Comparative transcriptomics of stem rust resistance in wheat NILs mediated by Sr24 rust resistance gene.

Stem rust of wheat is a deleterious fungal disease across the globe causing severe yield losses. Although, many stem rust resistance genes (Sr) are being used in wheat breeding programs, new emerging stem rust pathotypes are a challenge to important Sr genes. In recent years, multiple studies on leaf and yellow rust molecular mechanism have been done, however, for stem rust such studies are lacking. Current study investigated stem rust induced response in the susceptible wheat genotype C306 and its Near Isogenic Line (NIL) for Sr24 gene, HW2004, using microarray analysis to understand the transcriptomic differences at different stages of infection. Results showed that HW2004 has higher basal levels of several important genes involved in pathogen detection, defence, and display early activation of multiple defence mechanisms. Further Gene Ontology (GO) and pathway analysis identified important genes responsible for pathogen detection, downstream signalling cascades and transcription factors (TFs) involved in activation and mediation of defence responses. Results suggest that generation of Reactive Oxygen Species (ROS), cytoskeletal rearrangement, activation of multiple hydrolases, and lipid metabolism mediated biosynthesis of certain secondary metabolites are collectively involved in Sr24-mediated defence in HW2004, in response to stem rust infection. Novel and unannotated, but highly responsive genes were also identified, which may also contribute towards resistance phenotype. Furthermore, certain DEGs also mapped close to the Sr24-linked marker on Thinopyrum elongatum translocated fragment on wheat 3E chromosome, which advocate further investigations for better insights of the Sr24-mediated stem rust resistance.

Candidate effectors for leaf rust resistance gene Lr28 identified through transcriptome and in-silico analysis.

Puccinia spp. causing rust diseases in wheat and other cereals secrete several specialized effector proteins into host cells. Characterization of these proteins and their interaction with host's R proteins could greatly help to limit crop losses due to diseases. Prediction of effector proteins by combining the transcriptome analysis and multiple in-silico approaches is gaining importance in revealing the pathogenic mechanism. The present study involved identification of 13 Puccinia triticina (Pt) coding sequences (CDSs), through transcriptome analysis, that were differentially expressed during wheat-leaf rust interaction; and prediction of their effector like features using different in-silico tools. NCBI-BLAST and pathogen-host interaction BLAST (PHI-BLAST) tools were used to annotate and classify these sequences based on their most closely matched counterpart in both the databases. Homology between CDSs and the annotated sequences in the NCBI database ranged from 79 to 94% and with putative effectors of other plant pathogens in PHI-BLAST from 24.46 to 54.35%. Nine of the 13 CDSs had effector-like features according to EffectorP 3.0 (≥0.546 probability of these sequences to be effector). The qRT-PCR expression analysis revealed that the relative expression of all CDSs in compatible interaction (HD2329) was maximum at 11 days post inoculation (dpi) and that in incompatible interactions (HD2329 + Lr28) was maximum at 3 dpi in seven and 9 dpi in five CDSs. These results suggest that six CDSs (>0.8 effector probability as per EffectorP 3.0) could be considered as putative Pt effectors. The molecular docking and MD simulation analysis of these six CDSs suggested that candidate Lr28 protein binds more strongly to candidate effector c14094_g1_i1 to form more stable complex than the remaining five. Further functional characterization of these six candidate effectors should prove useful for a better understanding of wheat-leaf rust interaction. In turn, this should facilitate effector-based leaf rust resistance breeding in wheat.

Genome-wide identification and characterization of Puccinia striiformis-responsive lncRNAs in Triticum aestivum.

Wheat stripe rust (yellow rust) caused by Puccinia striiformis f. sp. tritici (Pst) is a serious biotic stress factor limiting wheat production worldwide. Emerging evidence demonstrates that long non-coding RNAs (lncRNAs) participate in various developmental processes in plants via post-transcription regulation. In this study, RNA sequencing (RNA-seq) was performed on a pair of near-isogenic lines-rust resistance line FLW29 and rust susceptible line PBW343-which differed only in the rust susceptibility trait. A total of 6,807 lncRNA transcripts were identified using bioinformatics analyses, among which 10 lncRNAs were found to be differentially expressed between resistance and susceptible lines. In order to find the target genes of the identified lncRNAs, their interactions with wheat microRNA (miRNAs) were predicted. A total of 199 lncRNAs showed interactions with 65 miRNAs, which further target 757 distinct mRNA transcripts. Moreover, detailed functional annotations of the target genes were used to identify the candidate genes, pathways, domains, families, and transcription factors that may be related to stripe rust resistance response in wheat plants. The NAC domain protein, disease resistance proteins RPP13 and RPM1, At1g58400, monodehydroascorbate reductase, NBS-LRR-like protein, rust resistance kinase Lr10-like, LRR receptor, serine/threonine-protein kinase, and cysteine proteinase are among the identified targets that are crucial for wheat stripe rust resistance. Semiquantitative PCR analysis of some of the differentially expressed lncRNAs revealed variations in expression profiles of two lncRNAs between the Pst-resistant and Pst-susceptible genotypes at least under one condition. Additionally, simple sequence repeats (SSRs) were also identified from wheat lncRNA sequences, which may be very useful for conducting targeted gene mapping studies of stripe rust resistance in wheat. These findings improved our understanding of the molecular mechanism responsible for the stripe rust disease that can be further utilized to develop wheat varieties with durable resistance to this disease.

Indian Wheat Genomics Initiative for Harnessing the Potential of Wheat Germplasm Resources for Breeding Disease-Resistant, Nutrient-Dense, and Climate-Resilient Cultivars.

Wheat is one of the major staple cereal food crops in India. However, most of the wheat-growing areas experience several biotic and abiotic stresses, resulting in poor quality grains and reduced yield. To ensure food security for the growing population in India, there is a compelling need to explore the untapped genetic diversity available in gene banks for the development of stress-resistant/tolerant cultivars. The improvement of any crop lies in exploring and harnessing the genetic diversity available in its genetic resources in the form of cultivated varieties, landraces, wild relatives, and related genera. A huge collection of wheat genetic resources is conserved in various gene banks across the globe. Molecular and phenotypic characterization followed by documentation of conserved genetic resources is a prerequisite for germplasm utilization in crop improvement. The National Genebank of India has an extensive and diverse collection of wheat germplasm, comprising Indian wheat landraces, primitive cultivars, breeding lines, and collection from other countries. The conserved germplasm can contribute immensely to the development of wheat cultivars with high levels of biotic and abiotic stress tolerance. Breeding wheat varieties that can give high yields under different stress environments has not made much headway due to high genotypes and environmental interaction, non-availability of truly resistant/tolerant germplasm, and non-availability of reliable markers linked with the QTL having a significant impact on resistance/tolerance. The development of new breeding technologies like genomic selection (GS), which takes into account the G × E interaction, will facilitate crop improvement through enhanced climate resilience, by combining biotic and abiotic stress resistance/tolerance and maximizing yield potential. In this review article, we have summarized different constraints being faced by Indian wheat-breeding programs, challenges in addressing biotic and abiotic stresses, and improving quality and nutrition. Efforts have been made to highlight the wealth of Indian wheat genetic resources available in our National Genebank and their evaluation for the identification of trait-specific germplasm. Promising genotypes to develop varieties of important targeted traits and the development of different genomics resources have also been highlighted.

Genetic Diversity and Population Structure Reveal Cryptic Genetic Variation and Long Distance Migration of Puccinia graminis f. sp. tritici in the Indian Subcontinent.

Stem rust caused by Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of wheat worldwide since time immemorial. Several wheat stem rust outbreaks have been reported worldwide including India. Approximately 7 mha wheat area in central and peninsular India is highly vulnerable to stem rust epidemics. In this study, a repository of 29 single genotype uredospore pathotypes, representing five geographical regions, was characterized by investigating their virulence phenotype and simple sequence repeat (SSR) genotypes using 37 reproducible polymorphic SSR markers, 32 of which had ≥ 0.50 polymorphic information content (PIC) value. Virulence phenotypes were used to evaluate the virulence frequency (VF) and construct a hypothetical evolutionary hierarchy of these pathotypes. We projected seven lineages to explain the evolutionary pattern of the Pgt population. The VF of these pathotypes ranged between 0% and 100%. The virulence-based neighbor-joining (NJ) cluster analysis grouped Pgt pathotypes into five virulence groups. Likewise, five molecular groups were categorized using molecular genotypes. The molecular grouping was supported by principal coordinate analysis (PCoA), which revealed 25% of the cumulative variance contributed by the first two axes. Analysis of molecular variance (AMOVA) revealed 8 and 92% of the variation among and within the populations, respectively. The Mantel test confirmed a positive but weak correlation (R 2 = 0.15) between virulence phenotypes and SSR genotypes. The highest and lowest values of different genetic diversity parameters (Na, Ne, I, He, uHe, and %P) revealed maximum and minimum variability in the Pgt population from Maharashtra and Uttar Pradesh, respectively. The population structure analysis clustered 29 Pgt pathotypes into two subpopulations and an admixture. Our results demonstrated that there was significant genetic diversity among Pgt pathotypes resulting from their long-distance dispersal ability complemented by gene flow. These findings provide insights into the virulence patterns, genetic variations, and possible evolution of Pgt pathotypes, which would support strategic stem rust resistance breeding.

Wheat spike blast: genetic interventions for effective management.

The fundamental concepts of the genetics, race classification and epidemiology of the Wheat spike blast causing fungus Magnaporthe oryzae pathotype Triticum (MoT) are still evolving despite of its discovery in 1985 in Brazil for the first time. The fungus seems to defy the research progress that is being made globally by continuously evolving into pathotypes which have already overcome the much celebrated 2NS resistance in wheat lines as well as few of the initially effective fungicides. The compartmentalized i.e. two speed genome of the MoT, conferring the fungus an evolutionary advantage, has emerged as a challenge for the wheat spike blast researchers complicating its already difficult management. The airborne fungus with a range of alternative hosts is finding new geographical niches situated on different continents and is a matter of great apprehension among the nations whose food security is primarily dependent on wheat. The wheat blast outbreak in Bangladesh during 2016 was attributed to an isolate from Latin America escaping through a seed import consignment while the latest Zambian outbreak is still to be studied in detail regarding its origin and entry. The challenges in dealing wheat spike blast are not only on the level of genetics and epidemiology alone but also on the levels of policy making regarding international seed movement and research collaborations. The present review deals with these issues mainly concerning the effective management and controlling the international spread of this deadly disease of wheat, with a particular reference to India. We describe the origin, taxonomy, epidemiology and symptomology of MoT and briefly highlight its impact and management practices from different countries. We also discuss the advances in genomics and genome editing technologies that can be used to develop elite wheat genotypes resistant against different stains of wheat spike blast.

Elucidating the Population Structure and Genetic Diversity of Indian Puccinia striiformis f. sp. tritici Pathotypes Based on Microsatellite Markers.

In India, systematic wheat yellow rust survey and pathotype (race) analysis work began in 1930. However, information on population structure and genetic diversity of yellow rust pathogen has not been available. To address this, we conducted studies on population structure and genetic diversity of Puccinia striiformis f. sp. tritici (Pst) pathotypes using 38 simple sequence repeat primer-pairs. Bayesian assignment and discriminant analysis of principal components indicated the presence of two distinct Pst subpopulations (Pop1 and Pop2) along with 37.9% admixed pathotypes. The unweighted pair-group method with arithmetic mean also categorized these pathotypes into two major clusters. Principal coordinates analysis explained 20.06 and 12.50% variance in horizontal and vertical coordinates, respectively. Index of association (IA) and the standardized index of association ([Formula: see text]) values showed that Pst subpopulations reproduced asexually (clonally). In total, 102 alleles were detected, with the expected heterozygosity (Hexp) per locus ranging from 0.13 to 0.73, with a mean of 0.47. The average polymorphic information content value of 0.40 indicated high genetic diversity among pathotypes. Analysis of molecular variance revealed 12% of the total variance between subpopulations, 11% among the pathotypes of each subpopulation, and 77% within pathotypes. A significant moderate level of genetic differentiation (FST = 0.122, P < 0.001) and gene flow (Nm = 1.80) were observed between subpopulations. The Pst virulence phenotypes showed a weak positive correlation (R2 = 0.027, P < 0.02) with molecular genotypes.

Application of Genomics Tools in Wheat Breeding to Attain Durable Rust Resistance.

Wheat is an important source of dietary protein and calories for the majority of the world's population. It is one of the largest grown cereal in the world occupying over 215 M ha. Wheat production globally is challenged by biotic stresses such as pests and diseases. Of the 50 diseases of wheat that are of economic importance, the three rust diseases are the most ubiquitous causing significant yield losses in the majority of wheat production environments. Under severe epidemics they can lead to food insecurity threats amid the continuous evolution of new races of the pathogens, shifts in population dynamics and their virulence patterns, thereby rendering several effective resistance genes deployed in wheat breeding programs vulnerable. This emphasizes the need to identify, characterize, and deploy effective rust-resistant genes from diverse sources into pre-breeding lines and future wheat varieties. The use of genetic resistance has been marked as eco-friendly and to curb the further evolution of rust pathogens. Deployment of multiple rust resistance genes including major and minor genes in wheat lines could enhance the durability of resistance thereby reducing pathogen evolution. Advances in next-generation sequencing (NGS) platforms and associated bioinformatics tools have revolutionized wheat genomics. The sequence alignment of the wheat genome is the most important landmark which will enable genomics to identify marker-trait associations, candidate genes and enhanced breeding values in genomic selection (GS) studies. High throughput genotyping platforms have demonstrated their role in the estimation of genetic diversity, construction of the high-density genetic maps, dissecting polygenic traits, and better understanding their interactions through GWAS (genome-wide association studies) and QTL mapping, and isolation of R genes. Application of breeder's friendly KASP assays in the wheat breeding program has expedited the identification and pyramiding of rust resistance alleles/genes in elite lines. The present review covers the evolutionary trends of the rust pathogen and contemporary wheat varieties, and how these research strategies galvanized to control the wheat killer genus Puccinia. It will also highlight the outcome and research impact of cost-effective NGS technologies and cloning of rust resistance genes amid the public availability of common and tetraploid wheat reference genomes.

Genome-Wide Association Studies in Diverse Spring Wheat Panel for Stripe, Stem, and Leaf Rust Resistance.

Among several important wheat foliar diseases, Stripe rust (YR), Leaf rust (LR), and Stem rust (SR) have always been an issue of concern to the farmers and wheat breeders. Evolution of virulent pathotypes of these rusts has posed frequent threats to an epidemic. Pyramiding rust-resistant genes are the most economical and environment-friendly approach in postponing this inevitable threat. To achieve durable long term resistance against the three rusts, an attempt in this study was made searching for novel sources of resistant alleles in a panel of 483 spring wheat genotypes. This is a unique and comprehensive study where evaluation of a diverse panel comprising wheat germplasm from various categories and adapted to different wheat agro-climatic zones was challenged with 18 pathotypes of the three rusts with simultaneous screening in field conditions. The panel was genotyped using 35K SNP array and evaluated for each rust at two locations for two consecutive crop seasons. High heritability estimates of disease response were observed between environments for each rust type. A significant effect of population structure in the panel was visible in the disease response. Using a compressed mixed linear model approach, 25 genomic regions were found associated with resistance for at least two rusts. Out of these, seven were associated with all the three rusts on chromosome groups 1 and 6 along with 2B. For resistance against YR, LR, and SR, there were 16, 18, and 27 QTL (quantitative trait loci) identified respectively, associated at least in two out of four environments. Several of these regions got annotated with resistance associated genes viz. NB-LRR, E3-ubiquitin protein ligase, ABC transporter protein, etc. Alien introgressed (on 1B and 3D) and pleiotropic (on 7D) resistance genes were captured in seedling and adult plant disease responses, respectively. The present study demonstrates the use of genome-wide association for identification of a large number of favorable alleles for leaf, stripe, and stem rust resistance for broadening the genetic base. Quick conversion of these QTL into user-friendly markers will accelerate the deployment of these resistance loci in wheat breeding programs.