The Central Research Institute for Field Crops, Turkey
Stem rust still remains an important threat to wheat with new races such as Ug99. In 2012 main season, some genotypes developed by Central Research Institute for Field Crops were sent to Kenya for screening to Ug99 and WWSRRN (winter wheat stem rust resistance nursery) was organized with resistant genotypes according to result. Addition to Ug99, the resistances to local stem rust, leaf rust and yellow rust races are important. The aim of this study was to determine reactions of 99 genotypes in WWSRRN to local rust population in the seedling stage (for PSt, Pgt and Pt) and adult plant stage (for Pst and Pgt) at the research facilities of CRIFC in Ankara and Kastamonu (stem rust) during 2014 season. For adult plant test; the genotypes were inoculated with local Pst populations (virulent on Yr2,6,7,8,9,25,27,Sd,Su,Avs) and local Pgt populations (avirulent on Sr24, Sr26, Sr27, and Sr31). Stripe and stem 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 and August 2014, respectively. Coefficients of infections were calculated and values below 20 were considered to be resistant. For seedling test; the seedling was inoculated with local Pgt, 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. Thirty seven (37%) (seedling) genotypes and 39 (39%) (adult stage) genotypes were resistant to local Pgt, 35 (35%) (seedling) were resistant to the local Pt, and 55 (56%) (seedling) and 59 (60%) (adult stage) genotypes were resistant to the local Pst populations. The resistance sources to stem, leaf, and stripe rust were determined with this research.
Agriculture and Agri-Food Canada, Lethbridge Research Centre, Canada
We earlier assembled the genomes of Pst isolates collected in western Canada using assembly of Illumina paired-end sequences. Two isolates, LSW3_2012_SP2 and SWS484_SPF, were assembled with ?15,150 and ?11,700 contigs each when compared to references North American PST-78 and Chinese CYR32, respectively. In order to reduce the number of contigs and therefore obtain a longer display of contiguous genes, we used the PacBio and Illumina Mate Pair (MP) technologies to achieve that goal using the two isolates. We had to modify our current protocol for DNA isolation from Pst spores to obtain DNA fragments of ?35 Kb suitable for construction of large insert genomic sequencing libraries. Libraries of 8-10 Kb and 3.5-6 Kb were used for PacBio and Illumina MP analyses, respectively. We obtained a 26x coverage of the Pst genome with the PacBio results with an mean size of 7,400 bp and 6,500 bp for the two libraries, and a 190x coverage with the Illumina MP sequencing information. We are using the Ray assembler with the datasets and the 50x Illumina paired end sequencing information from previously independently associated isolates LSW3_2012_SP2 and SWS484_SPF. The quality of our assembly will be compared to the contigs and supercontigs available for the reference isolates PST-78 and CYR32. These results will also enable us to establish the physical relationship among isolate-specific genes. Finally, the impact of the large insert libraries on the proportion of short paired-end unassembled reads will be discussed as it was 78% and 50% for LSW3_2012_SP2 and SWS484_SPF, respectively, after assembly of 100 bp paired-end reads.
Aarhus University, Denmark
In recent years Pgt race Ug99 has moved out of East Africa posing a threat to other regions in Africa, Central and West Asia, and other regions where this pathotype had not been found. In Europe a new Pst population (Warrior and Kranich races) replaced less diverse pathogen populations during the years 2011-2014. To enable the tracking and monitoring of the evolution of pathogen populations, a new web/database management and display system called the Wheat Rust Toolbox was developed. This platform, hosted by the Global Rust Reference Centre, supports two major initiatives: 1) the Durable Rust Resistance in Wheat project (DRRW), for managing and visualizing wheat rust pathogen data - mainly related to the stem rust pathogen (Pgt) from Africa, Central and West Asia; and 2) the ENDURE wheat rust network for managing and visualizing stripe rust, including Pst race, virulence and genotype data for Europe. The presentation will provide an overview of data and tools available in the Wheat Rust Toolbox, the research infrastructure behind it, and how data are disseminated via several information platforms such as wheatrust.org, eurowheat.org and http://rusttracker.cimmyt.org/. Opportunities available for analyzing genotypic data (SSR and SNP) online via a web-based version of the POPPR integrated with the Wheat Rust Toolbox will be presented. Overall the results show that the collation of data in a standardized way across many countries leads to more robust and fast conclusions that will stimulate closer collaborations between partners.
The University of Sydney Plant Breeding Institute Cobbitty, Faculty of Agriculture and Environment, Australia
Improvement of stripe rust resistance is one of the main aims of wheat breeding programs worldwide. Progress is dependent on the availability of genetically diverse and widely effective sources of resistance. This study focuses on genetic analysis of stripe rust resistance in landrace accession AWCC275 from the Watkins Collection. AWCC275 was scored resistant to moderately resistant under field conditions during three crop seasons and showed an intermediate seedling response (infection type 2C). AWCC275 was crossed with the susceptible genotype Avocet S and a population of 76 F3 families was generated. Twenty seedlings of each family were tested at the seedling stage with Puccinia striiformis f. sp. tritici pathotype 134 E16A+,Yr17+,Yr27+ under greenhouse conditions. Sixteen lines were homozygous resistant (HR), 43 segregated and 17 were homozygous susceptible (HS). Chi-squared analysis (?21:2:1 =1.34, non-significant at P=0.05 and 2 df) indicated segregation at a single locus. HR and HS lines were submitted for selective genotyping using the 90K SNP platform. The population is currently being advanced to F6 for detailed molecular mapping and the resistance gene is being backcrossed to three Australian wheat cultivars.
State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, P.R. China
A Pst pathotype group named V26, virulent to wheat lines possessing Yr26 (=Yr24) has become the third most frequent group in China after races CYR32 and CYR33. Twenty four near-isogenic lines (NILs) and 19 Chinese differentials were used to identify the avirulence/virulence spectra of 36 Yr26-virulent isolates from four provinces (Qinghai, Gansu, Sichuan and Ningxia). Eight races were identified when tested on the NIL set, and 7 races were identified on the Chinese set. There was no relationship with province of origin. Three races identified on the NILs occurred at relatively high frequencies (23, 3, and 3 isolates). Virulence differences existed for Yr1, Yr4, Yr6, Yr9, Yr17, Yr25, Yr32, YrSp, and YrTr1. Among the 7 races identified on the Chinese differentials, one (CYR32 + Yr26 virulence) was represented by 13 isolates and another (CYR33 + Yr26 virulence) included 15 isolates. Among the entire group there were virulence differences on Trigo-Eureka (Yr6+), Lovrin 13 (Yr9+), Kangyin 655, Fengchan 3 (Yr1+), Lovrin 10 (Yr9+), and Hybrid 46 (Yr4+). All isolates were avirulent on Zhong 4 and T. spelta. Using 18 polymorphic simple sequence repeat (SSR) markers, we identified 35 genotypes clustered into two molecular groups (MGs) at a similarity coefficient level of 0.70. SSR analysis also indicated a high level of recombination within the V26 group. The considerable diversity indicates a threat not only to cultivars carrying Yr26, but also to other currently resistant materials.
Agriculture and Agri-Food Canada, Lethbridge Research Centre, Canada
We evaluated Pst-wheat interaction at the transcriptome level between Pst isolate LSW3_2012_SP2 and Avocet/Yr5 (R), and Avocet S (S). For the compatible interaction we used a dataset of 9.3 M Illumina paired-end clean reads in which ?300,000 reads mapped against nearly 9,000 contigs of the PST-78 reference transcriptome, whereas 3.5 M reads mapped against a partial wheat transcriptome of 13,300 contigs. Pst transcripts in the infected wheat tissues amounted to about 10% of the mapped transcripts. In the incompatible reaction, we used a dataset of 13.2 M clean reads and ?27,000 reads mapped against nearly 2,900 Pst transcriptome contigs while 7.7 M reads mapped against 18,800 wheat transcripts. These results show an important differential regulation of genes in both the fungal pathogen and the wheat host. More than 3 times the number of distinct Pst transcripts was identified in the compatible reaction than the incompatible reaction. Genes differentially regulated between the incompatible and compatible reactions will be compared in terms of functionality and GO term classification. To validate Pst transcripts involved in the infection process, we adapted a leaf rust haustorial isolation protocol for characterization of proteins and modified it to protect the integrity of RNA in enriched Pst haustoria. A list of potential effectors present in LSW3_2012_SP2 and verified in haustoria-enriched tissues will be presented.
Agriculture and Agri-Food Canada, Cereal Research Centre, Canada
The stem rust resistance gene SrTmp carried by Triumph 64 confers resistance to Pgt race TTKSK and other members of the Ug99 race group. While some previous studies have mapped resistance postulated to be SrTmp, none used Triumph 64 as a parent. The purpose of this study was to genetically map SrTmp with DNA markers using a DH population from LMPG/Triumph 64 and compare the map position of SrTmp to previously mapped Sr genes. The DH population was tested with Pgt race TTKSK at the seedling stage. A single gene conditioned resistance to TTKSK (n = 144; χ21:1 = 0.44, p = 0.50). SrTmp was mapped to the distal region of chromosome 6DS with SSR markers. The map location of SrTmp was similar to SrCad and Sr42, which likely represent the same gene. In a concurrent study SNP markers were developed to fine-map SrCad. SNP markers were identified and/or developed using a public SNP database (http://www.cerealsdb.uk.net) and sequence information from an Aegilops tauschii genome sequencing project. Further SNP markers were developed by using resistance gene analogs from chromosome 6D to BLAST exome capture sequences from a set of Canadian wheat cultivars followed by searching for unique SNPs found in SrCad carriers. These SNP markers were added to the map of SrTmp. The map positions of SrTmp and SrCad/Sr42 are very similar. While preliminary data show functional differences between SrTmp and SrCad/Sr42, further analysis is needed to determine whether these genes are allelic or closely linked.
The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, Australia
Elite barley breeding lines from the Australian Northern Region Barley Breeding Program were evaluated at the seedling and adult growth stages for resistance to leaf rust (LR) caused by Puccinia hordei. F3:5 lines derived from parental germplasm of different geographic origins were screened in the glasshouse and field spanning four years of trials. The 2009 and 2011 breeding populations (BP1 and BP2) comprised 360 lines and were genotyped with 3,244 polymorphic diversity arrays technology (DArT) markers. The 2012 and 2013 breeding populations (BP3 and BP4) comprised 320 lines genotyped with the DArT GBS array (DArTseq), providing 15,400 high quality polymorphic markers. Association mapping (AM) using the DArT/DArT-seq datasets and phenotypic data from 15 independent LR response assays identified a number of genomic regions associated with resistance. The BP1 and BP2 study detected a total of 15 QTL; 5 QTL co-located with catalogued LR resistance genes (Rph1, Rph3/19, Rph8/14/15, Rph20, and Rph21), 6 QTL aligned with previously reported genomic regions and 4 QTL (3 on chromosome 1H and 1 on 7H) were novel. Markers in common between the DArT and DArTseq datasets enabled integration of mapping results for LR response across the four breeding populations and all QTL detected were visualised on a single map for validation. The adult plant resistance (APR) locus Rph20 was the only region detected in all field environments. Markers and their associated sequences identified in this study will be useful for building QTL combinations involving Rph20, thereby providing stable LR resistance in improved barley cultivars. We will also highlight the advantages of AM using breeding germplasm over traditional bi-parental mapping approaches that underutilise genetic diversity and divert valuable resources into populations of low breeding value.
Institute of Agricultural Environment and Resources, Yunnan Academy of Agricultural Sciences, China
Pathogen migration is a source of new pathogens and pathotypes to a particular region. Studies on origin and migration of pathogens were difficult in the past, but DNA sequence data and new analytical approaches now enable us to analyze population genetic structures from which we can determine possible migration routes. The Himalayan and neighboring regions (Gansu, China) were earlier suggested as putative centers of origin for Puccinia striiformis f. sp. tritici (Pst) and sampling locations for Chinese isolates were strongly focused in South Gansu. Previous field surveys indicated that Yunnan might be the primary source of the Gansu population in China. Using samples collected in 2008 and 2011 we compared the Pst population structures of Yunnan and south Gansu for SNPs in housekeeping genes Cdc2, Ef-1? and Mapk1, and the trajectories of upper air flow during wheat growing seasons from 2005 to 2013. The ancestral haplotype was detected in the Yunnan population, which had higher a mutation rate than the Gansu population, but the latter contained more recombination events. Both populations were highly diverse. There was massive air flow between Yunnan and Gansu with trajectories being mainly from Yunnan to Gansu. We propose a putative dispersal route of Pst from Yunnan to South Gansu, making Yunnan the center of origin for Pst in China, whereas Gansu is a secondary center of origin. It is proposed that Pst migrates from South Asia to Yunnan under the influence of westerly weather patterns, and subsequently spreads to other parts in China. DNA sequence comparisons should be undertaken to compare Pst populations of South Asia and China in order to confirm these hypotheses.
Management of the cereal rusts currently relies on the use of fungicides and varietal resistance. Host resistance can be rapidly overcome by mutations in the pathogen population and for this reason virulence surveys are undertaken worldwide to give early warning on any changes. The UK Cereal Pathogen Virulence Survey (UKCPVS) was established in 1967 following an outbreak of yellow rust on the variety Rothwell Perdix. The UKCPVS currently monitors the wheat yellow rust and brown rust pathogen populations as well as the wheat and barley powdery mildew pathogen populations. A watching brief is maintained on barley yellow rust. In 2011 a new Pst race was detected; named the Warrior race after the variety on which it was first found. Initial tests on differential host lines suggested that the race was another stepwise mutation with an added virulence for Yr7 in addition to a combination of virulences to Yr6, Yr9, Yr17 and Yr32. Other characteristics of this race, however, suggested something different from previous race changes with an increase in telial production seen under field conditions. In addition, this new race was seen simultaneously in multiple locations throughout Europe (www.wheatrust.org) in contrast to the more gradual appearance of new variants seen previously. Subsequent genotypic analysis of isolates (Hubbard et al. 2015, Genome Biol. 16:23) demonstrated that the new race was an exotic incursion. Results presented at the meeting will summarize some of the key changes in the pathogen populations, such as the arrival of the Warrior Pst race in 2011 and the re-emergence of the Glasgow race of the brown rust pathogen in 2014.