Rapid detection of micro-RNAs associated with APR to rust pathogens in wheat
BGRI 2015 Poster Abstract Singh
The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, Australia
The identification of R-genes using traditional map-based approaches is a long, laborious process, not to mention the time required for subsequent development of cultivars incorporating the new resistances. Breeders seek to reduce the length of breeding cycles, and researchers require new tools to accelerate discovery and understanding of mechanisms associated with durable resistance, especially adult plant resistance (APR). A new method for rapid generation advancement, known as ‘speed breeding’, significantly reduces the length of breeding cycles, provide increased recombination during line development and enable selection in early generations. The speed breeding protocol uses controlled temperature regimes and 24h light to accelerate plant growth and development. Phenotyping methods adapted for use in the speed breeding system permit year-round evaluation of APR to rust pathogens within 5 weeks from time of sowing. RNA sequencing (RNA-Seq) technology has revolutionized gene expression profiling in plants. We previously used RNAseq to identify novel transcripts and miRNAs associated with seedling resistance (Lr28) leading to identification of transcription factors and miRNA families (e.g. miR36, miR37 and miR39) involved in signalling and defense response (Kumar et al. J. Nuc. Acids 2014:570176). In this study we report the application of speed breeding and RNAseq technologies for the purpose of rapidly identifying transcripts and miRNA associated with APR. Wheat landraces harbouring novel sources of resistance were grown under speed breeding conditions and sampled for RNA at key growth stages, before and after inoculation, which enabled discovery of differentially expressed miRNAs. Our next steps are aimed at validating these genetic factors associated with APR in order to better understand the signalling pathways and deliver tools to assist the assembly of robust wheat cultivars for the future.
The Stubbs Pst Culture Collection: Recovery, avirulence/virulence phenotyping and past population structure at a global scale
BGRI 2015 Poster Abstract Thach
Department of Agroecology, Aarhus University, Denmark
The "Stubbs Collection", began in 1956 by the late Dutch plant pathologist R.W. Stubbs, refers to a unique historic collection of urediniospore samples of Puccinia striiformis that had been stored in liquid nitrogen for decades. Since 2010 the collection has been maintained by the Global Rust Reference Center (GRRC) in Denmark. Part of the collection is now being in a study of past pathogen diversity. A subset of samples collected between 1958 and 1991 from 35 countries was investigated to assess recovery rate, race identity, and previously undetected virulences. A new method for recovery using an airbrush sprayer and NovecTM 7100 fluid as dispersal agent in inoculating host plants was highly successful, resulting in a 96% recovery from 231 isolates. Phenotyping on the World and European differential host sets and additional wheat genotypes revealed 181 apparently uniform isolates, of which race identities were confirmed for 102. Race identities were updated for additional isolates based on improved resolution due to updated and more informative differential lines. Additional virulences corresponding to Yr17, Yr25, and Yr27 were added, as these were not assayed earlier. The past population structure was investigated by genotyping 212 isolates using 19 multilocus microsatellites. Seven distinct populations were detected, including clonal populations and recombinant populations. These results were compared with recent studies and demonstrated an overall consistent population subdivision at the global scale with clear migration events between populations. The outcome of the study facilitates conclusions about long-term temporal dynamics and overall migration patterns within and among world-wide populations of Pst.
Reactions of spring wheat genotypes in crossing block nursery to stem, leaf, and stripe rust
BGRI 2015 Poster Abstract Mert
The Central Research Institute for Field Crops, Turkey
Rusts (Puccinia spp.) are the most significant disease affecting wheat yield and quality in Turkey. Knowing the resistance status of wheat genotypes in crossing program is an important issue for breeding programs. The aim of the study was to determine of the resistance of the 106 wheat genotypes consisting of Crossing Block Spring Wheat (CBSW) nursery developed by the International Winter Wheat Improved Project (IWWIP). For this purpose, adult plant and seedling test were conducted for yellow rust while only seedling test were conducted for leaf and stem rust. Evaluations were carried out at the research facilities of CRIFC at İkizce and Yenimahalle in Ankara in the 2014 season. For adult plant reactions; the genotypes were inoculated with local Pst populations (virulent on Yr2,6,7,8,9,25,27,Sd,Su,Avs). Stripe rust development on each entry were scored using the modified Cobb scale when the susceptible check Little Club had reached 80S infection severity in June, 2014. Coefficients of infections were calculated and values below 20 were considered to be resistant. For seedling test; the seedling was inoculated with local Pgt (avirulent on Sr24, Sr26, Sr27, and Sr31), Pt (avirulent on Lr9, Lr19, Lr24, and Lr28) and Pst populations. Stripe, leaf and stem rust development on each entry were scored after 14 days with 0-4 and 0-9 scale for leaf-stem rust and yellow rust, respectively. In seedling stage, thirty nine (37%), 47 (44%), and 20 (19%) genotypes were resistant to local Pgt,Pt, and Pst populations, respectively. In adult plant test, 46 (43%) genotypes were resistant to Pst. The resistance genotypes to stem, leaf, and stripe rust were determined with this research.
Wheat stem rust effectors: Genomics and functional assay
BGRI 2015 Poster Abstract Upadhyaya
CSIRO Agriculture, Canberra, Australia
Puccinia graminis f. sp. tritici (Pgt) is one of the most destructive pathogens of wheat. Fungal secreted proteins termed effectors play an important role in modulating the host cellular environment and suppressing the plant defense response to enable fungal growth. They also become targets of plant resistance (R) proteins. We have taken a genomics approach to initially identify candidate effectors. We have built a draft genome for a founder Australian Pgt isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced North American Pgt isolate CDL 75-36-700-3 (p7a) and de novo assembly resulted in a 92 Mbp reference genome for Pgt isolate 21-0. This draft genome was subsequently used to build a pan-genome based on five Australian Pgt isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ~10% of the genes in germinated urediniospores as well as haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 586 were classified as haustorial secreted proteins (HSPs). We are currently exploring effector gene expression during infection of wheat to reduce this candidate list based on a common expression profile identified for Avr genes in the flax rust fungus. Comparison of 21-0 with two presumed clonal field derivatives (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (Sr5, Sr11, Sr27, SrSatu) identified mutations in 13 HSP effector candidates. These candidate effectors are being assessed for recognition in wheat accessions with the corresponding R genes using a bacterial type three secretion delivery system based on an engineered Pseudomonas fluorescence strain (Upadhyaya NM et al. Mol Plant Microbe Interact 27:255-264).
Targeting stem rust resistance genes Sr32 and Sr1644 for cloning by mutagenesis and sequence capture
BGRI 2015 Poster Abstract Wulff
John Innes Centre, UK
Resistance offers the best means of control of the cereal rusts, but must be strategically deployed so as to avoid exposure of single major genes, which have faltered so many times in the past. The pyramiding of multiple effective resistance genes is a strategy that has proven effective in a number of wheat production areas around the world. However, the process of incorporating multiple resistance genes into a single cultivar using standard breeding techniques is time consuming, laborious, and hampered by the problem of linkage drag. If a suite of effective resistance genes could be efficiently cloned and transferred into wheat as a cassette, it would accelerate the development of durably resistant varieties without the problem of linkage drag. Toward this end, we have developed a resistance gene cloning technology based on resistance gene enrichment sequencing (RenSeq) of EMS-derived mutant R gene alleles. As a proof of concept test, we successfully ‘re’-cloned the already characterized gene Sr33 and are now targeting the cloning of eight other effective resistance genes. For the identification of susceptible mutants for the cloning of Sr32 from Aegilops speltoides, we screened 1,109 M2 families with race TPMKC — as a surrogate for race TTKSK. Five susceptible M2 mutants were confirmed by progeny testing. These mutants were also susceptible to race TTKSK. For the population involving Sr1644 from Ae. sharonensis, 1,649 M2 families were screened, yielding 33 M2 families that appeared to segregate for susceptibility. Thirteen of 33 families were confirmed as bona fide susceptible mutants by progeny tests in the M3 generation. Identification of susceptible EMS mutants of Sr32 and Sr1644 suggests that the underlying resistance in these lines is conferred by single genes. We will report on progress to clone and characterize these genes using R gene exome capture and sequencing technology (RenSeq).
Introgression of genes for high grain protein content (Gpc-B1) and Lr24 into leading cultivars by marker assisted backcross breeding
BGRI 2015 Poster Abstract Mishra
Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, India
A wheat genotype PBW343+Gpc-B1+LR24 containing the high grain protein content (GPC) gene Gpc-B1 linked to marker Xucw108 was used as the donor parent to transfer Gpc-B1 and Lr24 into Eastern Gangetic Plains (EGP) cv. HUW234 and HUW468 that were released in 1986 and 1999, respectively. The backcrossing program involved the following steps: (i) foreground selection, (ii) marker selection, and (iii) recovery of recipient parent genome. Grain protein contents were recorded for all selected plants from the BC2F2:3 generation. The dominant marker Xucw108 was used for foreground selection, and heterozygous plants were identified through progeny testing. For RPG recovery, both genotypic and phenotypic selection was used. Introgression of the high GPC gene into the recipient background without yield loss was completed in 5 years, starting from 2009-10 (F1) and completed in 2013-14 (BC2F5). A conventional selection program would take the same time to reach BC2F5 but ensuring the transfer of GPC would not not be possible. Ten selected single plants from the BC2F3:4 generation had comparable yields of the parents with 26% higher GPC than the recurrent parent HUW 234. Eight selected plants had comparable yields and 34% higher GPC than HUW 468. Multi-row progenies (BC2F4 and BC2F5) of each selected plant were evaluated in yield traits with the donor and recipient parents as controls during 2012-13 and 2013-14. Two lines based on each recurrent parent were identified with significantly higher GPC with no yield penalty. The study reinforced the belief that MAS in combination with phenotypic selection could be a useful strategy to develop high GPC genotypes without sacrificing grain yield. These lines will be submitted to national trial where MAS derived lines require only two years of testing compared to four years for conventionally bred lines.
Global Pgt Initiative: An international genetic resource to combat stem rust
BGRI 2014 Plenary Abstract Les Szabo
USDA-ARS, Cereal Disease Laboratory
An important component of the management of wheat stem rust is an understanding of the population diversity of the pathogen, Puccinia graminis f. sp. tritici (Pgt). The discovery of “Ug99” resulted in renewed efforts on pathogen surveys, sample collections and pathotyping of Pgt, with a primary focus on Africa. In the last few years these efforts have been expanded to include other targeted regions, however a global effort is needed. The aims of the “Global Pgt Initiative” is: to capture and maintain living cultures that collectively reflect the entire global diversity of Pgt in the years 2014 - 2016; pathotype and genotype this collection; develop DNAbased diagnostic tools that will be able to rapidly detect shifts in Pgt populations, and provide an early warning system of the vulnerability of wheat to new virulent strains; and provide a genetic baseline for comparison of Pgt populations over time, both forward and backwards. This initiative will provide the wheat rust community with a geographically distributed, well characterized, living culture collection that represents the global diversity of Pgt; a global open access knowledge bank on Pgt pathotypes and genotypes; and advanced molecular diagnostic tools for rapid detection and tracking of Pgt populations. The Global Pgt Initiative represents the most comprehensive effort to capture and characterize the global diversity of Pgt and provide a unique resource to the global wheat rust community.
In 2010, Ethiopia experienced one of the largest stripe rust epidemics in recent history. Over 600,000 ha of wheat were affected, an estimated 60 million Ethiopian Birr ($US3.2 million) were spent on fungicides and large production losses were observed. Factors associated with the 2010 epidemic were conducive climatic conditions (prolonged rain and apparently optimal temperatures), large areas planted to susceptible cultivars, early infection and rapid spread of a virulent pathogen, a low level of awareness, and ineffective control measures. In 2013, highly favourable climatic conditions and early appearance of stripe rust showed remarkable similarity to the conditions observed in 2010, prompting fears of a similar major rust epidemic. However, no stripe rust epidemic developed in 2013. In contrast, only limited and localized outbreaks of stripe rust were observed in 2013; wheat crops remained in good condition and a good harvest was achieved. It seems that a series of positive and timely actions in Ethiopia contributed to the markedly different stripe rust situation in 2013 compared to 2010. The principle factors associated with the positive outcomes in 2013 are (i) effective promotion, plus rapid and widespread adoption of rust resistant wheat cultivars since 2010 - this dramatically reduced the vulnerability of the Ethiopian wheat crop; and (ii) timely and coordinated surveillance efforts, coupled to good information exchange amongst different stakeholders - this resulted in effective control and awareness campaigns that targeted emerging stripe rust outbreaks. A comparative analysis is presented which highlights the similarities and disparities between the 2010 and 2013 stripe rust situations in Ethiopia. The roles and contributions of different organisations are examined and an in-depth analysis of the biophysical conditions in the different years is presented.
There is emerging evidence that the geographical footprint of stripe rust is expanding, opening up prospects for an increase in economic losses attributable to this disease worldwide. Drawing on newly compiled data, along with insights obtained from a survey initiated at the BGRI meeting in New Delhi in August 2013, this talk will report on efforts to model the global occurrence and persistence of stripe rust in a geo-spatially sensitive fashion. Using the available data in conjunction with these newly developed climate suitability maps, I will present probabilistic crop production losses associated with the disease and place an economic value on the prospective losses. Given changes in the geographical spread of this disease, and the associated uncertainties about its likely wheat yield and economic effects, various scenarios will be assessed to inform and thereby help shape the research investment decisions regarding crop breeding and other options for ameliorating these prospective losses over the longer term.
Advances in breeding for resistance to stem rust caused by Ug99 and Ethiopian Pgt races in durum wheat
BGRI 2014 Plenary Abstract Karim Ammar
Stem rust (SR) resistance is required for CIMMYT durum germplasm to keep relevance in Ethiopia, where Ug99 and other Pgt races are a major yield-limiting constraint, and in countries along the possible dissemination paths of these races. Resistance to Ug99 is widespread in most durum germplasm groups when tested in Kenya, but resistance is lost when exposed to Ethiopian races; hence selection at the Debre Zeit site in Ethiopia is essential for durum wheat. Due to difficulties with shuttling segregating populations between Mexico and Ethiopia, we have adopted a strategy involving the identification of resistant/moderately resistant lines at Debre- Zeit, and inter-crossing in Mexico followed by selection for resistance to leaf rust and agronomic type and finally screening for SR reaction in the resulting F6 lines at Debre-Zeit at the same time as they are tested for yield and quality in preliminary yield trials in Mexico. This has generated a significant increase in the proportion of resistant and moderately resistant genotypes within outgoing CIMMYT germplasm, from less than 3% at the onset of the initiative in 2008 to 16% in 2011, and 38% in 2013. SR-resistant germplasm was characterized by similar frequency distributions to other traits in the overall germplasm such as yield potential, drought tolerance and industrial quality parameters. Advances have also been realized using marker-assisted selection (MAS) to introgress Sr22 from bread wheat and to combine it with Sr25, producing advanced lines with 2-gene stacks with confirmed outstanding resistance and superior quality attributes. Since the two genes are closely linked but from different sources bringing them together required a very rare recombination event finally detected via MAS among thousands of plants. They are now essentially inherited together with a very low likelihood of generating recombinant individuals with either gene. The yield potential and stability of these lines are under evaluation in Ethiopia and the best lines are being used in a second round of breeding.