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.

Next Generation Sequencing Based Forward Genetic Approaches for Identification and Mapping of Causal Mutations in Crop Plants: A Comprehensive Review.

The recent advancements in forward genetics have expanded the applications of mutation techniques in advanced genetics and genomics, ahead of direct use in breeding programs. The advent of next-generation sequencing (NGS) has enabled easy identification and mapping of causal mutations within a short period and at relatively low cost. Identifying the genetic mutations and genes that underlie phenotypic changes is essential for understanding a wide variety of biological functions. To accelerate the mutation mapping for crop improvement, several high-throughput and novel NGS based forward genetic approaches have been developed and applied in various crops. These techniques are highly efficient in crop plants, as it is relatively easy to grow and screen thousands of individuals. These approaches have improved the resolution in quantitative trait loci (QTL) position/point mutations and assisted in determining the functional causative variations in genes. To be successful in the interpretation of NGS data, bioinformatics computational methods are critical elements in delivering accurate assembly, alignment, and variant detection. Numerous bioinformatics tools/pipelines have been developed for such analysis. This article intends to review the recent advances in NGS based forward genetic approaches to identify and map the causal mutations in the crop genomes. The article also highlights the available bioinformatics tools/pipelines for reducing the complexity of NGS data and delivering the concluding outcomes.

Genome-wide association mapping revealed numerous novel genomic loci for grain nutritional and yield-related traits in rice (Oryza sativa L.) landraces.

A core set of 190 rice landraces were used to decipher the genetic structure and to discover the chromosomal regions containing QTLs, affecting the grain micro-nutrients, fatty acids, and yield-related traits by using 148 molecular markers in this study. Landraces were categorized into three sub-groups based on population stratification study and followed by neighbor-joining tree and principal component analysis. Analysis of variance revealed abundant variations among the landraces for studied traits with less influence of environmental factors. Genome Wide Association Studies (GWAS) revealed 22 significant and consistent QTLs through marker trait association (MTAs) for 12 traits based on 2 years and pooled analysis. Out of 22 QTLs, three have been reported earlier while 19 QTLs are novel. Interestingly, 13 QTLs out of 22 were explained more than 10% phenotypic variance. Association of RM1148 and RM205 with Days to 50% flowering was comparable with flowering control genes Ghd8/qDTH8 and qDTH9, respectively. Similarly, Zn content was associated with RM44, which is situated within the QTL qZn8-1. Moreover, significant association of RM25 with oleic acid content was closely positioned with QTL qOle8. Association of RM7434 with grain yield/plant; RM184 with spikelet fertility %; R3M10, R9M42 with hundred seed weight; RM536, RM17467, RM484, RM26063 with Fe content; RM44, RM6839 with Zn content are the major outcomes of this study. In addition, association of R11M23 with days to 50% flowering, panicle length and total spikelets per panicle are explained the possible occurrence of pleiotropism among these traits. Prominent rice landraces viz., Anjani (early maturity); Sihar (extra dwarf); Gangabaru (highest grain yield/plant); Karhani (highest iron content); Byalo-2 (highest zinc content) and Kadamphool (highest oleic acid) were identified through this study. The present study will open many avenues towards utilization of these QTLs and superior landraces in rice breeding for developing nutrition-rich high yielding varieties.

GLADS: A gel-less approach for detection of STMS markers in wheat and rice.

Sequence tagged microsatellite site (STMS) are useful PCR based DNA markers. Wide genome coverage, high polymorphic index and co-dominant nature make STMS a preferred choice for marker assisted selection (MAS), genetic diversity analysis, linkage mapping, seed genetic purity analysis etc. Routine STMS analysis involving low-throughput, laborious and time-consuming polyacrylamide/agarose gels often limit their full utility in crop breeding experiments that involve large populations. Therefore, convenient, gel-less marker detection methods are highly desirable for STMS markers. The present study demonstrated the utility of SYBR Green dye based melt-profiling as a simple and convenient gel-less approach for detection of STMS markers (referred to as GLADS) in bread wheat and rice. The method involves use of SYBR Green dye during PCR amplification (or post-PCR) of STMS markers followed by generation of a melt-profile using controlled temperature ramp rate. The STMS amplicons yielded characteristic melt-profiles with differences in melting temperature (Tm) and profile shape. These characteristic features enabled melt-profile based detection and differentiation of STMS markers/alleles in a gel-less manner. The melt-profile approach allowed assessment of the specificity of the PCR assay unlike the end-point signal detection assays. The method also allowed multiplexing of two STMS markers with non-overlapping melt-profiles. In principle, the approach can be effectively used in any crop for STMS marker analysis. This SYBR Green melt-profiling based GLADS approach offers a convenient, low-cost (20-51%) and time-saving alternative for STMS marker detection that can reduce dependence on gel-based detection, and exposure to toxic chemicals.

Expanding Avenue of Fast Neutron Mediated Mutagenesis for Crop Improvement.

: Fast neutron (FN) radiation mediated mutagenesis is a unique approach among the several induced mutagenesis methods being used in plant science in terms of impacted mutations. The FN mutagenesis usually creates deletions from few bases to several million bases (Mb). A library of random deletion generated using FN mutagenesis lines can provide indispensable resources for the reverse genetic approaches. In this review, information from several efforts made using FN mutagenesis has been compiled to understand the type of induced mutations, frequency, and genetic stability. Concerns regarding the utilization of FN mutagenesis technique for a plant with different level of ploidy and genome complexity are discussed. We have highlighted the utility of next-generation sequencing techniques that can be efficiently utilized for the characterization of mutant lines as well as for the mapping of causal mutations. Pros and cons of mapping by mutation (MutMap), mutant chromosome sequencing (MutChromSeq), exon capture, whole genome sequencing, MutRen-Seq, and different tilling approaches that can be used for the detection of FN-induced mutation has also been discussed. Genomic resources developed using the FN mutagenesis have been catalogued wooing to meaningful utilization of the available resources. The information provided here will be helpful for the efficient exploration for the crop improvement programs and for better understanding of genetic regulations.

InDel marker based genetic differentiation and genetic diversity in traditional rice (Oryza sativa L.) landraces of Chhattisgarh, India.

Rice has been cultivating and utilizing by humans for thousands of years under diverse environmental conditions. Therefore, tremendous genetic differentiation and diversity has occurred at various agro-ecosystems. The significant indica-japonica differentiation in rice provides great opportunities for its genetic improvement. In the present investigation, a total of 42 polymorphic InDel markers were used for differentiating 188 rice landraces and two local varieties of Chhattisgarh, India into indica and japonica related genotypes based on 'InDel molecular index'. Frequency of japonica alleles varied from 0.11 to 0.89 among landraces. Results revealed that 104 rice landraces have indica type genetic architecture along with three tested indica cultivars Swarna, Mahamaya and Rajeshwari. Another 60 landraces were placed under 'close to indica' type. It was found that three rice landraces i.e. Kalajeera, Kapri, Tulsimala were 'close to japonica' type and 21 landraces were 'intermediate' type. The result from the calculation of 'InDel molecular index' was further verified with STRUCTURE, AMOVA, PCA and cluster analysis. Population structure analysis revealed two genetically distinct populations within the 190 rice landraces/genotypes. Based on AMOVA, 'intermediate' type, 'close to japonica' type and Dongjinbyeo (a japonica cultivar from Republic of Korea) displayed significant genetic differentiation (ɸPT = 0.642, P = 0.000) from 'indica' and 'close to indica' groups. The PCA scatter plot and dendrogram demonstrated a clear pattern of two major group differentiations. 'Close to japonica' type and 'intermediate' type landraces/genotypes were grouped with Dongjinbyeo and formed a separate cluster at 30% Jaccard's similarity level from rest of the landraces/genotypes which were 'close to indica' or 'indica' type. Such a significant genetic differentiation among the locally adapted landraces could be exploited for the development of rice varieties introgressing higher yield potential and better plant types of japonica type as per the need of consumers and rice traders.