RNA-Seq Analysis of Developing Grains of Wheat to Intrigue Into the Complex Molecular Mechanism of the Heat Stress Response.

Heat stress is one of the significant constraints affecting wheat production worldwide. To ensure food security for ever-increasing world population, improving wheat for heat stress tolerance is needed in the presently drifting climatic conditions. At the molecular level, heat stress tolerance in wheat is governed by a complex interplay of various heat stress-associated genes. We used a comparative transcriptome sequencing approach to study the effect of heat stress (5°C above ambient threshold temperature of 20°C) during grain filling stages in wheat genotype K7903 (Halna). At 7 DPA (days post-anthesis), heat stress treatment was given at four stages: 0, 24, 48, and 120 h. In total, 115,656 wheat genes were identified, including 309 differentially expressed genes (DEGs) involved in many critical processes, such as signal transduction, starch synthetic pathway, antioxidant pathway, and heat stress-responsive conserved and uncharacterized putative genes that play an essential role in maintaining the grain filling rate at the high temperature. A total of 98,412 Simple Sequences Repeats (SSR) were identified from de novo transcriptome assembly of wheat and validated. The miRNA target prediction from differential expressed genes was performed by psRNATarget server against 119 mature miRNA. Further, 107,107 variants including 80,936 Single nucleotide polymorphism (SNPs) and 26,171 insertion/deletion (Indels) were also identified in de novo transcriptome assembly of wheat and wheat genome Ensembl version 31. The present study enriches our understanding of known heat response mechanisms during the grain filling stage supported by discovery of novel transcripts, microsatellite markers, putative miRNA targets, and genetic variant. This enhances gene functions and regulators, paving the way for improved heat tolerance in wheat varieties, making them more suitable for production in the current climate change scenario.

Uncovering Genomic Regions Associated With 36 Agro-Morphological Traits in Indian Spring Wheat Using GWAS.

Wheat genetic improvement by integration of advanced genomic technologies is one way of improving productivity. To facilitate the breeding of economically important traits in wheat, SNP loci and underlying candidate genes associated with the 36 agro-morphological traits were studied in a diverse panel of 404 genotypes. By using Breeders' 35K Axiom array in a comprehensive genome-wide association study covering 4364.79 cM of the wheat genome and applying a compressed mixed linear model, a total of 146 SNPs (-log10 P ≥ 4) were found associated with 23 traits out of 36 traits studied explaining 3.7-47.0% of phenotypic variance. To reveal this a subset of 260 genotypes was characterized phenotypically for six quantitative traits [days to heading (DTH), days to maturity (DTM), plant height (PH), spike length (SL), awn length (Awn_L), and leaf length (Leaf_L)] under five environments. Gene annotations mined ∼38 putative candidate genes which were confirmed using tissue and stage specific gene expression data from RNA Seq. We observed strong co-localized loci for four traits (glume pubescence, SL, PH, and awn color) on chromosome 1B (24.64 cM) annotated five putative candidate genes. This study led to the discovery of hitherto unreported loci for some less explored traits (such as leaf sheath wax, awn attitude, and glume pubescence) besides the refined chromosomal regions of known loci associated with the traits. This study provides valuable information of the genetic loci and their potential genes underlying the traits such as awn characters which are being considered as important contributors toward yield enhancement.

Development of model web-server for crop variety identification using throughput SNP genotyping data.

Crop varieties or genotypes of a given species are pivotal for agricultural production and ownership, management and improvement of their germplasm is a great challenge. Its morphological identification requires time, cost and descriptors are often compromised statistically due to phenotypic plasticity. Development of DNA based signature of varieties can overcome these limitations. There is a global need to implement world trade organization (WTO) and intellectual property rights (IPR) guidelines of Plant Breeders Rights (PBR) where DUS (distinctness, uniformity and stability) testing can be supplemented by DNA profile. Universalization and minimization of SNP number without compromising identification accuracy is the major challenge in development of varietal profile by rapid genotype assay. Besides this, there is no server-based approach reducing computational skill with global accessibility of referral phenotypic and genotypic data. We report world's first model web server for crop variety identification using >350 Indian wheat varieties and Axiom 35 K SNP chip data. Standard filtering and linkage disequilibrium approach were used to develop varietal signature in Linux using HTML, Java, PHP and MySQL with provision of QR code generator to facilitate bar-coding. Phylogenetic tree constructed by selected SNPs confirms six major trait based clusters of varieties and their pedigree. Our user friendly server based tool, VISTa (Variety Identification System of Triticum aestivum) ( http://webtom.cabgrid.res.in/vista ) can be used in DUS testing having dispute resolution of sovereignty and access benefit sharing (ABS) issues. This model approach can be used in other crops with pan-global level management of crop germplasm in endeavour of crop productivity.

Putative Microsatellite DNA Marker-Based Wheat Genomic Resource for Varietal Improvement and Management.

Wheat fulfills 20% of global caloric requirement. World needs 60% more wheat for 9 billion population by 2050 but climate change with increasing temperature is projected to affect wheat productivity adversely. Trait improvement and management of wheat germplasm requires genomic resource. Simple Sequence Repeats (SSRs) being highly polymorphic and ubiquitously distributed in the genome, can be a marker of choice but there is no structured marker database with options to generate primer pairs for genotyping on desired chromosome/physical location. Previously associated markers with different wheat trait are also not available in any database. Limitations of in vitro SSR discovery can be overcome by genome-wide in silico mining of SSR. Triticum aestivum SSR database (TaSSRDb) is an integrated online database with three-tier architecture, developed using PHP and MySQL and accessible at http://webtom.cabgrid.res.in/wheatssr/. For genotyping, Primer3 standalone code computes primers on user request. Chromosome-wise SSR calling for all the three sub genomes along with choice of motif types is provided in addition to the primer generation for desired marker. We report here a database of highest number of SSRs (476,169) from complex, hexaploid wheat genome (~17 GB) along with previously reported 268 SSR markers associated with 11 traits. Highest (116.93 SSRs/Mb) and lowest (74.57 SSRs/Mb) SSR densities were found on 2D and 3A chromosome, respectively. To obtain homozygous locus, e-PCR was done. Such 30 loci were randomly selected for PCR validation in panel of 18 wheat Advance Varietal Trial (AVT) lines. TaSSRDb can be a valuable genomic resource tool for linkage mapping, gene/QTL (Quantitative trait locus) discovery, diversity analysis, traceability and variety identification. Varietal specific profiling and differentiation can supplement DUS (Distinctiveness, Uniformity, and Stability) testing, EDV (Essentially Derived Variety)/IV (Initial Variety) disputes, seed purity and hybrid wheat testing. All these are required in germplasm management as well as also in the endeavor of wheat productivity.

Mutations in the plastidic ACCase gene endowing resistance to ACCase-inhibiting herbicide in Phalaris minor populations from India.

Littleseed canarygrass (Phalaris minor Retz.) is one of the most common and troublesome weeds infesting wheat crop in India. Repeated use during the last two decades of the ACCase-inhibiting herbicide (clodinafop) to control this weed has resulted in the occurrence of resistance. Fifty-three P. minor populations were collected from wheat fields in Haryana and Punjab states of India. The dose-response assays indicated that 29 populations were resistant, 23 populations were susceptible and one population was moderately resistant to clodinafop. Sequence analysis of the CT domain of ACCase gene among resistant and susceptible populations revealed two non-synonymous mutations, Trp2027 to Cys and Ile2041 to Asn in the resistant populations. Allele-specific PCR markers were developed to differentiate between wild-type and resistant codons at positions 2027 and 2041 of ACCase in P. minor which enables molecular assays for rapid detection and resistance diagnosis for efficient weed management in wheat. This is the first report from India of a target site mutation corresponding to resistance to clodinafop in P. minor.

Molecular genotyping of herbicide resistance in P. minor: ACCase resistance.

Little seed canary grass (Phalaris minor Retz.) populations resistant to herbicides that inhibit acetyl-CoA carboxylase (ACCase) represent an increasingly important weed control problem in northern India. The objective of this study was to develop DNA-based markers to differentiate herbicide-resistant and herbicide-susceptible population of P. minor. Primers were designed to amplify the conserved region carrying two reported mutations Trp2027 to Cys and Ile2041 to Asn conferring ACCase inhibitor resistance in several grass weeds and subjected to single-strand conformational polymorphism (SSCP) to detect the mutations. Five distinctive electrophoretic patterns on non-denaturing PAGE were observed, and four patterns were found to be associated with ACCase herbicide resistance in P. minor. The PCR-SSCP test developed in this study confirmed 17 resistant populations to contain mutations in CT domain of ACCase gene. This is the first report of rapid and easy molecular diagnosis of ACCase herbicide-resistant and herbicide-sensitive population of P. minor through PCR-SSCP analysis.