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.
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.
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.
Department of Agriculture, Sunsari, Nepal
Wheat (Triticum aestivum L.) is one of the major cereal crops vital for global food supply. Most of the wheat crop in developing world including that of Nepal is either grown with limited irrigation or under rainfed conditions and thus face moisture stress at one or more growth stages limiting grain yield. An experiment was carried out at the Institute of Agriculture and Animal Science, Rampur to evaluate the genetic variability of selected drought adaptive traits in Nepalese wheat germplasm. The wheat genotypes evaluated comprised of Nepalese landraces and commercial cultivars, CIMMYT (International Center for Maize and Wheat Improvement) derived advanced introduction lines and three checks with differential drought adaptability. The wheat genotypes were grown in pots (single plant) arranged in a replicated split plot design in greenhouse under two contrasting moisture regimes, optimum and moisture stressed. The genotypes were evaluated for water use, water use efficiency, relative leaf water content and biomass production. The ANOVA (Analysis of Variance) revealed significant variation between environments and among the wheat genotypes for most of the traits studied. A wide range of variability was observed for water use, water use efficiency, biomass yield and relative leaf water content in moisture stressed and non-stressed environments. Nepalese cultivar Gautam showed a number of favorable drought adaptive traits, whereas, Bhrikuti was average in this respect. Based on the scores of drought adaptive traits recently released Cultivar (cv). Vijay was characterized as drought sensitive. A number of landraces and advanced breeding lines showed high level of water use efficiency and other positive traits for drought adaptation.
Department of Plant Molecular Biology, University of Delhi South Campus
L-myo-inositol phosphate synthase (MIPS; EC 18.104.22.168) have been involved in abiotic stress tolerance and its disruption leads to spontaneous cell death and enhanced tolerance to pathogen. However, its molecular mechanism underlying role of MIPS in growth, immunity and abiotic stress tolerance remains unknown. To delve deeper into the conserved molecular mechanism of MIPS action during growth and stress condition, we characterized the overexpression transgenic of TaMIPS and mutant lines of AtMIPS1. Subsequent, transcriptome analysis revealed the activation of ET/JA dependent immune response in transgenic and SA defense response in mutant. Pull-down analysis revealed the interaction of TaMIPS2 with ethylene synthesis (ACO) and signaling protein (CTR1) component. Due to the established role of ethylene during the skotomorphogenesis, we investigated the effect of myo-inositol phosphate synthase role in ethylene response during hook formation. Our results thus suggest the requirement of MIPS for ethylene response and regulating the growth and immunity.
Lake Chad Research Institute, Maiduguri, Borno State-Nigeria
Dattijo Aminu, Zakari Goji Silas Turaki, Fatima Henkrar, Udupa Sripada
The research was conducted at ICARDA, Rabat. Twenty-four accessions were obtained from LCRI for marker analysis. Wizard Genomic DNA Purification Kit was used for DNA extraction. DNA was extracted by CTAB method and quantified using 1.0 % (w/v) agarose gels. Total of 12 loci, 5 functional and 7 linked random DNA markers to the traits of interest were used. PowerMarker and DARwin software were used to calculate the No. of alleles and values of genetic diversity, PIC, genetic distance, and NJ dendrogram. The total No. of detected alleles was 39; and mean No. of alleles was 3.25. No. of alleles range from 1 (Dreb-B1) to 9 (Xgwm577). Genetic diversity index ranged from 0.0000 in Dreb-B1 to 0.8471 in Xgwm577. The PIC value was also varied from 0.0000 (Dreb-B1) to 0.8296 (Xgwm577). The frequency of biotic resistance linked random DNA marker allele at Xgwm144 and Xwmc44, associated with yellow and leaf rust gene was 25% each. Marker alleles Xgwm577 and Xgwm533 linked to Stb2 and Stb8 at 150 and 120bp have frequencies of 21 and 4%. The frequency of abiotic resistance showed 50% of accessions had 1R segment (1BL.1RS translocation) and 58% of accessions showed presence of 120bp allele of Xwmc89, associated with QTL for drought tolerant. Functional marker alleles of Dreb-B1 associated with drought tolerant genes showed alleles frequency in all accessions. Linked marker allele Xgwm111 linked to heat tolerant gene showed 17% allele frequency at 220bp. Rht1 and Rht2, the allele frequencies were 92 and 4%. 92% of the cultivars had photoperiod insensitive allele at Ppd-D1 locus. VrnA1a and VrnA1c primer pair amplified at 965, 876, and 484bp, allele frequency of 13 and 87%. Cluster analysis had grouped the accessions into 5 at a genetic distance level 0.15.
John Innes Centre
Burkhard,Steuernagel, Caixia, Lan, Miroslava, Karafi?tov?, Ksenia, Krasileva, Jaroslav, Dole?el, Evans, Lagudah, Ravi, Singh, Brande, Wulff, , , , , , , , , , , , , ,
Adult Plant Resistance (APR) genes are broad-spectrum, partial-resistance genes that have the potential to contribute to sustainable control of wheat rust diseases. However, their isolation and characterization are complicated by the lack of precise molecular markers required for their identification, and therefore their use in plant breeding programs has been limited. Recent developments including the falling cost of sequencing and the increasing use of sequence capture methods to reduce genome complexity have enabled previously intractable methods such as mutational genomics to clone genes in wheat. Despite their increasing ease of use, many of these approaches require prior knowledge of the gene space and, in some cases, the gene family of the target gene to be cloned. As the APRs cloned so far do not belong to any common gene family, it is not possible to use general features of these identified APRs to conduct biased searches for novel APRs. This project aims to use an unbiased gene isolation technique called MutChromSeq, which combines chromosome flow-sorting and mutational genomics, and is independent of fine mapping, to rapidly clone the recently discovered APR gene Lr68 (Leaf Rust 68). Cloning APRs allows breeders to trace genes cheaply and quickly using gene-specific markers, enabling them to build effective and durable resistance gene pyramids. It also allows us to elucidate any common mechanism of action they have, helping researchers and breeders understand better the basis of their durable resistance. At the same time, the generation time of wheat has become one of the major limiting factors for the response time of breeders to rust epidemics. Thus, this project also aims to combine marker-assisted selection with accelerated generation advancement ('speed breeding') for rapid germplasm structuring and field performance evaluation.
Wheat Research Institute, Faisalabad, Pakistan
Javed Ahmad, Abid Mahmood
Wheat is an important cereal crop and staple food in Pakistan. Most of the wheat is cultivated late after cotton, rice and sugarcane. Introduction of long duration Bt cotton varieties further pushed its sowing to late December or even early January. Late sowing of wheat crop results in yield loss in the Punjab province. A study has been conducted in experimental fields of Wheat Research Institute, AARI, Faisalabad, Pakistan to find out the possible reasons of low grain yields in late sown crop. Twelve experimental wheat lines were planted on seven sowing dates starting from 1st November to 30th December with ten days interval. The experimental design was a factorial combination of seven sowing dates as main plots and twelve varieties/ genotypes as subplots in a split-plot design with three replications. Effect of temperature on several crop growth stages was studied. Mean minimum temperature during the month of December, 2016 and January, 2017 remained below 5?C and mean maximum was more than 30?C during March 2017. Weather conditions experienced by the crop at each developmental stage were compared with the optimum conditions required on that specific stage in each sowing date. It was revealed that in late sown crop, different phonological/growth stages of the crop and yield components and grain yield were affected negatively. It was concluded from the study that the late sown crop suffered from two types of temperature stresses. The late sown crop faced low temperature stress at starting phase which result in delayed germination and low tillering. At caryopsis formation and grain filling the same crop face high temperature stress causing reduced grain formation and shriveled grains due to enforced maturity. Sowing of wheat at proper time i.e., by the end of November was recommended to fetch maximum yields.
John Innes Centre
Christopher,Judge, Francesca, Minter, Nik, Cunniffe, Richard, Morris, Diane, Saunders, , , , , , , , , , , , , , , , , , , ,
Wheat yellow rust is a disease caused by the fungus Puccinia striiformis f. sp tritici (PST) that is a significant threat to wheat production worldwide. Recently, a novel approach called "Field Pathogenomics" was developed that allows acquisition of genotypic data from field samples of PST-infected wheat. This has enabled us to study the re-emergence of this pathogen in the UK and understand the different races that form the current PST population. However, the dynamics of pathogen transmission and dispersal still remain unknown and understanding this is essential for designing effective surveillance. The objective of this project is to develop a spatially-explicit model for the spread of PST that can contribute to better management of the disease and be used as a warning system for wheat yellow rust infection in the UK. The first aim is to study how PST spreads at the field level and determine whether there are differences between PST races in terms of disease dynamics. To this end, a set of markers have been designed that can be used to genotype field-collected isolates and determine which race they belong to. Field trials were also undertaken across the UK using wheat varieties that are known to be susceptible to the disease, with PST-infected wheat samples collected during the 2015-2016 and 2016-2017 seasons. These samples will be genotyped to study the prevalence of different PST races and determine whether PST genotypes identified early in the season are predictive of dominant genotypes found later in the season. Understanding PST dynamics within a field is key to build an epidemiological model that can predict how this disease behaves. This would improve disease management, targeting of chemical sprays and optimize pathogen surveillance.
College of Plant Protection, Northwest A&F University, China
Yuanyuan Zhao, Shuxia Zuo, Dan Zheng, Lili Huang, Zhengshen Kang
Wheat stripe rust, caused by basidiomycete fungus Puccinia striiformis f. sp. tritici (Pst), is a damaging disease worldwide. The recent discovery demonstrated the fungus depends on living wheat and aecial hosts, mainly barberry (Berberis) species, to complete its life cycle. In China, we determined that, under natural conditions, the sexual cycle of Pst occurs based on collections of Pst isolates from the diseased barberry in the past three years. However, no direct evidence to support whether barberry plays a role in spreading inoculums to wheat field to cause stripe rust was detected. In the present study, we recovered 103 Pst samples from natural-infected B. shensiana in the western Shaanxi in spring 2016, and also collected 107 Pst isolates from neighboring wheat fields. Phenotype and genotype of the two Pst populations were tested using a set of Chinese differential hosts for Pst and SSR markers, respectively. The phenotype tests showed that 57 race types produced from the barberry-derived Pst populations, consisting of 58 known races, such as CYR 34, CYR32, G22-14, and Su11-14-3, and 45 new races. Many of the two Pst populations shared the same race types. The genotype tests indicated the barberry-derived Pst population produced a rich genotype, obviously higher than the wheat-derived Pst populations. The seven same genotypes were found on 40 isolates of the former and 26 of the latter. Our results provide evidence to support that sexual cycle of Pst occurs regularly in nature in China and that barberry provides inoculums to neighboring wheat fields, triggering stripe rust infections in the spring. This could be a reason why the Chinese Pst populations represent extreme genetic diversity.