Whole Genome Characterization of a Few EMS-Induced Mutants of Upland Rice Variety Nagina 22 Reveals a Staggeringly High Frequency of SNPs Which Show High Phenotypic Plasticity Towards the Wild-Type

The Indian initiative, in creating mutant resources for the functional genomics in rice, has been instrumental in the development of 87,000 ethylmethanesulfonate (EMS)-induced mutants, of which 7,000 are in advanced generations. The mutants have been created in the background of Nagina 22, a popular drought- and heat-tolerant upland cultivar. As it is a pregreen revolution cultivar, as many as 573 dwarf mutants identified from this resource could be useful as an alternate source of dwarfing. A total of 541 mutants, including the macromutants and the trait-specific ones, obtained after appropriate screening, are being maintained in the mutant garden. Here, we report on the detailed characterizations of the 541 mutants based on the distinctness, uniformity, and stability (DUS) descriptors at two different locations. About 90% of the mutants were found to be similar to the wild type (WT) with high similarity index (>0.6) at both the locations. All 541 mutants were characterized for chlorophyll and epicuticular wax contents, while a subset of 84 mutants were characterized for their ionomes, namely, phosphorous, silicon, and chloride contents. Genotyping of these mutants with 54 genomewide simple sequence repeat (SSR) markers revealed 93% of the mutants to be either completely identical to WT or nearly identical with just one polymorphic locus. Whole genome resequencing (WGS) of four mutants, which have minimal differences in the SSR fingerprint pattern and DUS characters from the WT, revealed a staggeringly high number of single nucleotide polymorphisms (SNPs) on an average (16,453 per mutant) in the genic sequences. Of these, nearly 50% of the SNPs led to non-synonymous codons, while 30% resulted in synonymous codons. The number of insertions and deletions (InDels) varied from 898 to 2,595, with more than 80% of them being 1–2 bp long. Such a high number of SNPs could pose a serious challenge in identifying gene(s) governing the mutant phenotype by next generation sequencing-based mapping approaches such as Mutmap. From the WGS data of the WT and the mutants, we developed a genic resource of the WT with a novel analysis pipeline. The entire information about this resource along with the panicle architecture of the 493 mutants is made available in a mutant database EMSgardeN22 (http://14.139.229.201/EMSgardeN22)

Genome-Wide Distribution and Organization of Microsatellites in Plants: An Insight into Marker Development in Brachypodium

Plant genomes are complex and contain large amounts of repetitive DNA including microsatellites that are distributed across entire genomes. Whole genome sequences of several monocot and dicot plants that are available in the public domain provide an opportunity to study the origin, distribution and evolution of microsatellites, and also facilitate the development of new molecular markers. In the present investigation, a genome-wide analysis of microsatellite distribution in monocots (Brachypodium, sorghum and rice) and dicots (Arabidopsis, Medicago and Populus) was performed. A total of 797,863 simple sequence repeats (SSRs) were identified in the whole genome sequences of six plant species. Characterization of these SSRs revealed that mono-nucleotide repeats were the most abundant repeats, and that the frequency of repeats decreased with increase in motif length both in monocots and dicots. However, the frequency of SSRs was higher in dicots than in monocots both for nuclear and chloroplast genomes. Interestingly, GC-rich repeats were the dominant repeats only in monocots, with the majority of them being present in the coding region. These coding GC-rich repeats were found to be involved in different biological processes, predominantly binding activities. In addition, a set of 22,879 SSR markers that were validated by e-PCR were developed and mapped on different chromosomes in Brachypodium for the first time, with a frequency of 101 SSR markers per Mb. Experimental validation of 55 markers showed successful amplification of 80% SSR markers in 16 Brachypodium accessions. An online database ‘BraMi’ (Brachypodium microsatellite markers) of these genome-wide SSR markers was developed and made available in the public domain. The observed differential patterns of SSR marker distribution would be useful for studying microsatellite evolution in a monocot–dicot system. SSR markers developed in this study would be helpful for genomic studies in Brachypodium and related grass species, especially for the map based cloning of the candidate gene(s)

Aquaporins: Dynamic Role and Regulation

Aquaporins (AQPs), a class of integral membrane proteins, form channels facilitating movement of water and many other solutes. In solute transport systems of all living organisms including plants, animals and fungi, AQPs play a vital role. Plants contain a much higher number of AQP genes compared to animals, the likely consequence of genome duplication events and higher ploidy levels. As a result of duplication and subsequent diversification, plant AQPs have evolved several subfamilies with very diverse functions. Plant AQPs are highly selective for specific solutes because of their unique structural features. For instance, ar/R selectivity filters and NPA domains have been found to be key elements in governing solute permeability through the AQP channels. Combination of conserved motifs and specific amino acids influencing pore morphology appears to regulate the permeability of specific solutes such as water, urea, CO2, H2O2, boric acid, silicic acid and many more. The discovery of novel AQPs has been accelerated over the last few years with the increasing availability of genomic and transcriptomic data. The expanding number of well characterised AQPs provides opportunities to understand factors influencing water transport, nutritional uptake, and elemental balance. Homology-based search tools and phylogenetic analyses offer efficient strategies for AQP identification. Subsequent characterization can be based on different approaches involving proteomics, genomics, and transcriptomic tools. The combination of these technological advances make it possible to efficiently study the inter-dependency of AQPs, regulation through phosphorylation and reversible phosphorylation, networking with other transporters, structural features, pH gating systems, trafficking and degradation. Several studies have supported the role of AQPs in differential phenotypic responses to abiotic and biotic stress in plants. Crop improvement programs aiming for the development of cultivars with higher tolerance against stresses like drought, flooding, salinity and many biotic diseases, can explore and exploit the finely tuned AQP-regulated transport system. For instance, a promising approach in crop breeding programs is the utilization of genetic variation in AQPs for the development of stress tolerant cultivars. Similarly, transgenic and mutagenesis approaches provide an opportunity to better understand the AQP transport system with subsequent applications for the development of climate-smart drought-tolerant cultivars. The contributions to this Frontiers in Plant Science Research Topic have highlighted the evolution and phylogenetic distribution of AQPs in several plant species. Numerous aspects of regulation that seek to explain AQP-mediated transport system have been addressed. These contributions will help to improve our understanding of AQPs and their role in important physiological aspects and will bring AQP research closer to practical applications

Role of Silicon in Plants

Silicon (Si) is gaining increased attention in the farming sector because of its beneficial effects observed in several crop species, particularly under stress conditions. The magnitude of benefits is predominantly observed in plant species that can accumulate Si above a certain threshold. Therefore, deciphering the molecular mechanisms and genetic factors conferring a plant ability to take up silicon is necessary. Along these lines, several efforts have been made to identify the specific genes regulating Si uptake and distribution in plant tissues. This information finds its usefulness in identifying Si-competent species, and could eventually lead to improving this ability in low-accumulating species. The successful exploitation of Si in agriculture depends highly on the understanding of different Si properties including plant-available Si from the soil, transport within tissues, deposition in planta, and Si effect on different metabolic and physiological processes. In addition, a better comprehension of external factors influencing Si uptake and deposition in plant tissue remains important. A plant can take up Si efficiently only in the form of silicic acid and most soils, despite containing high concentrations of Si, are deficient in plant-available Si. Consequently, soil amendment with fertilizers rich in plant-available Si is now viewed as an affordable option to protect plants from the biotic and abiotic stresses and achieve more sustainable cropping management worldwide. Articles compiled in the present research topic touch upon several aspects of Si properties and functionality in plants. The information will be helpful to further our understanding of the role of Si and contribute to exploit the benefits plants derive from it

Cloning of fibrinolytic protease-0 (<i>Efp-0</i>) gene from diverse earthworm individuals

270-273Fibrinolytic enzyme has potential role as therapeutic agents for the treatment of some blood clotting diseases. It is a novel oral-administered fibrinolytic agent used for the prevention and treatment of cardiac and cerebrovascular diseases. As a drug, it has been widely studied and used for clinical treatment. In the present study, we have cloned and sequenced partial Eisenia fetida fibrinolytic protease (Efp-0) gene from three earthworm strains collected from diverse habitats. The gene was amplified by using polymerase chain reaction and cloned in pGEM-T easy vector. The size of Efp-0 gene, cloned from the two strains of E. fetida and one strain of Eudrilus eugeniae, was 658 bp, 658 bp and 660 bp, respectively after sequencing,. Multiple sequence alignment of the cloned gene along with gene sequences available in public database showed 21 SNPs and 5 InDels in the coding region. However, diversity observed in Efp-0 gene cloned from different earthworm strains will provide basis for allele mining of this gene. It is concluded that Efp-0 is a novel gene and can be utilized for industrial applications. The gene sequences obtained in this study have been submitted in the GenBank (Accession no. GQ385193, GQ385194, GQ385195)

Drought and Oxidative Stress in Flax (<i>Linum usitatissimum</i> L.) Entails Harnessing Non-Canonical Reference Gene for Precise Quantification of qRT-PCR Gene Expression

Flax (Linum usitatissimum L.) is a self-pollinating, annual, diploid crop grown for multi-utility purposes for its quality oil, shining bast fiber, and industrial solvent. Being a cool (Rabi) season crop, it is affected by unprecedented climatic changes such as high temperature, drought, and associated oxidative stress that, globally, impede its growth, production, and productivity. To precisely assess the imperative changes that are inflicted by drought and associated oxidative stress, gene expression profiling of predominant drought-responsive genes (AREB, DREB/CBF, and ARR) was carried out by qRT-PCR. Nevertheless, for normalization/quantification of data obtained from qRT-PCR results, a stable reference gene is mandatory. Here, we evaluated a panel of four reference genes (Actin, EF1a, ETIF5A, and UBQ) and assessed their suitability as stable reference genes for the normalization of gene expression data obtained during drought-induced oxidative stress in flax. Taking together, from the canonical expression of the proposed reference genes in three different genotypes, we report that EF1a as a stand-alone and EF1a and ETIF5A in tandem are suitable reference genes to be used for the real-time visualization of cellular impact of drought and oxidative stress on flax