Pyramiding of genes for grain protein content, grain quality, and rust resistance in eleven Indian bread wheat cultivars: a multi-institutional effort.

Improvement of grain protein content (GPC), loaf volume, and resistance to rusts was achieved in 11 Indian wheat cultivars that are widely grown in four different agro-climatic zones of India. This involved use of marker-assisted backcross breeding (MABB) for introgression and pyramiding of the following genes: (i) the high GPC gene Gpc-B1; (ii) HMW glutenin subunits 5 + 10 at Glu-D1 loci, and (iii) rust resistance genes, Yr36, Yr15, Lr24, and Sr24. GPC increased by 0.8 to 3.3%, although high GPC was generally associated with yield penalty. Further selection among high GPC lines allowed identification of progenies with higher GPC associated with improvement in 1000-grain weight and grain yield in the backgrounds of the following four cultivars: NI5439, UP2338, UP2382, and HUW468. The high GPC progenies (derived from NI5439) were also improved for grain quality using HMW glutenin subunits 5 + 10 at Glu-D1 loci. Similarly, progenies combining high GPC and rust resistance were obtained in the backgrounds of following five cultivars: Lok1, HD2967, PBW550, PBW621, and DBW1. The improved pre-bred lines developed following multi-institutional effort should prove a valuable source for the development of cultivars with improved nutritional quality and rust resistance in the ongoing wheat breeding programmes. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01277-w.

Comparative analysis of nine Tilletia indica genomes for the development of novel microsatellite markers for genetic diversity and population structure analysis.

Karnal bunt (KB; Tilletia indica) is the prime quarantine concern for quality wheat production throughout the world. The most effective approach to dealing with this biotic stress is to breed KB-resistant wheat varieties, which warrants a better understanding of T. indica genome architecture. In India, the North Western Plain Zone is the prime hot spot for KB disease, but only limited efforts have been made to decipher T. indica diversity at the genomic level. Microsatellites offer a powerful and robust typing system for the characterization and genetic diversity assessment of plant pathogens. At present, inadequate information is available with respect to the development of genome-derived markers for revealing genetic variability in T. indica populations. In current research, nine complete genome sequences of T. indica (PSWKBGH_1, PSWKBGH_2, PSWKBGD_1_3, RAKB_UP_1, TiK_1, Tik, DAOMC236408, DAOMC236414, and DAOMC236416) that exist in the public domain were explored to know the dynamic distribution of microsatellites. Comparative genome analysis revealed a high level of relative abundance and relative density of microsatellites in the PSWKBGH_1 genome in contrast to other genomes. No significant correlation between microsatellite distribution for GC content and genome size was established. All the genomes showed the dominance of tri-nucleotide motifs, followed by mono-, di-, tetra-, hexa-, and penta-nucleotide motifs. Out of 50 tested markers, 36 showed successful amplification in T. indica isolates and produced 52 different alleles. A PCR assay along with analysis of the polymorphic information content (PIC) revealed 10 markers as neutral and polymorphic loci (PIC 0.37). The identified polymorphic SSR loci grouped a geographically distinct T. indica population of 50 isolates representing seven Indian regions (Jammu, Himachal Pradesh, Punjab, Haryana, Uttarakhand, Uttar Pradesh, and Rajasthan) into four distinct clusters. The results of the analysis of molecular variance identified 94% genetic variation within the population and 6% among the population. Structure analysis also confirmed the existence of four genetically diverse groups containing admixtures of T. indica isolates across populations. In nutshell, the current study was successful in identifying novel, neutral and polymorphic microsatellite markers that will be valuable in offering deep insight into the evolutionary relationship and dynamics of the T. indica population for devising effective KB management strategies in wheat.

Molecular diagnostic assay for pre-harvest detection of Tilletia indica infection in wheat plants.

The current study describes a new diagnostic method for the rapid and accurate detection of Tilletia indica, the pathogen accountable for causing Karnal bunt (KB) disease in wheat. This method uses quantitative real-time polymerase chain reaction (qPCR) and a primer set derived from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of T. indica to identify the presence of the pathogen. The qPCR assay using this primer set was found highly sensitive, with a limit of detection (LOD) value of 4 pg of T. indica DNA. This level of sensitivity allows for the detection of the pathogen even in cases of different growth stages of wheat, where no visible symptoms of infection on the wheat plants can be seen by naked eyes. The study also validated the qPCR assay on ten different wheat cultivars. Overall, this study presents a valuable molecular tool for rapid, specific and sensitive detection of KB fungus in wheat host. This method has practical applications in disease management, screening of wheat genotypes against KB and can aid in the development of strategies to mitigate the impact of Karnal bunt disease on wheat production.

Molecular diversity, haplotype distribution and genetic variation flow of Bipolaris sorokiniana fungus causing spot blotch disease in different wheat-growing zones.

Bipolaris sorokiniana (BS) is an economically important fungal pathogen causing spot blotch of wheat (Trtiticum aestivum) and found in all wheat-growing zones of India. Very scanty and fragmentary information is available on its genetic diversity. The current research is the first detailed report on the geographic distribution and evolution of BS population in five geographically distinct wheat-growing zones (North Western Plain Zone (NWPZ), North Eastern Plain zone (NEPZ), North Hill Zone (NHZ), Southern Hill Zone (SHZ) and Peninsular Zone (PZ)) of India, studied by performing nucleotide sequence comparison of internal transcribed spacer region of 528 isolates. A moderate to low levels of haplotypic diversity was noticed in different wheat-growing zones. Phylogenetic analysis suggests that B. sorokiniana exist in two distinct lineages as all isolates under study were grouped in two different clades and found analogous to the findings of haplotypic and TCS network analysis. The genetic parameters revealed the existence of 40 haplotypes with three major haplotypes (H-1, H-2 and H-3) which showed star-like structure network surrounded by several single haplotypes, revealing high frequency of the mutations (Eta = 2 - 158) in total analyzed population. H-1 was observed as a predominant haplotype and prevalent in all the five zones. Moderate level of genetic differentiation was found between NHZ and other zones like NWPZ (Fst = 0.332) and SHZ (Fst = 0.382) and PZ (Fst = 0.299), whereas it was low between NEPZ and PZ (Fst = 0.034). Higher transfer rate of genetic variation was noticed between NEPZ and PZ (Nm = 7.06), while it was found minimum between NHZ and SHZ (Nm = 0.40). Moreover, negative score of neutrality statistics (Tajima's D and Fu's FS test) for NWPZ population suggested recent population expansion. However, positive score for both the neutrality tests observed in NEPZ indicated the dominance of balancing selection in structuring their population. Recombination events were observed in the NWPZ and NHZ population, while it was absent in SHZ, NEPZ and PZ population. Thus, the lack of any specific genetic population structure in all the zones indicates for the expansion history only from one common source population, i.e. NWPZ, a mega zone of wheat production in India. Overall, it seems that the predominance of individual haplotypes with a moderate level of genetic variation and human-mediated movement of contaminated seed and dispersal of inoculum, mutations and recombination as prime evolutionary processes play essential role in defining the genetic structure of BS population.

Molecular Diagnostic Assay for Rapid Detection of Flag Smut Fungus (Urocystis agropyri) in Wheat Plants and Field Soil.

Flag smut incited by Urocystis agropyri has the potential to cause substantial reduction in yield and quality of wheat production. An early and precise diagnosis is a key component in the successful management of flag smut of wheat. Therefore, a simple molecular assay for the rapid detection of U. agropyri was developed for the first time. To detect U. agropyri, species specific primers were developed by comparing the partial sequences of internal transcribed spacer (ITS) DNA region of U. agropyri with related and unrelated phytopathogenic fungi. The clear amplicons of 503 and 548 bp were obtained with the two sets of designed primers (UA-17F/UA-519R and UA-15F/UA-562R) from the genomic DNA of 50 geographic distinct isolates of U. agropyri. However, no amplicon was obtained from the DNA of other 21 related and unrelated phytopathogenic fungi which showed the specificity of the primers for the U. agropyri. PCR reaction was also set up to confirm the presence of U. agropyri spores in six different wheat varieties along with eleven distinct regional soil samples as template DNA. The presence of U. agropyri in all the soil samples collected from an infected field and plant tissue of diseased plants collected at two different stages (20 and 40 days post sowing) and the absence in the soils and plants of healthy plots indicated 100% reliability for detection of U. agropyri. This simple and rapid test can be employed for the detection of U. agropyri from enormous wheat and soil samples in very short time with less man power. Thus, the reported molecular assay is very specific for U. agropyri and requires less time and man power over conventional diagnosis which is often confused by coinciding morphological features of closely related fungal pathogens, and therefore, it can be used for quarantine surveillance of flag smut.

Identification of Novel Microsatellite Markers to Assess the Population Structure and Genetic Differentiation of Ustilago hordei Causing Covered Smut of Barley.

Barley covered smut (CS) pathogen Ustilago hordei genome was mined for microsatellite distribution and their application in defining population structure and genetic variation. To dissect the molecular variation and genetic structure of U. hordei, 59 fungal isolates representing two distinct agro-ecological zones of India were analyzed by employing simple sequence repeats (SSRs). Using bioinformatic approaches, a total of 100,239 and 137,442 microsatellites were identified from 20.13 and 26.94 Mb of assembled genomic sequences of Uh364 and Uh4857-4 isolates of U. hordei, respectively. Penta-nucleotides (31.29 and 29.75%) followed by tri-nucleotide (28.27 and 29.88%) were most prevalent in both the genomes. Out of them, 15 polymorphic microsatellites showing conservancies in both the genomes were selected for exploring population genetic structure of U. hordei. An average of two alleles per microsatellite marker was generated with band size ranging from 180 to 850 bp. Polymorphic information content (PIC) varied between 0.095 and 0.37. Fifty-nine isolates were distributed in two distinct groups with about 65% genetic similarity according to UPGMA clustering and population structure analysis (K = 2). Gene flow analysis (Nm = 1.009) reflected moderate gene flow among the analyzed population. An analysis of molecular variance (AMOVA) displayed high level of genetic variation within population (87%) and low variation among populations (13%). Linkage disequilibrium (LD) analysis indicated positively significant but relatively low standardized index of association (SIA) value in both the population sets (SIA = 0.181), advocating a state of LD with epidemic population structure. In conclusion, the newly developed neutral SSR markers are highly polymorphic within U. hordei and will be useful for revealing evolutionary history and providing deep insight into the population dynamics of U. hordei in India as well as facilitating developing management strategies for CS of barley.

Phylogeography and Population Structure Analysis Reveal Diversity by Gene Flow and Mutation in Ustilago segetum (Pers.) Roussel tritici Causing Loose Smut of Wheat.

Ustilago segetum (Pers.) Roussel tritici (UST) causes loose smut of wheat account for considerable grain yield losses globally. For effective management, knowledge of its genetic variability and population structure is a prerequisite. In this study, UST isolates sampled from four different wheat growing zones of India were analyzed using the second largest subunit of the RNA polymerase II (RPB2) and a set of sixteen neutral simple sequence repeats (SSRs) markers. Among the 112 UST isolates genotyped, 98 haplotypes were identified. All the isolates were categorized into two groups (K = 2), each consisting of isolates from different sampling sites, on the basis of unweighted paired-grouping method with arithmetic averages (UPGMA) and the Bayesian analysis of population structure. The positive and significant index of association (IA = 1.169) and standardized index of association (rBarD = 0.075) indicate population is of non-random mating type. Analysis of molecular variance showed that the highest variance component is among isolates (91%), with significantly low genetic differentiation variation among regions (8%) (Fst = 0.012). Recombination (Rm = 0) was not detected. The results showed that UST isolates have a clonal genetic structure with limited genetic differentiation and human arbitrated gene flow and mutations are the prime evolutionary processes determining its genetic structure. These findings will be helpful in devising management strategy especially for selection and breeding of resistant wheat cultivars.