Long-read genome sequencing of bread wheat facilitates disease resistance gene cloning.

The cloning of agronomically important genes from large, complex crop genomes remains challenging. Here we generate a 14.7 gigabase chromosome-scale assembly of the South African bread wheat (Triticum aestivum) cultivar Kariega by combining high-fidelity long reads, optical mapping and chromosome conformation capture. The resulting assembly is an order of magnitude more contiguous than previous wheat assemblies. Kariega shows durable resistance to the devastating fungal stripe rust disease1. We identified the race-specific disease resistance gene Yr27, which encodes an intracellular immune receptor, to be a major contributor to this resistance. Yr27 is allelic to the leaf rust resistance gene Lr13; the Yr27 and Lr13 proteins show 97% sequence identity2,3. Our results demonstrate the feasibility of generating chromosome-scale wheat assemblies to clone genes, and exemplify that highly similar alleles of a single-copy gene can confer resistance to different pathogens, which might provide a basis for engineering Yr27 alleles with multiple recognition specificities in the future.

Population Structure of Pyrenophora teres f. teres Barley Pathogens from Different Continents.

Net form net blotch disease, caused by Pyrenophora teres f. teres, results in significant yield losses to barley industries. Up-to-date knowledge of the genetic diversity and structure of pathogen populations is critical for elucidating the disease epidemiology and unraveling pathogen survival and dispersal mechanisms. Thus, this study investigated long-distance dispersal and adaptation by analyzing the genetic structure of 250 P. teres f. teres isolates collected from Australia, Canada, Hungary, and Republic of South Africa (RSA), and historical isolates from Canada, Denmark, Japan, and Sweden. The population genetic structure detected by discriminant analysis of principal components, with the use of 5,890 Diversity Arrays Technology markers, revealed the presence of four clusters. Two of these contained isolates from all regions, and all isolates from RSA were grouped in these two. Australia and Hungary showed three clusters each. One of the Australian clusters contained only Australian isolates. One of the Hungarian clusters contained only Hungarian isolates and one Danish isolate. STRUCTURE analysis indicated that some isolates from Australia and Hungary shared recent ancestry with RSA, Canada, and historical isolates and were thus admixed. Subdivisions of the neighbor joining network indicated that isolates from distinct countries were closely related, suggesting that multiple introduction events conferred genetic heterogeneity in these countries. Through a neighbor joining analysis and amplification with form-specific DNA markers, we detected two hybrid isolates, CBS 281.31 from Japan and H-919 from Hungary, collected in 1931 and 2018, respectively. These results provide a foundation for exploring improved management of disease incursions and pathogen control through strategic deployment of resistance.

Mapping a Resistance Gene to Puccinia graminis f. sp. tritici in the Bread Wheat Cultivar 'Matlabas'.

Puccinia graminis f. sp. tritici race TTKSF+ was collected from the South African wheat cultivar 'Matlabas' in 2010. F2 and F3 populations derived from a Matlabas × Line 37-07 cross segregated for a single resistance gene to race TTKSF that is avirulent to Matlabas. In screening genomic DNA bulks of susceptible or resistant F2 plants with simple sequence repeat (SSR) markers, three chromosome arm 2BS markers and one multilocus marker amplified alleles present only in the resistant bulks and Matlabas. Additional 2B-specific SSR markers, incorporating markers spanning regions containing Sr9h, SrWLR, Sr28, and Sr47, were screened in the parental lines and mapped in the F2 population. Linkage and QTL mapping showed that the gene is located between Xbarc160 in the centromeric region and Xgwm47 on the long arm of chromosome 2B. When 2B-specific SNP markers were mapped, the area of interest was delimited to a 15.3 cM region on chromosome arm 2BL, with XIWA543-HRM and Xgwm47 as flanking loci. Matlabas, Webster, and related Sr9h lines all produced a similar, low infection type to race TTKSF, but were susceptible to race TTKSF+. Phenotypic data and allelic studies suggested that stem rust resistance in Matlabas was derived from an Sr9h source.

Point Inoculation Method for Measuring Adult Plant Response of Wheat to Stripe Rust Infection.

Depending on the pathogenicity of the stripe rust fungus Puccinia striiformis f. sp. tritici, the nature of resistance in the wheat host plant, and the environment, a broad range of disease phenotypes can be expressed. Therefore, the phenotyping of partial adult plant stripe rust resistance requires reliable and repeatable procedures, especially under controlled conditions. In this study, the development of a flag leaf point inoculation method, which resulted in a 100% initial infection rate, is reported. Flag leaf inoculations were achieved by placing 6-mm antibiotic test paper discs, dipped into a urediniospore and water suspension and covered with water-proof plastic tape, on the adaxial side of leaves. Results from independent trials allowed for the statistical comparison of stripe rust lesion expansion rate in wheat entries that differ in resistance. The technique is inexpensive, reliable, and applicable to routine screening for adult plant response type, quantitative comparison of stripe rust progress, environmental influences, and pathogenicity of different isolates.

Yield Loss Associated with Different Levels of Stem Rust Resistance in Bread Wheat.

Puccinia graminis f. sp. tritici race Ug99 (syn. TTKSK) has been identified as a major threat to wheat production based on its broad virulence. Despite its importance, the effect of Ug99 on different types of resistance in wheat has not been thoroughly researched. In field trials conducted with P. graminis f. sp. tritici race PTKST (Ug99 race group) over 2 years, AUDPC differentiated the moderately susceptible variety SC Stallion (515) and susceptible entries SC Nduna (995) and Line 37-07 (1634) from those with adult plant resistance (APR). AUDPC of APR varieties W1406 (256), W6979 (399), and Kingbird (209) was higher than the mean of 25 recorded for the all stage resistant (ASR) variety SC Sky. In fungicide-protected and unprotected plots, race PTKST resulted in a mean yield loss of 21.3%, with susceptible Line 37-03 recording a 47.9% decrease in grain yield. Yield reduction in APR varieties reached 19.5% in W1406, whereas the ASR control SC Sky showed a mean loss of 6.4%. Although APR reduced the effects of stem rust on yield and yield components under conditions of high disease pressure, it did not provide the same protection as effective ASR.

Genome Targeted Introgression of Resistance to African Stem Rust from Aegilops sharonensis into Bread Wheat.

Many accessions of the wheat wild relative Sharon goatgrass ( Eig., ) are resistant to African races of the stem rust pathogen (i.e., Ug99 group races), which currently threaten wheat production worldwide. A procedure was designed to introgress the respective resistances to specific bread wheat genomes by producing plants homozygous for the A and B genomes and hemizygous for the D and S genomes or homozygous for the A and D genomes and hemizygous for the B and S genomes. In these genotypes, which lack the allele, homeologous pairing was expected mainly between chromosomes of the D and S genomes or B and S genomes, respectively. An antigametocidal (AG) wheat mutant () was used to overcome gametocidal effects. Wheat lines initially found resistant at the seedling stage were also highly resistant at the adult plant stage in rust nurseries established in the field. DNA of 41 selected homozygous resistant lines, analyzed by the Axiom wheat 820K SNP array, showed alien chromatin mainly in wheat chromosomes 1B, 1D, and 5B. This work suggests that, in most cases, it is possible to target introgressions into the homeologous chromosome of a selected genome of bread wheat.

Stem Rust Resistance in a Geographically Diverse Collection of Spring Wheat Lines Collected from Across Africa.

Following the emergence of the Ug99 lineage of Puccinia graminis f. sp. tritici (Pgt) a collective international effort has been undertaken to identify new sources of wheat stem rust resistance effective against these races. Analyses were undertaken in a collection of wheat genotypes gathered from across Africa to identify stem rust resistance effective against the Pgt races found in Eastern and Southern Africa. The African wheat collection consisted of historic genotypes collected in Kenya, South Africa, Ethiopia, Sudan, Zambia, Morocco, and Tunisia, and current South African breeding lines. Both Bayesian cluster and principal coordinate analyses placed the wheat lines from Sudan in a distinct group, but indicated a degree of genetic relatedness among the other wheat lines despite originating from countries across Africa. Seedling screens with Pgt race PTKST, pedigree information and marker haplotype analysis confirmed the presence of Sr2, Sr36, Sr24, Sr31, and Lr34/Yr18/Sr57 in a number of the lines. A genome-wide association study (GWAS) undertaken with Diversiry Arrays Technology (DArT) and stem rust (Sr) gene associated markers and Stem Area Infected (SAI) and Reaction Type (RT) field phenotypes, collected from trials carried out across two seasons in Kenya in 2009 and in South Africa in 2011, identified 29 marker-trait associations (MTA). Three MTA were in common between SAI and RT, with the biggest effect MTA being found on chromosome 6AS. Two wheat lines, W1406 and W6979 that exhibited high levels of adult plant stem rust resistance were selected to generate bi-parental mapping populations. Only the MTA on chromosomes 6AS and 3BS, and the locus Lr34/Yr18/Sr57 were confirmed following QTL mapping. Additional stem rust resistance QTL, not detected by the GWAS, were found on chromosomes 2BS, 2DL, 3DL, and 4D.

C-reactive protein as an adverse prognostic marker for men with castration-resistant prostate cancer (CRPC): confirmatory results.

We previously reported that higher serum concentrations of C-reactive protein (CRP) are associated with shorter survival in men with castration-resistant prostate cancer (CRPC). To confirm this finding in an independent data set, we used 119 CRPC patients enrolled in 6 phase II clinical trials and examined the relationship of CRP, alkaline phosphatase, hemoglobin, age, ECOG PS, and prostate specific antigen (PSA) with survival. Median follow-up was 19.7 months (0.9-98.5 months), and 89% have died. After analyzing the form of the risk function using the generalized additive model method, univariate and multivariate Cox proportional hazard models were used to assess associations between baseline individual categorical and continuous variables. Quartiles of CRP were: 0-1.0, 1.1-4.9, 5.0-17.0, and 17.1-311 mg/L. In a Cox multivariate model, log(2) (CRP) (HR 1.106, P = 0.013) as well as hemoglobin and alkaline phosphatase were independently associated with survival, confirming that higher CRP is associated with shorter survival in CRPC. Since CRP is a marker of inflammation, this finding suggests that inflammation may play an important role in the natural history of advanced prostate cancer. CRP is a readily measurable biomarker that has the potential to improve prognostic models and should be validated in a prospective clinical trial.

Histopathology and PR-protein markers provide insight into adult plant resistance to stripe rust of wheat.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a serious disease of wheat. The spring wheat cultivar Kariega expresses complete adult plant resistance to stripe rust, whereas Avocet S is susceptible. In former studies, quantitative trait loci (QTL) analysis of doubled haploid lines derived from a Kariega x Avocet S cross revealed two major QTL (QYr.sgi-7D and QYr.sgi-2B.1) and two minor QTL (QYr.sgi-1A and QYr.sgi-4A.1) responsible for the adult resistance of Kariega in the field. Avocet S contains none of these QTL. In the present study, stripe rust development was compared, by means of fluorescence and confocal laser scanning microscopy, in flag leaves of Kariega, Avocet S and six doubled haploid (DH) lines, containing all four, none or one QTL. Depending on the QTL present, the infection types of the DH lines ranged from resistant to fully susceptible. No differences in fungal growth were observed during the first 5 days post inoculation (dpi), whereas the mean length of the fungal colonies started to differ at 6 dpi. Interestingly, MP 51 carrying QYr.sgi-7D responded with lignification to the fungal growth without restricting it, whereas MP 35 containing QYr.sgi-2B.1 did not show lignified host tissue, but fungal growth was restricted. RT PCR experiments with sequences of pathogenesis-related (PR) proteins resulted in a slightly stronger induction of PR 1, 2 and 5, known markers for the hypersensitive reaction, and peroxidases in MP 51, whereas a second band for chitinases was detected in MP 35 only.