The Yr5 gene confers resistance to all races of the stripe rust pathogen ( Puccinia striiformis f. sp. tritici) of wheat in the United States. To develop molecular markers for Yr5, a BC(7):F(3) population was developed by backcrossing the Yr5 donor ' Triticum spelta album' (TSA) with the recurrent parent 'Avocet Susceptible' (AVS). Seedlings of the Yr5 near-isogenic lines (AVS/6* Yr5), AVS, TSA, and the BC(7):F(3) lines were tested with North American races of P. striiformis f. sp. tritici under controlled greenhouse conditions. The single gene was confirmed by a 1:2:1 segregation ratio for homozygous-resistant, heterozygous and homozygous-susceptible BC(7):F(3) lines. Genomic DNA was extracted from the parents (the Yr5 near-isogenic line and AVS) and 202 BC(7):F(3) lines. The resistance gene-analog polymorphism (RGAP) technique was used to identify molecular markers. The parents and the homozygous-resistant and homozygous-susceptible BC(7):F(3) bulks were used to identify putative RGAP markers for Yr5. Association of the markers with Yr5 was determined using segregation analysis with DNA from the individual BC(7):F(3) lines. Of 16 RGAP markers confirmed by segregation analysis with 109 BC(7):F(3) lines, and nine of the markers confirmed with an additional 93 BC(7):F(3) lines, three markers co-segregated with the resistance allele and three markers co-segregated with the susceptibility allele at the Yr5 locus. The other four markers were tightly linked to the locus. Analysis of a set of Chinese Spring nulli-tetrasomic lines with three markers that co-segregated with, or were linked to, the susceptibility allele confirmed that the Yr5 locus is on chromosome 2B. Of five RGAP markers that were cloned and sequenced, markers Xwgp-17 and Xwgp-18 that co-segregated with the Yr5 locus were co-dominant and had 98% homology with each other in both DNA and translated amino-acid sequences. The two markers had 97% homology with a resistance gene-like sequence from Aegilops ventricosa and had significant homology with many known plant resistance genes, resistance gene analogs and expressed sequence tags (ESTs) from wheat and other plant species. The markers Xwgp-17 and Xwgp-18 also had significant homology with the NB-ARC domain that is in several genes for plant resistance to diseases, nematode cell death and human apoptotic signaling. These markers should be useful to clone Yr5 and combine Yr5 with other genes for durable and superior resistance for the control of stripe rust.

A species-specific polymerase chain reaction (PCR) method was developed to detect and identify the root-lesion nematodes Pratylenchus neglectus and P. thornei from soil. A primer set was designed from Pratylenchus 28S rRNA gene sequences of the D3 expansion domain. Primer specificity was confirmed with 23 isolates of 15 nematode species and other plant-parasitic and non-plant-parasitic nematodes typically present in the soil communities, and with six fungal species commonly associated with wheat root rot. DNA obtained using a commercially available kit and a method developed in our laboratory gave comparable amplification. PCR conditions were optimized and the two species were differentiated by PCR products of 144 bp for P. neglectus and 288 bp for P. thornei. With this assay, we detected a single juvenile in 1 g of sterile, inoculated soil. Examination of 30 field soil samples revealed that this method was applicable to a range of soils naturally infested with these two pathogens in Oregon. This PCR-based method is rapid, efficient, and reliable, does not require expertise in nematode taxonomy and morphology, and could be used as a rapid diagnostic tool for commercial and research applications for disease forecasting and management.

Stripe rust (caused by Puccinia striiformis Westend. f. sp. tritici Eriks.) is a serious disease of wheat (Triticum aestivum L.). Resistance genes Yr5 and Yr15 are the only known all‐stage resistance genes that defeat all stripe rust races currently found in the United States. Previously mapped markers for these genes, however, show limited polymorphism across diverse genotypes and/or map at a distance from the genes, reducing the effectiveness of marker‐assisted selection. Our objective was to create new linkage maps for both genes using sequence tagged site (STS) and simple sequence repeat (SSR) loci and to evaluate closely linked markers across a diverse panel of wheat genotypes. Two recombinant inbred populations created using Avocet‐Susceptible as a susceptible parent and Triticum aestivum L. ssp. spelta (L.) Thell. ‘Album’ and Triticum dicoccoides Koern. as the Yr5 and Yr15 donors, respectively, were evaluated for resistance to multiple races of stripe rust. Molecular markers that had been previously mapped to wheat chromosome 1B (Yr5) or 2B (Yr15) were mapped on the appropriate population. Markers most closely linked to each gene were evaluated against a panel of genotypes collected from active introgression programs in the United States. The Yr5 gene is flanked on the distal side by STS7/8 marker and by Xbarc349 and Xbarc167 on the proximal side, although none of these markers were diagnostic in all backgrounds. For Yr15, Xbarc8 and Xgwm413 appear to be completely linked with the gene in our population, along with resistance gene analog polymorphism marker Xwgp34. These two SSR markers also appear to be diagnostic in all backgrounds tested with one exception (Zak). We have developed linkage maps for both genes and identified several useful SSR and STS markers for introgression of Yr5 and Yr15

The Yr5 gene confers resistance to all races of the wheat stripe rust pathogen [Puccinia striiformis Westend. f. sp. tritici Eriks. (P. s. tritici)] identified so far in the USA. Cosegregating resistance gene analog polymorphism (RGAP) markers for Yr5 are available but their use requires skills in polyacrylamide gel electrophoresis and may not be polymorphic across various varieties. To develop better markers to be used in marker‐assisted selection for the Yr5 resistance, sequence tagged site (STS) primers were designed on the basis of the sequences of RGAP markers Xwgp‐18 (AY167598) from the spring wheat (Triticum aestivum L.) ‘Avocet Susceptible’ (AVS) and Xwgp‐17 (AY167597) from the Yr5 near isogenic line (NIL) in the AVS background carrying the Yr5 gene from T. aestivum subsp. spelta (L.) Thell. cv. Album (TSA). Three sets of STS markers (two codominant and one dominant) were developed to amplify a region including a polymorphic 6‐base pairs (bp) insertion–deletion (indel). The cosegregation of the STS markers with Yr5 was confirmed with 114 BC7:F3 lines developed from the cross between AVS and TSA. The STS markers worked well in five out of 17 non‐Yr5 wheat varieties, but the remaining varieties had a similar size of fragment to the Yr5 marker. Because the codominant STS markers were based on a 6‐bp indel, they could not be separated by agarose gel electrophoresis. Cleaved amplified polymorphic sequence (CAPS) markers were then developed on the basis of a DpnII restriction site that is present in all non‐Yr5 varieties and absent in the Yr5 NIL. The CAPS markers for the Yr5 NIL and non‐Yr5 varieties can be separated by agarose gel electrophoresis. The codominant STS markers are easier to score than the original RGAP markers. The CAPS markers are not only easier to score, but also can be used in crosses of an Yr5 donor with a much wider range of wheat germplasms. These markers should be valuable tools to accelerate the introgression of Yr5 into commercial cultivars and to combine Yr5 with other genes for durable resistance to stripe rust.

The root-lesion nematode Pratylenchus thornei is one of the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). It is laborious and difficult to use microscopy to count and identify the nematodes in soils. A SYBR Green I-based real-time polymerase chain reaction (PCR) assay was developed to detect and quantify this species from DNA extracts of soil. A primer set, designed from the internal transcribed spacer region (ITS1) of rDNA, was highly specific to P. thornei and did not amplify DNA from 27 isolates of other Pratylenchus spp., other nematodes, and six fungal species present in PNW wheat fields. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. The standard curve was supported by a high correlation between the numbers of P. thornei added to soil and the numbers quantified using real-time PCR. Examination of 15 PNW dryland field soils and 20 greenhouse samples revealed significant positive correlations between the numbers determined by real-time PCR and by the Whitehead tray and microscopic method. Real-time PCR is a rapid, sensitive alternative to time-consuming nematode extractions, microscopic identification, and counting of P. thornei from field and greenhouse soils.