Identification and characterisation of stripe rust resistance genes Yr66 and Yr67 in wheat cultivar VL Gehun 892

Wheat cultivar VL Gehun 892 has shown a high level of resistance against Australian Puccinia striiformis f. sp. tritici (Pst) pathotypes. In this study, it was crossed with Westonia, a susceptible wheat cultivar, and digenic segregation was observed in the derived population against Pst pathotype 134 E16A+Yr17+Yr27+. Single-gene recombinant inbred line (RIL) populations were developed from F3 families (VL Gehun 892/Westonia#1 and VLGehun 892/Westonia#4) that showed monogenic segregations with two distinct phenotypes. Single-gene segregation against Pst pathotype 134 E16A+Yr17+Yr27+ was confirmed in both F6 RIL populations. Bulked segregant analysis using a 90K Infinium SNP array placed YrVL1 in the short arm of chromosome 3D and YrVL2 in the long arm of chromosome 7B. Kompetitive allele specific polymerase chain reaction (KASP) assays were developed for the SNPs linked with YrVL1 and YrVL2 and were mapped onto the respective populations. KASP_48179 (0.6 cM proximal) and KASP_18087 (2.1 cM distal) flanked YrVL1, whereas YrVL2 was mapped between KASP_37096 (1.2 cM proximal) and KASP_2239 (3.6 cM distal). Based on their pathotypic specificities, map locations, and stages of expression, YrVL1 and YrVL2 were demonstrated to be unique loci and named Yr66 and Yr67, respectively. Markers linked with these genes showed more than 85% polymorphism when tested on a set of 89 Australian cultivars and hence could be used for the marker-assisted selection of these genes in wheat breeding programs, following checks of parental polymorphisms

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

Stripe rust (Pucinia striformis f.sp. tritici) 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 G25 accession carries Yr15, a gene on chromosome arm 1BS. Yr15 confers resistance to all known stripe rust isolates; it is also effective in introgressed durum and bread wheat. Retrotransposons generate polymorphic insertions, which can be scored as Mendelian markers with techniques including REMAP and IRAP. Six REMAP and IRAP-derived SCAR markers were developed using 1256 F2 plants derived from crosses of the susceptible T. durum accession D447 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 independent 500 F2 plants derived from crosses of B9 and B10 with the susceptible cultivar Langdon. SCAR270 and SCAR790, surrounding Yr15 at 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 aiming to incorporate Yr15 into elite wheat lines and cultivars for durable and broad-spectrum resistance against stripe rust.Peer reviewe

Resistance gene cloning from a wild crop relative by sequence capture and association genetics

Disease resistance (R) genes from wild relatives could be used to engineer broad-spectrum resistance in domesticated crops. We combined association genetics with R gene enrichment sequencing (AgRenSeq) to exploit pan-genome variation in wild diploid wheat and rapidly clone four stem rust resistance genes. AgRenSeq enables R gene cloning in any crop that has a diverse germplasm panel

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

Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture

Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5–15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution1. If several cloned R genes were available, it would be possible to pyramid R genes2 in a crop, which might provide more durable resistance1. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize

Development and validation of molecular markers linked with stem rust resistance gene Sr13 in durum wheat

Stem rust resistance gene Sr13, found frequently in tetraploid wheats, was tested effective against Puccinia graminis f. sp. tritici pathotype Ug99 (TTKSK) and its derivatives. It remains a candidate for developing new cultivars with diverse combinations of stem rust resistance genes. To combine Sr13 with other genes that produce a similar phenotype, linked markers would be required. We used the AFLP approach to identify markers linked closely with Sr13. The STS marker AFSr13, derived from an AFLP fragment, mapped at 3.4-6.0 cM proximal to Sr13 across three mapping populations. Marker dupw167, previously reported to be linked with Sr13, mapped 2.3-5.7 cM distal to Sr13 in four F-3 populations. Marker gwm427 mapped proximal to AFSr13 in two populations, and these markers were monomorphic on one population each. The map order dupw167-Sr13-AFSr13-gwm427 was deduced from the recombination data. Markers dupw167 and AFSr13 were validated on 21 durum wheat genotypes. Combination of dupw167 and AFSr13 would facilitate marker-assisted selection of Sr13 in segregating populations. At the hexaploid level, only gwm427 showed polymorphism and differentiated the presence of Sr13 in 10 of the 15 backcross derivatives carrying Sr13 from their Sr13-lacking recurrent parents

Molecular mapping of all-stage stripe rust resistance in Indian wheat (Triticum aestivum) cultivar ‘VL404’

Puccinia striiformis f. sp. tritici (Pst), the causal agent of stripe rust of wheat, is a highly evolving fungal pathogen and several widely deployed stripe rust resistance genes have been overcome worldwide often through single step increase in virulence. Development of stripe rust resistant cultivars depends on the availability of widely effective sources of resistance. An Indian wheat cultivar ‘VL404’ exhibited high level of resistance against Australian Pst pathotypes. ‘VL404’ was crossed with a susceptible genotype Avocet ‘S’ (AvS) and an F6 recombinant inbred line (RIL) population was developed. The VL404/AvS RIL population was evaluated at the seedling stage against three Pst pathotypes differing in their virulence profiles. Monogenic segregation for stripe rust response variation was observed in this population and the resistance locus was tentatively named YrVL. Incorporation of stripe rust data into the VL404/AvS genetic map constructed using 40K Wheat-Barley Illumina XT single-nucleotide polymorphism (SNP) array placed YrVL in the long arm of chromosome 2B in the 769.1–779.3 Mb region of the Chinese Spring physical map. YrVL was aligned with the previously reported genes in chromosome 2BL (Yr43, Yr72 and YrAW12) using Pretzel, and it was placed distal to all these genes. Hence, YrVL appears to represent a new resistance locus

Association mapping of rust resistance in pre-green revolution wheat accessions

Association mapping detects correlations between genotypes and phenotypes in a sample of individuals based on the linkage disequilibrium and can be used to uncover new genetic variation among germplasm collections. Two hundred and five landraces collected by the English botanist A. Watkins in the 1920s were screened for rust response variation under field conditions during three crop seasons. An integrated map of 350 polymorphic DArT markers was developed. Association mapping identified the involvement of several genomic regions in controlling resistance to three rust diseases. Seven, eight and nine genomic regions, respectively, appeared to carry yet uncharacterized leaf rust, stripe rust and stem rust resistance. Three dimensional analyses indicated genetic association of leaf rust and stripe rust resistance in some accessions, whereas no such association was observed between stem rust resistance and resistance to either of the other two rust diseases. A new stripe rust resistance locus, Yr47, has been named