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Leaf rust and stripe rust caused by the fungi Puccinia triticina and P. striiformis f. sp. tritici, respectively, are important diseases of wheat and represent a significant threat in most wheat producing regions worldwide. Growing resistant varieties and the identification and characterization of new sources of resistance are necessary to combat the threat from the evolving pathogen population. Bread wheat (Triticum aestivum L.) line 'Kijil' developed at CIMMYT showed adult plant resistance (APR) to leaf rust (LR) and stripe rust (YR). The genetic basis of the resistance was investigated using 198 recombinant inbred lines (RILs) derived from the cross of susceptible Apav#1 and resistant Kijil. Field phenotyping of parents and RILs were conducted at El Batón, Toluca and Ciudad Obregon, Mexico during 2016 and 2017. Pearson correlation coeffcients (P< 0.0001) were high for disease severities between two years of evaluations: LR (r= 0.90) and YR (r= 0.83). Correlations (r= 0.30-0.76) were also significant between LR and YR in all environments. Genetic analyses indicated that 3 to 5 genes of additive effects governed resistance to both rusts. RILs carrying the pleiotropic APR gene Lr46/Yr29/Sr58 showed 23 and 41% of disease severity for LR and YR respectively, whereas lines lacking it had 55 and 78% severities. RILs positive for Sr2/Yr30 showed 66% YR severity, whereas those negative displayed 78%. In addition, lines carrying the race-specific gene Yr17/Sr38 showed 28% YR severity in contrast to non-carriers that displayed 78% severity. We conclude that Kijil possesses a complex nature of resistance.
Wheat leaf rust (LR) and stripe rust (YR), caused by the air-borne fungi Puccinia triticina (Pt) and Puccinia striiformis f. sp. tritici (Pst), respectively, are considered the primary biotic threats to bread wheat and durum wheat production globally. Growing resistant wheat varieties is a key method of minimizing the extent of yield losses caused by these diseases. Bread wheat lines Francolin #1, Kenya Kongoni, Kundan and Sujata, and CIMMYT-derived durum wheat lines Bairds and Dunkler display an adequate level of adult plant resistance (APR) to both leaf rust and stripe rust in Mexican field environments. Six recombinant inbred line (RIL) populations developed from crosses Avocet/Francolin #1, Avocet/Kenya Kongoni, Avocet/Kundan, Avocet/Sujata, Atred#1/Bairds and Atred#1/Dunkler were phenotyped for leaf rust response at Ciudad Obregon, Mexico, and the bread wheat populations for stripe rust response at Toluca for under artificial inoculations for multiple seasons. The RIL populations and their parents were genotyped with the 50 K diversity arrays technology (DArT) sequence system and simple sequence repeat (SSR) markers. Known pleotropic APR genes Lr46/Yr29 mapped in all of six populations, and explained 7.4-65.1% and 7.7-66.1% severity variations for LR and YR across different bread wheat populations and accounted for 12.4-60.8% of LR severity variations over two durum wheat populations. In addition, several new APR loci identified on chromosomes 1AS, 1DS, 2BS, 2BL, 3D and 7BL in bread wheat and QTL on chromosome 6BL in durum wheat. Among these loci, QTL on chromosomes 1AS, 3D and 7BL might be represent new co-located/pleotropic loci conferring APR to LR and YR. RILs combining these APR loci can be used as sources of complex APR in both bread wheat and durum wheat breeding. In addition, the closely linked single nucleotide polymorphism (SNP) markers have been converted into breeder-friendly kompetitive allele specific PCR (KASP) markers and their diagnostic verified.
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.
Leaf rust is the most widely occurring disease of wheat worldwide. Resistance is the most practical and effective way to control the disease. Most leaf rust resistance genes are race-specific (“R”, qualitative resistance) and a relatively few are adult plant resistance genes, some of which have been described as slow rusting (“APR”, quantitative resistance). Due to limited knowledge, most resistance genes have been deployed in cultivars by an inefficient “blind” approach. This results in the well known “boom and bust cycle” (resistance followed by susceptibility) because the pathogen evolves rapidly and migrates over long distances. Therefore, a breeding-by-design approach is needed to achieve durable resistance. Pyramiding multiple R, APR or APR+R genes has been used successfully over many years to achieve durable resistance to leaf rust in Canada and some other countries. To further enhance this strategy we seek to understand the molecular mechanisms underlying key resistance genes. To identify the molecular mechanisms underlying rust resistance conferred by major R and APR genes, we performed an integrated systemic transcriptome analysis via RNA-seq on the Thatcher NILs with Lr16, Lr22a, Lr21, Lr34, Lr34+Lr16, and Lr67 challenged with Pt race BBBD. Sampling was conducted over a time series during the infection process of both seedlings and adult plants. Through RNA-seq we were able to capture the dynamic interactome of host-pathogen interactions conferred by these R and APR genes. Preliminary results revealed that resistance reactions conferred by R gene Lr21 and APR gene Lr67 were significantly different compared to other R and APR genes. Significantly, the Thatcher NIL line with Lr34+Lr16 showed the combines defense reactions of Lr16 and Lr34.
Resistance is the most economically viable approach to curb the threat of rusts in wheat. The defeat of Sr31 and vulnerability of other resistance genes to the highly virulent Pgt race Ug99 and variants led to renewed efforts to discover and deploy resistance genes/QTLs in new durably resistant varieties. Akuri is a CIMMYT-developed bread wheat line exhibiting adult plant resistance (APR) in field trials in Kenya despite susceptibility to many races at the seedling stage. This study was designed to identify genomic regions contributing APR to stem rust in Akuri. One hundred and forty one RILs and parents of an F2:5 Akuri x PBW343 population were evaluated in Njoro for APR to stem rust over three seasons. Composite interval mapping was implemented on Windows QTL Cartographer to detect QTLs at a LOD threshold of 2.5 utilizing 910 high quality SNPs previously typed on the DArT-GBS platform. Preliminary QTL analyses revealed loci on chromosomes 1B, 2B and 3B consistently contributing to stem rust resistance. These QTL respectively explained ~7, 9, and 8% of the phenotypic variation. A comparison with the recently reported QTL consensus map revealed that the QTL herein discovered are probably novel. Work is underway to saturate the identified genomic regions with microsatellite markers to identify candidate, linked markers for use in marker assisted selection (MAS)
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.
Wheat stripe rust can be managed using adult plant resistance (APR). Knowledge of the growth stage at onset of APR is crucial for integrated management of this disease. Wheat varieties Annuello, Baxter, EGA Kidman, GBA Sapphire, Janz, Kennedy, Livingston, Spitfire, Sunstate, Sunvale, Wyalkatchem, Yitpi, and Avocet NILs carrying Yr18 and Yr18+29, all carrying adult plant stripe rust resistance, were compared for expression of APR under greenhouse conditions. Weekly plantings of all genotypes and susceptible controls Mace and Morocco permitted simultaneous comparisons of infection at the seedling, tillering, jointing, flag leaf, and head emergence stages. Ten replicates of each genotype at the five different growth stages were inoculated with Pst pathotype 134 E16,A+,17+,27+. Percentages of leaf area affected by stripe rust and host response were recorded at 14, 17, 20, 24, 28 and 31 days post inoculation to establish the latent period. With one exception all genotypes including the controls showed latent periods of less than 14 days when inoculated at the seedling, tillering and stem elongation, or jointing growth stages; Spitfire had a latent period of more than 17 days when inoculated at jointing. At the flag leaf and head emergence stages, all genotypes except the controls Mace and Morocco had latent periods of greater than 20 days. The results indicate that the onset of APR in Spitfire was earlier (at jointing) than the other genotypes, and that the resistance remained effective until head emergence and beyond. The information generated shows the importance of information regarding stripe rust development within specific wheat genotypes, and in supporting decisions on chemical intervention to control this disease.
A set of 63 wheat landraces obtained from Institute of Agri-Biotechnology and Genetic Resources, NARC-Islamabad was screened for adult plant resistance (APR) at two inoculated locations during the 2012-13 cropping season, i.e. Wheat Research Institute (WRI), Faisalabad, for leaf rust and Cereal Crops Research Institute (CCRI), Pirsabak, for stripe rust. Responses based on coefficients of infection (CI) were recorded. Five landraces were susceptible (CI >60) and 39 were resistant to both rusts at the adult plant stage; 47 lines were resistant to leaf rust and 51 were resistant to stripe rust, with CI values of 0-20. Four landraces had moderate levels of APR (CI 21-40) to both rusts, 12 to leaf rust and 8 to stripe rust. Only two landraces (accessions 10975 and 11029) showed MR/MS reactions, and one (11438) had an MS/S reaction; the remaining produced S reactions to leaf rust at WRI. At CCRI Pirsabak, the majority of lines responded with MR/MRMS reactions, the remaining lines were susceptible. The 39 landraces identified to have resistance to both diseases may carry new APR gene(s) to one or both rusts, but must be further characterized prior to use as parents in national wheat breeding programs.
The Lr34 resistance gene from Triticum aestivum encodes a putative ABC transporter protein that confers broad spectrum, partial adult plant resistance to all three rusts species and powdery mildew. It has remained a durable source of resistance for over 100 years in which time no increased virulence towards Lr34 has been observed. This gene is located on chromosome 7D and consequently cannot be readily transferred to durum wheat by traditional breeding. A transgenic approach was used to transfer Lr34 to durum wheat cultivar Stewart by Agrobacterium transformation. Homozygous progeny from a number of independent Stewart lines expressing Lr34 under regulatory control of its endogenous promoter showed high levels of rust resistance at the seedling stage. A correlation between seedling resistance and transgene expression levels was observed in these plants. In contrast seedlings from a near isogenic line of hexaploid wheat cultivar Thatcher containing Lr34 showed only a minor difference in rust growth when compared with Thatcher seedlings, typical of this adult plant resistance gene in hexaploid wheat. Little is known about how the Lr34 gene product functions; however, the seedling resistance of these durum transgenics enables functional assays to be readily undertaken without the need for adult plant material. By infecting seedlings we have shown that day length has an effect on Lr34 resistance to leaf rust, with higher levels of resistance observed under long days (16 h light) compared with short days (8 h light). This study demonstrates that Lr34 provides strong and presumably durable seedling resistance to rust in durum plants that can be used to further understand how this gene confers resistance.
Effective control of stripe rust (YR) requires deployment of resistant cultivars. Adult plant resistance (APR) is preferred over all-stage resistance because of its putatively durable nature. Discovery of new sources of resistance is a priority to combat rapidly evolving Pst races. Genebanks contain untapped genetic diversity that likely harbor novel resistance genes. We examined a diverse panel of 300 lines sourced from the Vavilov Institute, including landraces, cultivars and breeding lines from 28 countries. The most virulent Pst pathotype in Australia (134 E16 A+,Yr17+,Yr27) was used for all experiments, where YR reactions were determined on seedlings in a greenhouse and on adult plants in a field disease nursery. A total of 54% of accessions displayed all-stage resistance and 33% displayed moderate to high levels of APR. Accessions were genotyped using the DArTseq genotyping platform and using an association mapping approach we identified genomic regions associated with YR resistance. These were aligned with previously reported QTL and cataloged resistance genes on a consensus map. This enabled identification of novel genomic regions. Accessions carrying high levels of APR were screened using markers linked to well-known APR genes (i.e. Yr18, Yr29 and Yr46). Twenty two accessions carrying potentially novel sources of APR to YR were identified. Our current efforts are aimed at further characterizing and validating these genetic resources against a wide array of pathotypes and environments around Australia.