CSIRO Agriculture and Food, Australia
Timothy Hewitt, Peng Zhang, Zacharias A. Pretorius, Narayana Upadhyaya, Rohit Mago, Sambasivam Periyannan, Xiuying Kong, Burkhard Steuernagel, Brande H. Wulff, Evans S. Lagudah
Multiple rust resistance gene combinations are considered as a practical solution for providing durable rust resistance and preventing resistance breakdown arising from single gene deployment. The stem rust resistance locus Sr26, originally derived from Thinopyrum ponticum and introgressed into wheat as a chromosome translocation, is one of the very few genes conferring durable resistance for almost 40 years to all known races of stem rust, including the highly virulent stem rust race Ug99 (TTKSK) and its derivatives (Dundas et al. 2015). To understand the underlying mechanisms of its unusual long-term effectiveness and to explore allelic diversity in different Th. ponticum accessions for other functional alleles that may offer new sources of resistance, we used comparative genomics and gene capture techniques (Resistance gene enrichment sequencing, RenSeq) as complementary strategies for isolating the target gene (Steuernage et al. 2016). Sr26 region was first mapped using NB-LRR (Nucleotide-binding site and leucine-rich repeat) sequences from the orthologous gene members located on the long arm of chromosome 6D from Aegilops tauschii (the D-genome donor of wheat) reference genome. Subsequently, we revealed a cluster of NB-LRR sequences located at the distal end of the Th. ponticum introgression segment that were absent in the smallest interstitial Sr26 deletion mutant. Therefore, we substantially narrowed down the genetic interval for Sr26. In addition to this approach, we subjected the mutant population to RenSeq pipeline. A candidate gene of Sr26 has been successfully identified to be a NBS-LRR type resistance gene. Validation of the gene candidate by complementation studies is currently in progress. In order to enhance durable resistance, genetic stocks of Sr26 from different backgrounds as well as a panel of Sr26-APR (Adult Plant Resistance) gene combinations have been generated to further investigate the resistance response of Sr26 in combination with different multi-pathogen APR genes.
Agriculture & Agri-Food Canada
Wheat cultivar McNair 701 carries resistance gene SrMcN and is used as a differential line to identify Pgt races using the international letter code nomenclature. The inheritance and location of the resistance gene has not been characterized. We developed a doubled haploid (DH) population from cross LMPG/McNair 701 to study the genetics and chromosomal location of SrMcN. A DH population inoculated with race QCCJB segregated 100 resistant : 94 susceptible, a 1:1 ratio (?2=0.186, P=0.666, NS) indicative of segregation at a single locus. This gene was mapped to chromosome 2DL using the Infinium 90k platform. The map position of SrMcN was similar to that of Sr54, one of two genes previously found in Norin 40. Comparison of stem rust seedling reactions using 12 diverse Pgt races indicated that McNair 701 and an Sr54 line derived from Norin 40 had an identical pattern of responses and similar low infection types (IT=12-) to races LCBNB and QCCJB. Based on the chromosomal location on 2DL and identical seedling responses to Sr54, it is likely that the resistance gene in McNair 701 formerly known as SrMcN is Sr54. This finding will be confirmed by a test of allelism.
Cereal Laboratory, Wheat Research Institute, Faisalabad, Punjab, Pakistan
Hira Shair, Anjum Javed, Muhammad Abdullah, Makhdoom Hussain, Javed Ahmed
Globally, more than two billion people are undernourished in the world and deficient in key vitamins and minerals, making it the world's greatest health risk factor. Among these, iron and zinc are of greater significance from human nutrition perspective, ranking them 5th and 6th in developing countries. The population most vulnerable to these micronutrient deficiencies is women and children. Iron deficiency results about 1.62 billion people as anemic, largely preschool children (47%). It is responsible for approximately 20854 deaths and two million disability adjusted life years (DALYs) among children under five years old, whereas, zinc deficiency is responsible for approximately 4% of deaths and 16 million DALYs, among children under age five. This leads to malnutrition ultimately leading to a disabled society.
Widespread accessibility of these nutrients is the solution to cater malnutrition. Wheat, the "staff of life," consumed by masses can help eradicate "hidden hunger." For this, fortification and bio-fortification are highly talked about, but one having limitations in reaching the masses and other a long term intervention, respectively, suitability of planting times to screen out varieties high in zinc and iron, is an on-field solution. In a study, wheat varieties; Punjab-11, Millat-11 and Galaxy-13 were selected from three planting times, with an interval of one month. Results reveal varieties exhibited their natural genotypic response but planting time impact on Zn and Fe were visibly significant. 30th December gave higher contents of Fe and Zn as compared to previous planting dates of the same year. Iron on an overall basis ranged from (135.0-147.0) ppm, while Zinc gave a confined range of (30.2-33.2) ppm. Thus, concluded that comparatively delayed sowing favours the mineral content concentration in wheat grains. And these creamed out varieties can readily be used in crosses with high yielding varieties, in order to make our wheat mineral sufficient.
Crop Development Centre/Department of Plant Science, University of Saskatchewan, Saskatoon, Canada
Sajid Ali, Dinah Qutob, Steve Ambrose, Ron Maclachlan, Kun Lou, Curtis Pozniak, Yong-Bi Fu, Andrew Sharpe, Randy Kutcher
Puccinia striiformis f. sp. tritici (Pst), the cause of wheat stripe rust, is one of the most important pathogens of wheat. Attempts have been made in the past to characterize the worldwide genetic structure of Pst populations, excluding Canada. Characterization of 59 isolates identified 33 races with three most common races representing half of the population and subtle differences in races of eastern and western prairies. For molecular characterization, 48 isolates were sequenced to obtain SNPs and genotyped with Pst-specific SSR markers. Isolates that were suspected of recombination based on SNP data were examined for their telia production ability as a proxy for sexual recombination. The study revealed that the majority of the population was clonal, however, not exclusively clonal, with the existence of four genetic lineages. Two lineages previously reported were identified: PstS0, representing an old northwestern-European and PstS1, an invasive warmer-temperature adapted lineage. Additionally, two new lineages, PstPr and PstS1-related, were detected that have not been reported previously. The PstPr and PstS1-related lineages produced more telia than the other lineages and had double the number of unique recombination events compared to PstS0 and PstS1. PstPr was concluded to be a sexual recombinant and an exotic incursion, which was closely associated with PstS5, PstS7 (Warrior), and PstS8 (Kranich) lineages, all of which arose by sexual recombination in the center of diversity - the Himalayan region. The total phenotypic variation in the population could not be explained solely by molecular genotypes, and a hypothesis on existence of epigenetic machinery in the Pst genome was tested. Homologs of the DNMTases class (DNMT1) were identified, providing compelling evidence of a role for DNA methylation. As a first report of DNA methylation, an average of ~5%, 5-methyl cytosine (5-hmC) in the Puccinia epigenome indicated the possibility of epigenetic regulation, which merits further investigation.
The University of Sydney
Davinder Singh, Peter Dracatos
Following the introduction of wheat stripe rust into Australia in 1979, an uncharacterized resistance (YrA) was identified in both Australian and International spring wheats. Genetic analyses of YrA indicated it was a pair of complementary genes, which were mapped to chromosomes 3DL and 5BL and designated Yr73 and Yr74, respectively. While selection Avocet 'R' carries both genes, selection Avocet 'S' carries Yr73 only. P. triticina pathotype (pt.) 104-1,2,3,(6),(7),11 +Lr37 ("104-VPM"), first detected in Australia in 2002, most likely arose via mutation from pt. 104-1,2,3,(6),(7),11 ("104"), with added virulence for Lr37. Interestingly, while both pathotypes are avirulent on Lr13, 104-VPM shows a much lower Infection Type (IT, ";1") than pt. 104 ("X++3") on several genotypes carrying Lr13 (e.g.Avocet 'R', Avocet 'S'). Other Lr13 genotypes (e.g. cv. Hereward) respond similarly to both pts ("X++3"). Genetic analyses of 4 doubled haploid (DH) populations based on intercrosses between Avocet 'R' and genotypes lacking Lr13 segregated in a 1:7 ratio to pt. 104-VPM (";1" : all other ITs). Two populations fixed for Lr13 (viz. Hereward/ Avocet 'R' and Estica/Avocet 'R') segregated 1:3 to pt. 104-VPM (";1" : all other ITs). This segregation pattern fitted a model where two complementary genes interact with Lr13 to generate the low (IT ";1") IT. Mapping of a Teal/Avocet 'R' DH population using 92 lines and 9,035 DArT-Seq markers identified three QTLs: chromosome 2BS (Lr13); chromosome 3DL (co-located with Yr73); chromosome 1DS. These results suggest that Yr73 acts in a complementary manner with a gene on chromosome 1DS to confer leaf rust resistance (IT "X"), and that these complementary genes are additive with Lr13. It appears that Yr73 is a modifier of two independent genes in wheat, one conferring resistance to stripe rust (Yr74 on chromosome 5BL), and one conferring resistance to leaf rust (LrAv on chromosome 1DS).
University of Queensland, St. Lucia
Adnan Riaz, Jonathan Powell, Timothy Fitzgerald, Kemal Kazan, Neena Mitter, Evans Lagudah, Lee T. Hickey
The Lr34/Yr18/Sr57/Pm38/Ltn1 multi-resistance locus has been deployed and remained effective in wheat cultivars for more than 100 years. The durability and pleiotropic nature makes Lr34 a unique and highly valuable resource for rust resistance breeding. Despite its functional annotation as an ABC transporter, the mode of action is unknown. Considering this, we aimed to decipher molecular factors and signaling components essential for Lr34 function using RNA-seq of Chara resistant (Lr34) and Chara mutant (heavy ion irradiation, HII) susceptible wheat lines. Screening of Chara and Chara HII lines with Lr34-specific markers confirmed the integrity of Lr34 in both lines; however, phenotyping confirmed rust and powdery mildew susceptibility in the Chara HII lines. Plants were grown under controlled conditions and infected with Puccinia triticina pathotype 76-1,3,5,7,9,10,12,13+Lr37 at the flag leaf stage. Flag leaves were sampled at 0, 24, 48, 72, 96 and 168 hours post inoculation (hpi) from mock and infected plants. Based on real-time PCR analysis of basal defense genes and the Lr34 gene, we selected 72 hpi for RNA-seq with four biological replicates per condition. The samples were sequenced on an Illumina Hiseq 4000 at the Beijing Genomics Institute, China. A total of 9.0 Gb of sequence (2.25 Gb/library) from 16 libraries for four conditions was obtained. Differential expression analysis was performed using the Tuxedo analysis pipeline with standard parameters. Analysis revealed deletion of DNA fragments with collinear gene order on chromosomes 1A, 2D, 5A, 5B, 5D and 7D of Chara HII mutants. To determine the significance of the deletions we performed bulk segregant analyses on segregating F2 populations of Chara ? Chara HII crosses. Analyses revealed key genomic regions associated with Lr34-functional resistance and we are in the process of validating candidate genes using qPCR.
Institute of Plant Biology and Biotechnology
Makpal Atishova, Aygul Madenova, Kanat Galymbek, Jenis Keyshilov, Hafiz Muminjanov, Alexey Morgounov
Wheat rust diseases are a major cause of yield losses of this crop. Yellow (Puccinia striiformis f. sp. tritici) rust is of the most widespread and dangerous disease of wheat and is the major factor that adversely affects wheat yield and quality. The use of genetic host resistance is the most effective, economical and environmentally safe method of controlling stripe rust that allows elimination of fungicides and minimize crop losses from this disease. Due to the threat of the development of epiphytoties of rust disease it is necessary to identify new donors of resistance to yellow rust and to develop resistant wheat breeding material. In the present study, attention was drawn to the effective yellow rust resistance genes Yr5, Yr10 and Yr15, which were identified in the process of molecular screening of wheat germplasm. Genetic analysis using S23M41 molecular marker linked to Yr5 revealed the presence of this gene in 17 out of 136 promising lines. Thirteen genotypes screened with Xbarc8 generated the DNA fragment associated with Yr15. Three advanced lines with Yr10 were identified using the SCAR marker. Three lines carrying two Yr genes (Yr5 and Yr15) were detected. Combination of Yr5 and Yr10 were found in 15 wheat lines. We identified a number of wheat genotypes highly resistant to stripe rust, which could be further evaluated to release new resistant varieties or to be used in the breeding program.
National Research Council of Canada (NRC)-Saskatoon
Kerry Boyle, Tammy Francis, Peng Gao, Brittany Polley, Christine Sidebottom, Brent McCallum, Harpinder Randhawa, Tom Fetch, Randy Kutcher, Sylvie Cloutier, Pierre R. Fobert
Most rust resistant genes in wheat are race-specific (R), with relatively few genes conferring resistance only at the adult stage that have been described as slow rusting genes (APR). Pyramiding multiple R, APR or APR+R genes has been used successfully over many years to achieve durable rust resistance. To further enhance this strategy, a genetic genomics approach was exploited to identify genes with different resistant mechanisms and the most effective gene pyramids.
Several new combinations of rust genes were created and tested in the Thatcher background, revealing synergistic ("booster") effects involving Lr21 with Lr16. With QTL mapping approach, we found that genes combined from 7D, 1B and 7B conferred an almost immune response to leaf rust, while genes from 7D, 1B and 3B provided an almost immune response to stripe rust. With a genomics approach, a large scale transcriptome analysis was conducted on key rust resistant genes including six R genes, three APR genes and one gene pyramid with Lr34+Lr16 over a time series during the infection process of both seedlings and adult plants. Detailed transcriptome analysis of gene expression associated with different major and minor leaf rust genes, alone or in combination, identified common and unique aspects of defense responses. For example, Lr9 is different from the other three leaf rust genes tested, with resistance triggered at a very early stage, consistent with pre-haustorial resistance. R genes Lr21 and Lr16 were also significantly different compared to other R and APR genes. With gene co-expression network analysis, a shared unique gene module mediated by Lr34 and Lr67 was also identified. This large transcriptome dataset also allowed the development of a rust-wheat interactome atlas for rust functional genomics research in wheat.
James Winans, Julian Garcia, Kellie Damann, Gary Bergstrom
In addition to causing Fusarium head blight of wheat and other cereals, Fusarium graminearum is associated with dozens of wild or weedy grass species. Their role in the disease cycle and evolution of the pathogen has not been established despite their widespread distribution. A three-year survey of wild grasses in New York (USA) found that inflorescences and overwintered stems were frequently colonized by F. graminearum. Through a series of controlled laboratory experiments, wheat and five common grass species were compared for their potential to support inoculum production. Artificially infested stem tissue from several grasses both retained F. graminearum at higher rates through a single winter and supported greater ascospore production per dry gram than wheat. Susceptibility of these species to root and crown rot was measured with a modified seed germination assay and a diverse panel of F. graminearum isolates. Differences were seen between host species, and some grasses were resistant to infection. Our results indicate that wild grass species may support significant F. graminearum inoculum production while differing in their suitability for root and crown colonization. Studying interactions between F. graminearum and alternative host plants can improve our understanding of evolution in a broad host range pathogen and our ability to predict the risk of crop epidemics. We are currently evaluating isolates collected from wild grasses for mycotoxin production and aggressiveness on wheat.
Genetic Resources Institute, Azerbaijan
N. Aminov, A. Jahangirov, H. Hamidov, Aigul Abugalieva, Vladmir Shamanin, Alexey Morgunov
Hexaploid synthetics have become widely used in bread wheat improvement in recent years, enabling the introduction of specific traits as well as enhancing genetic diversity and development of valuable germplasm. This study demonstrated the difference between two groups of primary synthetics in terms of development rate, plant height, rust reactions, and productivity components. During 2015 and 2016, three groups of synthetics were studied in Azerbaijan (3 sites): Baku (0 masl) under irrigated conditions, Gobustan (850 masl) under dry rainfed conditions and Ujar (20 masl) under irrigated conditions with high salinity. Germplasm was also evaluated for diseases and agronomic traits in Omsk (Russia) in 2016.
All primary synthetics were resistant to leaf rust, several to stem rust, and few to stripe rust. Stripe rust occurred in all years at all sites, proving its importance as major wheat pathogen. Its severity reached intermediate levels in Baku in 2016 (33.7%) and in Gobustan in 2015 (26.8%), and epidemic level in Gobustan in 2016 (72.7%). Gobustan also experienced high levels of stem rust in 2016. These two diseases substantially reduced grain productivity in Gobustan in 2016, especially 1000 kernel weight (30.2 g) and grain weight per spike (1.17 g). . Superior genotypes from all three groups were identified that combine high expression of spike productivity traits and stress tolerance index. Five superior synthetics were selected from each of the three groups, based on grain weight per spike. Only four of these demonstrated resistance to stripe rust (entries 13, 15, 31, and 32). Japanese synthetics (group 3) were susceptible to stripe rust but all demonstrated resistance to stem rust. Synthetics from groups 1 and 3 were all resistant to leaf rust when tested under severe disease pressure in Omsk in 2016.