APR) and ASR genes in new wheat cultivars is desirable. Adult plant resistance gene Lr49 was previously mapped on the long arm of chromosome 4B of cultivar VL404 and flanked by microsatellite markers barc163 (8.1 cM) and wmc349 (10.1 cM), neither of which was sufficiently closely linked for efficient marker assisted selection. This study used high-density SNP genotyping and flow sorted chromosome sequencing to fine-map the Lr49 locus as a starting point to develop a diagnostic marker for use in breeding and to clone this gene. Marker sunKASP_21 was mapped 0.4 cM proximal to Lr49, whereas a group of markers including sunKASP_24 were placed 0.6 cM distal to this gene. Testing of the linked markers on 75 Australian and 90 European cultivars with diverse genetic backgrounds showed that sunKASP_21 was most strongly associated with Lr49. Our results also show that the Lr49 genomic region contains structural variation relative to the reference stock Chinese Spring, possibly an inverted genomic duplication, which introduces a new set of challenges for the Lr49 cloning.

Characterisation and mapping of adult plant stripe rust resistance in wheat accession Aus27284.

KEY MESSAGE: A new adult plant stripe rust resistance gene, Yr80, was identified in a common wheat landrace Aus27284. Linked markers were developed and validated for their utility in marker-assisted selection. Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is among the most important constraints to global wheat production. The identification and characterisation of new sources of host plant resistance enrich the gene pool and underpin deployment of resistance gene pyramids in new cultivars. Aus27284 exhibited resistance at the adult plant stage against predominant Pst pathotypes and was crossed with a susceptible genotype Avocet S. A recombinant inbred line (RIL) population comprising 121 lines was developed and tested in the field at three locations in 2016 and two in 2017 crop seasons. Monogenic segregation for adult plant stripe rust response was observed among the Aus27284/Avocet S RIL population and the underlying locus was temporarily designated YrAW11. Bulked-segregant analysis using the Infinium iSelect 90K SNP wheat array placed YrAW11 in chromosome 3B. Kompetitive allele specific PCR (KASP) primers were designed for the linked SNPs and YrAW11 was flanked by KASP_65624 and KASP_53292 (3 cM) proximally and KASP_53113 (4.9 cM) distally. A partial linkage map of the genomic region carrying YrAW11 comprised nine KASP and two SSR markers. The physical position of KASP markers in the pseudomolecule of chromosome 3B placed YrAW11 in the long arm and the location of markers gwm108 and gwm376 in the deletion bin 3BL2-0.22 supported this conclusion. As no other stripe rust resistance locus has been reported in chromosome 3BL, YrAW11 was formally designated Yr80. Marker KASP_ 53113 was polymorphic among 94% of 81 Australian wheat cultivars used for validation.

Tight repulsion linkage between Sr36 and Sr39 was revealed by genetic, cytogenetic and molecular analyses.

KEY MESSAGE: The shortening of Aegilops speltoides segment did not facilitate recombination between stem rust resistance genes Sr36 and Sr39 . Robustness of marker rwgs28 for marker-assisted selection of Sr39 was demonstrated. Stem rust resistance genes Sr39 and Sr36 were transferred from Aegilops speltoides and Triticum timopheevii, respectively, to chromosome 2B of wheat. Genetic stocks RL6082 and RWG1 carrying Sr39 on a large and a shortened Ae. speltoides segments, respectively, and the Sr36-carrying Australian wheat cultivar Cook were used in this study. This investigation was planned to determine the genetic relationship between these genes. Stem rust tests on F3 populations derived from RL6082/Cook and RWG1/Cook crosses showed tight repulsion linkage between Sr39 and Sr36. The genomic in situ hybridization analysis of heterozygous F3 family from the RWG1/Cook population showed that the translocated segments do not overlap. Meiotic analysis on the F1 plant from RWG1/Cook showed two univalents at the metaphase and anaphase stages in a majority of the cells indicating absence of pairing. Since meiotic pairing has been reported to initiate at the telomere, pairing and recombination may be inhibited due to very little wheat chromatin in the distal end of the chromosome arm 2BS in RWG1. The Sr39-carrying large Ae. speltoides segment transmitted preferentially in the RL6082/Cook F3 population, whereas the Sr36-carrying T. timopheevii segment over-transmitted in the RWG1/Cook cross. Genotyping with the co-dominant Sr39- and Sr36-linked markers rwgs28 and stm773-2, respectively, matched the phenotypic classification of F3 families. The RWG1 allele amplified by rwgs28 was diagnostic for the shortened Ae. speltoides segment and alternate alleles were amplified in 29 Australian cultivars. Marker rwgs28 will be useful in marker-assisted pyramiding of Sr39 with other genes.

Mapping of a new stem rust resistance gene Sr49 in chromosome 5B of wheat.

KEY MESSAGE: A new stem rust resistance gene Sr49 was mapped to chromosome 5BL of wheat. Usefulness of the closely linked markers sun209 and sun479 for marker-assisted selection of Sr49 was demonstrated. Landrace AUS28011 (Mahmoudi), collected from Ghardimaou, Tunisia, produced low stem rust response against Australian pathotypes of Puccinia graminis f. sp. tritici (Pgt) carrying virulence for several stem rust resistance genes deployed in modern wheat cultivars. Genetic analysis based on a Mahmoudi/Yitpi F3 population indicated the involvement of a single all-stage stem rust resistance gene and it was temporarily named SrM. Bulked segregant analysis using multiplex-ready SSR technology located SrM on the long arm of chromosome 5B. Since there is no other all-stage stem rust resistance gene located in chromosome 5BL, SrM was permanently designated Sr49. The Mahmoudi/Yitpi F3 population was enhanced to generate F6 recombinant inbred line (RIL) population for detailed mapping of Sr49 using publicly available genomic resources. Markers sun209 and sun479 flanked Sr49 at 1.5 and 0.9 cM distally and proximally, respectively. Markers sun209 and sun479 amplified PCR products different than the Sr49-linked alleles in 146 and 145 common wheat cultivars, respectively. Six and seven cultivars, respectively, carried the resistance-linked marker alleles sun209 148bp and sun479 200bp ; however, none of the cultivars carried both resistance-linked alleles. These results demonstrated the usefulness of these markers for marker-assisted selection of Sr49 in breeding programs.

A haplotype map of allohexaploid wheat reveals distinct patterns of selection on homoeologous genomes.

BACKGROUND: Bread wheat is an allopolyploid species with a large, highly repetitive genome. To investigate the impact of selection on variants distributed among homoeologous wheat genomes and to build a foundation for understanding genotype-phenotype relationships, we performed population-scale re-sequencing of a diverse panel of wheat lines. RESULTS: A sample of 62 diverse lines was re-sequenced using the whole exome capture and genotyping-by-sequencing approaches. We describe the allele frequency, functional significance, and chromosomal distribution of 1.57 million single nucleotide polymorphisms and 161,719 small indels. Our results suggest that duplicated homoeologous genes are under purifying selection. We find contrasting patterns of variation and inter-variant associations among wheat genomes; this, in addition to demographic factors, could be explained by differences in the effect of directional selection on duplicated homoeologs. Only a small fraction of the homoeologous regions harboring selected variants overlapped among the wheat genomes in any given wheat line. These selected regions are enriched for loci associated with agronomic traits detected in genome-wide association studies. CONCLUSIONS: Evidence suggests that directional selection in allopolyploids rarely acted on multiple parallel advantageous mutations across homoeologous regions, likely indicating that a fitness benefit could be obtained by a mutation at any one of the homoeologs. Additional advantageous variants in other homoelogs probably either contributed little benefit, or were unavailable in populations subjected to directional selection. We hypothesize that allopolyploidy may have increased the likelihood of beneficial allele recovery by broadening the set of possible selection targets.

Development of IRAP- and REMAP-derived SCAR markers for marker-assisted selection of the stripe rust resistance gene Yr15 derived from wild emmer wheat.

KEY MESSAGE: Yr15 provides broad resistance to stripe rust, an important wheat disease. REMAP- and IRAP-derived co-dominant SCAR markers were developed and localize Yr15 to a 1.2 cM interval. They are reliable across many cultivars. Stripe rust [Pucinia striiformis f.sp. tritici (Pst)] is one of the most important fungal diseases of wheat, found on all continents and in over 60 countries. Wild emmer wheat (Triticum dicoccoides), which is the tetraploid progenitor of durum wheat, is a valuable source of novel stripe rust resistance genes for wheat breeding. T. dicoccoides accession G25 carries Yr15 on chromosome 1BS. Yr15 confers resistance to virtually all tested Pst isolates; it is effective in durum and bread wheat introgressions and their derivatives. Retrotransposons generate polymorphic insertions, which can be scored as Mendelian markers using techniques such as REMAP and IRAP. Six REMAP- and IRAP-derived SCAR markers were mapped using 1,256 F2 plants derived from crosses of the susceptible T. durum accession D447 (DW1) with its resistant BC3F9 and BC3F10 (B9 and B10) near isogenic lines, which carried Yr15 introgressed from G25. The nearest markers segregated 0.1 cM proximally and 1.1 cM distally to Yr15. These markers were also mapped and validated at the same position in another 500 independent F2 plants derived from crosses of B9 and B10 with the susceptible cultivar Langdon (LDN). SC2700 and SC790, defining Yr15 on an interval of 1.2 cM, were found to be reliable and robust co-dominant markers in a wide range of wheat lines and cultivars with and without Yr15. These markers are useful tags in marker-assisted wheat breeding programs that aim to incorporate Yr15 into elite wheat lines and cultivars for durable and broad-spectrum resistance to stripe rust.

Genomic prediction for rust resistance in diverse wheat landraces.

KEY MESSAGE: We have demonstrated that genomic selection in diverse wheat landraces for resistance to leaf, stem and strip rust is possible, as genomic breeding values were moderately accurate. Markers with large effects in the Bayesian analysis confirmed many known genes, while also discovering many previously uncharacterised genome regions associated with rust scores. Genomic selection, where selection decisions are based on genomic estimated breeding values (GEBVs) derived from genome-wide DNA markers, could accelerate genetic progress in plant breeding. In this study, we assessed the accuracy of GEBVs for rust resistance in 206 hexaploid wheat (Triticum aestivum) landraces from the Watkins collection of phenotypically diverse wheat genotypes from 32 countries. The landraces were genotyped for 5,568 SNPs using an Illumina iSelect 9 K bead chip assay and phenotyped for field-based leaf rust (Lr), stem rust (Sr) and stripe rust (Yr) responses across multiple years. Genomic Best Linear Unbiased Prediction (GBLUP) and a Bayesian Regression method (BayesR) were used to predict GEBVs. Based on fivefold cross-validation, the accuracy of genomic prediction averaged across years was 0.35, 0.27 and 0.44 for Lr, Sr and Yr using GBLUP and 0.33, 0.38 and 0.30 for Lr, Sr and Yr using BayesR, respectively. Inclusion of PCR-predicted genotypes for known rust resistance genes increased accuracy more substantially when the marker was diagnostic (Lr34/Sr57/Yr18) for the presence-absence of the gene rather than just linked (Sr2). Investigation of the impact of genetic relatedness between validation and reference lines on accuracy of genomic prediction showed that accuracy will be higher when each validation line had at least one close relationship to the reference lines. Overall, the prediction accuracies achieved in this study are encouraging, and confirm the feasibility of genomic selection in wheat. In several instances, estimated marker effects were confirmed by published literature and results of mapping experiments using Watkins accessions.

New slow-rusting leaf rust and stripe rust resistance genes Lr67 and Yr46 in wheat are pleiotropic or closely linked.

The common wheat genotype 'RL6077' was believed to carry the gene Lr34/Yr18 that confers slow-rusting adult plant resistance (APR) to leaf rust and stripe rust but located to a different chromosome through inter-chromosomal reciprocal translocation. However, haplotyping using the cloned Lr34/Yr18 diagnostic marker and the complete sequencing of the gene indicated Lr34/Yr18 is absent in RL6077. We crossed RL6077 with the susceptible parent 'Avocet' and developed F(3), F(4) and F(6) populations from photoperiod-insensitive F(3) lines that were segregating for resistance to leaf rust and stripe rust. The populations were characterized for leaf rust resistance at two Mexican sites, Cd. Obregon during the 2008-2009 and 2009-2010 crop seasons, and El Batan during 2009, and for stripe rust resistance at Toluca, a third Mexican site, during 2009. The F(3) population was also evaluated for stripe rust resistance at Cobbitty, Australia, during 2009. Most lines had correlated responses to leaf rust and stripe rust, indicating that either the same gene, or closely linked genes, confers resistance to both diseases. Molecular mapping using microsatellites led to the identification of five markers (Xgwm165, Xgwm192, Xcfd71, Xbarc98 and Xcfd23) on chromosome 4DL that are associated with this gene(s), with the closest markers being located at 0.4 cM. In a parallel study in Canada using a Thatcher × RL6077 F(3) population, the same leaf rust resistance gene was designated as Lr67 and mapped to the same chromosomal region. The pleiotropic, or closely linked, gene derived from RL6077 that conferred stripe rust resistance in this study was designated as Yr46. The slow-rusting gene(s) Lr67/Yr46 can be utilized in combination with other slow-rusting genes to develop high levels of durable APR to leaf rust and stripe rust in wheat.

A robust molecular marker for the detection of shortened introgressed segment carrying the stem rust resistance gene Sr22 in common wheat.

Stem rust resistance gene Sr22 transferred to common wheat from Triticum boeoticum and T. monococcum remains effective against commercially prevalent pathotypes of Puccinia graminis f. sp. tritici, including Ug99 and its derivatives. Sr22 was previously located on the long arm of chromosome 7A. Several backcross derivatives (hexaploid) possessing variable sized Sr22-carrying segments were used in this study to identify a closely linked DNA marker. Expressed sequenced tags belonging to the deletion bin 7AL-0.74-0.86, corresponding to the genomic location of Sr22 were screened for polymorphism. In addition, RFLP markers that mapped to this region were targeted. Initial screening was performed on the resistant and susceptible DNA bulks obtained from backcross derivatives carrying Sr22 in three genetic backgrounds with short T. boeoticum segments. A cloned wheat genomic fragment, csIH81, that detected RFLPs between the resistant and susceptible bulks, was converted into a sequence tagged site (STS) marker, named cssu22. Validation was performed on Sr22 carrying backcross-derivatives in fourteen genetic backgrounds and other genotypes used for marker development. Marker cssu22 distinguished all backcross-derivatives from their respective recurrent parents and co-segregated with Sr22 in a Schomburgk (+Sr22)/Yarralinka (-Sr22)-derived recombinant inbred line (RIL) population. Sr22 was also validated in a second population, Sr22TB/Lakin-derived F(4) selected families, containing shortened introgressed segments that showed recombination with previously reported flanking microsatellite markers.

Pathogenic and molecular variation support the presence of genetically distinct clonal lineages in Australian populations of Puccinia graminis f. sp. avenae.

Previous studies of the causal agent of stem rust of oats (Puccinia graminis f. sp. avenae; P. g. avenae) in Australia have demonstrated a high level of pathogenic variability. In this work, the pathotypic structure of the Australian P. g. avenae population in 1999 was investigated, as well as the pathotypic and genetic diversity of a collection of 26 Australian isolates representing a 25-year period (1971-1996). In the 1999 sample, 16 races belonging to six international standard races were identified from 97 isolates, with standard race 94 predominant in all regions. Race 94+Pg-13,Pg-Sa,Pg-a, detected in southern New South Wales (sNSW) and northern New South Wales (nNSW), was virulent on all of the differential genotypes used. Detailed analyses of pathogenicity and AFLP variability among 26 isolates collected from 1971-1996 revealed that isolates of standard race 94 collected in 1999 were genetically distinct from other Australian races of P. g. avenae. This evidence, along with data from annual pathogenicity surveys, suggests that the group to which standard race 94 belongs appeared during the late 1980s, and that it increased in frequency to dominate P. g. avenae pathogen populations throughout Australia from 1992 onward. The existence of groups of P. g. avenae isolates in Australia that differ in pathogenicity and AFLP phenotype suggests that current populations have evolved from a number of isolates of the fungus that differ in their genetic backgrounds, which may have originated from independent introductions or from asexual hybridisational events.

The expression and genetics of resistance to stripe (yellow) rust in three European and four New Zealand wheat cultivars.

Adult plant resistance (APR) to stripe rust in three European (Pegaso, Victo and Aztec) and four New Zealand cultivars (Weka, Kopara, Kokart and Takahe) was characterised using hybrid analysis and tests of allelism. In agreement with earlier work, the APR in most of these cultivars appeared to be controlled by two or more genes with additive effects. It was suggested that heavy selection pressure should be avoided in early generations in breeding programs utilising APR, because lines in which APR genes are heterozygous may display lower levels of resistance due to the incompletely dominant and interactive nature of many APRs. Such lines are capable of generating more resistant progenies following selfing. It was also demonstrated that it is possible to misclassify F2 plants as susceptible if APR genes are in a heterozygous condition, especially in the case of gene(s) conferring intermediate levels of resistance. The presence of a common APR gene in Kopara and Takahe, and perhaps Weka, was suggested because all shared a common parent in their pedigree and no susceptible plants were observed in F2 populations derived from intercrossing them. The difficulties inherent in conducting genetic studies on APRs and the need for large population sizes for such studies were emphasised.