Relocation of Sr48 to chromosome 2D using an alternative mapping population and development of a closely linked marker using diverse molecular technologies

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 F5:7 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

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

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