Discovery and characterization of two new stem rust resistance genes in Aegilops sharonensis
Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goatgrass species Aegilops sharonesis (Sharon goatgrass) as a rich reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F6 recombinant inbred line and an F2 population, two genes were identified that mapped to the short arm of chromosome 1Ssh, designated as Sr-1644-1Sh, and the long arm of chromosome 5Ssh, designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (a member of the Ug99 race group), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors.
Guotai Yu, Nicolas Champouret, Burkhard Steuernagel, Pablo D. Olivera, Jamie Simmons, Cole Williams, Ryan Johnson, Matthew J. Moscou, Inmaculada Hernández-Pinzón, Phon Green, Hanan Sela, Eitan Millet, Jonathan D. G. Jones, Eric R. Ward, Brian J. Steffenson, Brande B. H. Wulff
Theoretical and Applied Genetics · Volume 130 · Issue 6 · 2017
Comparative Analysis Highlights Variable Genome Content of Wheat Rusts and Divergence of the Mating Loci
Three members of the Puccinia genus, P. triticina (Pt), P. striiformis f.sp. tritici (Pst), and P. graminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; by comparison repeats occupy 31.5% for Pst and 36.5% for Pgt. We find all three genomes are highly heterozygous, with Pst (5.97 SNPs/kb) nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt. Of 1,358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate degradation enzymes. Two allelic homeodomain pairs, HD1 and HD2, were identified in each dikaryotic Puccinia species along with three pheromone receptor (STE3) mating-type genes, two of which are likely representing allelic specificities. The HD proteins were active in a heterologous Ustilago maydis mating assay and host induced gene silencing of the HD and STE3 alleles reduced wheat host infection.
Christina A. Cuomo, Guus Bakkeren, Hala Badr Khalil, Vinay Panwar, David Joly, Rob Linning, Sharadha Sakthikumar, Xiao Song, Xian Adiconis, Lin Fan, Jonathan M. Goldberg, Joshua Z. Levin, Sarah Young, Qiandong Zeng, Yehoshua Anikster, Myron Bruce, Meinan Wang, Chuntao Yin, Brent McCallum, Les J. Szabo, Scot Hulbert, Xiaming Chen, John P. Fellers
G3 · Volume 7 · Issue 2 · 2017
Microsatellite markers for the Triticum timopheevi-derived leaf rust resistance gene Lr18 on wheat 5BL chromosome
Leaf rust, caused by Puccinia triticina, is a common wheat disease worldwide. Developing resistant cultivars through deploying new or pyramiding resistance genes in a suitable line, is the most effective approach to control this disease. However, to stack genes in a genotype, efficient and reliable markers are required. In the present study, F2 plants and their corresponding F3 families from a cross between the resistant line; Thatcher (Tc) Lr18, and the susceptible cultivar ‘Boolani’ were used to map rust resistance gene, Lr18 using SSR markers on chromosome 5BL of hexaploid wheat. The P. triticina pathotype no 15 was used to inoculate plants. Out of 20 primers tested, eight showed polymorphism between the two parents and were subsequently genotyped in the entire F2 population. The markers Xgpw7425 and Xwmc75 flanked the locus at a distance of 0.3 and 1.2 cM, respectively. Analysis of 81 genotypes from different backgrounds with these two markers confirmed their usefulness in screening absence or presence of Lr18. Therefore, these markers can be used for gene postulation and marker-assisted selection (MAS) of this gene in wheat breeding programs in future.
Ali Aliakbari Sadeghabad, Ali Dadkhodaie, Bahram Heidari, Hooman Razi, Reza Mostowfizadeh-Ghalamfarsa
Breeding Science · Volume 67 · Issue 2 · 2017
Identification and validation of single nucleotide polymorphic markers linked to Ug99 stem rust resistance in spring wheat
Wheat stem rust (Puccinia graminis f. sp. tritici Eriks. and E. Henn.) is one of the most destructive diseases world-wide. Races belonging to Ug99 (or TTKSK) continue to cause crop losses in East Africa and threaten global wheat production. Developing and deploying wheat varieties with multiple race-specific genes or complex adult plant resistance is necessary to achieve durability. In the present study, we applied genome-wide association studies (GWAS) for identifying loci associated with the Ug99 stem rust resistance (SR) in a panel of wheat lines developed at the International Maize and Wheat Improvement Center (CIMMYT). Genotyping was carried out using the wheat 9K iSelect single nucleotide polymorphism (SNP) chip. Phenotyping was done in the field in Kenya by infection of Puccinia graminis f. sp. tritici race TTKST, the Sr24-virulent variant of Ug99. Marker-trait association identified 12 SNP markers significantly associated with resistance. Among them, 7 were mapped on five chromosomes. Markers located on chromosomes 4A and 4B overlapped with the location of the Ug99 resistance genes SrND643 and Sr37, respectively. Markers identified on 7DL were collocated with Sr25. Additional significant markers were located in the regions where no Sr gene has been reported. The chromosome location for five of the SNP markers was unknown. A BLASTN search of the NCBI database using the flanking sequences of the SNPs associated with Ug99 resistance revealed that several markers were linked to plant disease resistance analogues, while others were linked to regulatory factors or metabolic enzymes. A KASP (Kompetitive Allele Specific PCR) assay was used for validating six marker loci linked to genes with resistance to Ug99. Of those, four co-segregated with the Sr25-pathotypes while the rest identified unknown resistance genes. With further investigation, these markers can be used for marker-assisted selection in breeding for Ug99 stem rust resistance in wheat.
Long-Xi Yu, Shiaoman Chao, Ravi P. Singh, Mark E. Sorrells
PLoS One · 2017
Speed breeding for multiple disease resistance in barley
To respond faster to the changing climate, evolving pathogens and to feed a global population of 9–10 billion by 2050, plant breeders are exploring more efficient crop improvement strategies. In this study, we applied novel methodology for rapid trait introgression to the European two-rowed barley cultivar Scarlett. Scarlett is widely-grown in Argentina and is preferred for malting and brewing, yet lacks adequate disease resistance. We used four donor lines combining multiple disease resistance (i.e. leaf rust, net and spot forms of net blotch and spot blotch) in a modified backcross strategy, which incorporated both multi-trait phenotypic screens and the rapid generation advance technology ‘speed breeding’, to develop 87 BC1F3:4 Scarlett introgression lines (ILs) within two years. Phenotyping this set of lines in disease nurseries located in Australia and Uruguay revealed the ILs had high levels of multiple disease resistance. Preliminary yield testing of the 12 most promising ILs in Argentina identified three ILs that were significantly higher yielding than Scarlett at Balcarce, whereas all 12 ILs displayed yield equivalent to Scarlett at Tres Arroyos. We propose that this approach is useful to rapidly transfer genes for multiple target traits into adapted cereal cultivars or pyramiding desirable traits in elite breeding material.
Lee T. Hickey, Silvia E. Germán, Silvia A. Pereyra, Juan E. Diaz, Laura A. Ziems, Ryan A. Fowler, Greg J. Platz, Jerome D. Franckowiak, Mark J. Dieters
Euphytica · Volume 213 · Issue 64 · 2017
Vulnerability of Barley to African Pathotypes of Puccinia graminis f.sp.tritici and Sources of Resistance
The emergence of widely virulent pathotypes (e.g., TTKSK in the Ug99 race group) of the stem rust pathogen (Puccinia graminis f.sp. tritici) in Africa threatens wheat production on a global scale. Although intensive research efforts have been advanced to address this threat in wheat, few studies have been conducted on barley, even though pathotypes such as TTKSK are known to attack the crop. The main objectives of this study were to assess the vulnerability of barley to pathotype TTKSK and identify possible sources of resistance. From seedling evaluations of more than 1,924 diverse cultivated barley accessions to pathotype TTKSK, more than 95% (1,844) were found susceptible. A similar high frequency (910 of 934 = 97.4%) of susceptibility was found for the wild progenitor (Hordeum vulgare subsp. spontanewn) of cultivated barley. Additionally, 55 barley lines with characterized or putative introgressions from various wild Hordeum spp. were also tested against pathotype TTKSK but none was found resistant. In total, more than 96% of the 2,913 Hordeum accessions tested were susceptible as seedlings, indicating the extreme vulnerability of the crop to the African pathotypes of P. graminis f. sp. tritici. In total, 32 (1.7% of accessions evaluated) and 13 (1.4%) cultivated and wild barley accessions, respectively, exhibited consistently highly resistant to moderately resistant reactions across all experiments. Molecular assays were conducted on these resistant accessions to determine whether they carried rpg4/Rpg5, the only gene complex known to be highly effective against pathotype TTKSK in barley. Twelve of the 32 (37.5%) resistant cultivated accessions and 11 of the 13 (84.6%) resistant wild barley accessions tested positive for a functional Rpg5 gene, highlighting the narrow genetic base of resistance in Hordeum spp. Other resistant accessions lacking the rpg4/Rpg5 complex were discovered in the evaluated germplasm and may possess useful resistance genes. Combining rpg4/Rpg5 with resistance genes from these other sources should provide more durable resistance against the array of different virulence types in the Ug99 race group.
B. J. Steffenson, A. J. Case, Z. A. Pretorius, V. Coetzee, F. J. Kloppers, H. Zhou, Y. Chai, R. Wanyera, G. Macharia, S. Bhavani, and S. Grando
Phytopathology · Volume 107 · Issue 8 · 2017
Mining Vavilov’s Treasure Chest of Wheat Diversity for Adult Plant Resistance to Puccinia triticina
Leaf rust (LR) caused by Puccinia triticina, is among the most important diseases of wheat (Triticum aestivum L.) crops globally. Deployment of cultivars incorporating genetic resistance, such as adult plant resistance (APR) or all-stage resistance, is considered the most sustainable control method. APR is preferred for durability because it places lower selection pressure on the pathogen and is often polygenic. In the search for new sources of APR, here we explored a diversity panel sourced from the N. I. Vavilov Institute of Plant Genetic Resources. Based on DNA marker screening, 83 of the 300 lines were deemed to carry known APR genes; namely, Lr34, Lr46, and Lr67. Interestingly, lines carrying Lr67 were mostly landraces from India and Pakistan, reconfirming the likely origin of the gene. Rapid phenotypic screening using a method that integrates assessment at both seedling and adult growth stages under accelerated growth conditions (i.e., constant light and controlled temperature) identified 50 lines carrying APR. Levels of APR corresponded well with phenotypes obtained in a field nursery inoculated using the same pathotype (R2 = 0.82). The second year of field testing, using a mixture of pathotypes with additional virulence for race-specific APR genes (Lr13 and Lr37), identified a subset of 13 lines that consistently displayed high levels of APR across years and pathotypes. These lines provide useful sources of resistance for future research. A strategy combining rapid generation advance coupled with phenotyping under controlled conditions could accelerate introgression of these potentially novel alleles into adapted genetic backgrounds.
Riaz, A.; Athiyannan, N.; Periyannan, S.; Afanasenko, O.; Mitrofanova, O.; Aitken, E. A. B.; Lagudah, E.; Hickey, L. T.
Plant Disease · Volume 101 · Issue 2 · 2017
Genetic analysis of resistance to stripe rust in some Iranian bread wheat cultivars and elite lines
Stripe rust is the most important disease of wheat in many wheat growing areas in Iran. Good knowledge of the genetic basis of resistance to stripe rust in commercial bread wheat cultivars and selected elite lines is an important objective in wheat breeding programs. This study aimed to identify resistance genes and modes of inheritance of stripe rust resistance in recently released Iranian commercial wheat cultivars (Aflak, Parsi, Sivand, Uroum, and Pishgam) and elite bread wheat lines (M-84-14 and M-83-6). Crosses were made between these cultivars and elite lines with Avocet S and the F1, F2, and F3 generations were developed. Two F3-derived families (one adult plant stage and one seedling stage), as well as parents and controls, were grown under field and greenhouse conditions and inoculated with stripe rust pathotypes 134E158 A+, 166E150 A+, and 6E150 A+, Yr27. The adult plant responses of parental cultivars Aflak, Uroum, Parsi, Pishgam, Sivand, and elite lines M-84-14 and M-83-6 to stripe rust in the field were 40 MR, 10R, 50 M, 10R, 50 MS, respectively, in 2011, and 60 MR, 5R, 40 MR, 30 MR, and 40 M, respectively, in 2013. Cultivars and elite lines were resistant to stripe rust at the seedling stage test. Avocet S was susceptible at both adult plant and seedling stages. In addition to the seedling resistance responses of the parents, frequencies of F3 lines for each of the crosses in both adult plant and seedling stages conformed well with those expected for segregating for a trait at two loci, indicating that all five cultivars and two elite lines carry two dominant seedling resistance genes that have so far been effective for controlling stripe rust in Iran.
A. Zakeri, F. Afshari, M. Yasaie, A. R. Nikzad, S. Rajaei
Crop Breeding Journal · 2017
Genetic mapping of stem rust resistance to Puccinia graminis f. sp. tritici race TRTTF in the Canadian wheat cultivar harvest
Stem rust, caused by Puccinia graminis f. sp. tritici, is a destructive disease of wheat that can be controlled by deploying effective stem rust resistance (Sr) genes. Highly virulent races of P. graminis f. sp. tritici in Africa have been detected and characterized. These include race TRTTF and the Ug99 group of races such as TTKSK. Several Canadian and U.S. spring wheat cultivars, including the widely grown Canadian cultivar ‘Harvest’, are resistant to TRTTF. However, the genetic basis of resistance to TRTTF in Canadian and U.S. spring wheat cultivars is unknown. The objectives of this study were to determine the number of Sr genes involved in TRTTF resistance in Harvest, genetically map the resistance with DNA markers, and use markers to assess the distribution of that resistance in a panel of Canadian cultivars. A doubled haploid (DH) population was produced from the cross LMPG-6S/Harvest. The DH population was tested with race TRTTF at the seedling stage. Of 92 DH progeny evaluated, 46 were resistant and 46 were susceptible which perfectly fit a 1:1 ratio indicating a single Sr gene was responsible for conferring resistance to TRTTF in Harvest. Mapping with single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers placed the resistance gene distally on the chromosome 6AS genetic map, which corresponded to the location reported for Sr8. SSR marker gwm459 and 30 cosegregating SNP markers showed the closest linkage, mapping 2.2 cM proximal to the Sr gene. Gene Sr8a confers resistance to TRTTF and may account for the resistance in Harvest. Testing a panel of Canadian wheat cultivars with four SNP markers closely linked to resistance to TRTTF suggested that the resistance present in Harvest is present in many Canadian cultivars. Two of these SNP markers were also predictive of TRTTF resistance in a panel of 241 spring wheat lines from the United States, Canada, and Mexico.
Hiebert, C. W.; Rouse, M. N.; Jayaveeramuthu Nirmala; Fetch, T.
Phytopathology · Volume 107 · Issue 2 · 2017
First Report of Virulence to Sr25 in Race TKTTF of Puccinia graminis f. sp. tritici Causing Stem Rust on Wheat
Puccinia graminis f. sp. tritici (Pgt) race TKTTF was reported as the dominant race in the wheat stem rust epidemics in Ethiopia during 2014–15 (Olivera et al. 2015). The race and variants hereof have also been recorded elsewhere in Africa, the Middle East, and Europe (www.wheatrust.org/stem-rust-tools-maps-and-charts/race-frequency-map). Here, we report the presence of additional virulence to Sr25 in the TKTTF population, a resistance gene transferred to several Australian and CIMMYT wheat genotypes. At the seedling stage, Sr25 confers infection type (IT) 2 or lower for isolates in the Ug99 race group and up to IT 2+ toward race TKTTF (Newcomb et al. 2016; Olivera et al. 2015). Our results are based on Pgt isolates of the TKTTF race from Ethiopia (2012, 2013, 2015), Egypt (2014), Azerbaijan (2014), Iran (2009, 2011, 2014), Iraq (2014), Lebanon, Sudan, and Turkey (2012), Denmark and Germany (2013), and Sweden (2014). Race typing was carried out at the Global Rust Reference Center according to Jin et al. (2008), except that we scored IT on both leaf 1 and 2; additional single pustule isolates of each sample were raised and stored in liquid nitrogen (–196°C). Sr25 response was assayed using seedling leaves and stems of adult plants of Misr1 (Oasis/Skauz//4*BCN/3/2*Pastor) and Agatha/9*LMPG (Sr25 carriers) along with two reference lines, Triumph 64 (SrTmp) and NA101/MqSr7a (Sr7a), and Morocco as a control. Seedling ITs were scored 17 days post-inoculation at 18 ± 2°C using a 0 to 4 scale (McIntosh et al. 1995). Isolates showing ITs of 33+ to 4 on Misr1, Agatha/9*LMPG, and susceptible check were considered Sr25 virulent, and clearly different from ITs conferred by Sr25 avirulent isolates. Results were confirmed for each isolate by race typing additional single-pustule isolates derived from cultivars Misr1 and/or Agatha, along with avirulent reference isolates. Virulence for Sr25 was observed in race TKTTF isolates from Azerbaijan, Egypt, Ethiopia, Iran, Iraq, and Sweden, collected in 2014 or 2015, but not in any sample collected earlier than 2014. The results were confirmed on adult plants of Misr1 and Agatha/9*LMPG by Sr25 virulent and avirulent isolates of TKTTF, TTKSK, and TTKST, respectively. Spore suspensions of ∼0.5 ml at concentration of ∼3 × 105 spores/ml were injected into the stem internodes at Zadoks 45. The adult plant and seedling tests were carried out concurrently using the environmental conditions described above. The plants containing Sr25 were susceptible to the Sr25 virulent isolate and moderately resistant to moderately susceptible to the Sr25-avirulent isolates of TKTTF, TTKSK, and TTKST. The experiments were repeated two times with three replicates, using cv. Morocco as a susceptible check. Emergence of virulence to Sr25 in the race TKTTF is considered significant due to its spread into new areas and the potential loss of a significant source of resistance against Ug99.
M. Patpour, M. S. Hovmøller, D. Hodson
Plant Disease · Volume 101 · Issue 9 · 2017