Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes

Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCR - detection and expression analysis of gene(s) in real-time - has revolutionized the 21(st) century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to name a few. We comprehensively review various aspects of real-time PCR, including technological refinement and application in all scientific fields ranging from medical to environmental issues, and to plant.     

The DNA Replication,Repair, and Recombination Pathway Genes Modulating Yield and Stress Tolerance Traits in Chickpea

DNA replication, repair, and recombination (DRRR) are the fundamental processes required for faithful transmission of genetic information within and between generations. The DRRR genes protect the cells from potential mutations and damage during the developmental phases and stress conditions. Thus, these genes indirectly regulate diverse important agronomic traits in a crop plant. A genome-wide survey of six DRRR pathway genes, namely, DNA replication, Base Excision Repair, Nucleotide Excision Repair, Homologous Recombination, Mismatch Excision Repair, and Non-Homologous End-Joining, identified 157 DRRR genes in chickpea. Phylogenetic analysis of these genes within the legume clades and model plant Arabidopsis identified 42 conserved DRRR genes exhibiting clade-specific evolutionary patterns. Integrating the gene-based association mapping with differential expression profiling identified the natural alleles of the potential DRRR genes, primarily regulating flowering and maturation time and involved in drought tolerance of chickpea. Identifying and understanding DRRR genes’ roles in regulating yield and stress tolerance traits in a vital grain legume like chickpea is requisite for its future crop improvement endeavors. Manipulation of promising functionally relevant DRRR genes will pave the way for simultaneous improvement in multiple beneficial agronomic traits in chickpea.

     

Differential transcriptional activation of copia family of different plant retrotransposons

Journal of Plant Biochemistry and Biotechnology - Plant genomes contain a sizeable fraction, ranging from 14 to 75% of retrotransposons (class I elements), predominantly comprising LTR (Long...     

A study on biosynthesis of “citral” in lemongrass (<Emphasis Type="Italic">C. flexuosus</Emphasis>) cv. Suvarna

Incorporation of [1-¹³C]-glucose and fosmidomycin was achieved in young and rapidly expanding (aged 15 days) leaves of lemongrass (C. flexuosus) cv. suvarna to elucidate biosynthetic origin of citral (3,7-dimethyl-2,6-octadienal). Analyses of the resultant ¹³C-labeling patterns of citral by quantitative ¹³C-NMR spectroscopy revealed significant %¹³C enrichment at carbons C-3, C-5, C-7 and C-9 in citral. This labeling pattern of the citral is in accordance with their biosynthesis via 2C-methyl-d-erythritol-4-phosphate (MEP) pathway. However, incorporation of [1-¹³C]-glucose achieved in the presence of fosmidomycin resulted in a ¹³C-labeling pattern of citral which did not match with labeling pattern characteristic of the MEP pathway. In addition, we studied the activity pattern of the DXR enzyme following fosmidomycin (25, 50, 75 and 100 μM concentrations) treatment of the young (aged 15 days) leaves for 48 h. The results revealed that fosmidomycin (100 μM) caused drastic inhibition (>50 %) of the DXR enzyme activity. The levels of the citral measured in the fosmidomycin treated leaves were also found to be reduced with decrease the activity of DXR enzyme. In conclusion, the results of the present work revealed the presence of the MEP pathway and its role in the biosynthesis of citral in lemongrass. In addition, the critical role of the DXR enzyme in the citral biosynthesis is highlighted. This is the first report on elucidation of the MEP pathway in lemongrass and may help in deeper understanding of the monoterpene biosynthesis and regulation in the genus Cymbopogon of high industrial significance.     

A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis

The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4∆ and bud6∆ alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis.     

Deploying QTL-seq for rapid delineation of a potential candidate gene underlying major trait-associated QTL in chickpea

A rapid high-resolution genome-wide strategy for molecular mapping of major QTL(s)/gene(s) regulating important agronomic traits is vital for in-depth dissection of complex quantitative traits and genetic enhancement in chickpea. The present study for the first time employed a NGS-based whole-genome QTL-seq strategy to identify one major genomic region harbouring a robust 100-seed weight QTL using an intra-specific 221 chickpea mapping population (desi cv. ICC 7184 × desi cv. ICC 15061). The QTL-seq-derived major SW QTL (CaqSW1.1) was further validated by single-nucleotide polymorphism (SNP) and simple sequence repeat (SSR) marker-based traditional QTL mapping (47.6% R² at higher LOD >19). This reflects the reliability and efficacy of QTL-seq as a strategy for rapid genome-wide scanning and fine mapping of major trait regulatory QTLs in chickpea. The use of QTL-seq and classical QTL mapping in combination narrowed down the 1.37 Mb (comprising 177 genes) major SW QTL (CaqSW1.1) region into a 35 kb genomic interval on desi chickpea chromosome 1 containing six genes. One coding SNP (G/A)-carrying constitutive photomorphogenic9 (COP9) signalosome complex subunit 8 (CSN8) gene of these exhibited seed-specific expression, including pronounced differential up-/down-regulation in low and high seed weight mapping parents and homozygous individuals during seed development. The coding SNP mined in this potential seed weight-governing candidate CSN8 gene was found to be present exclusively in all cultivated species/genotypes, but not in any wild species/genotypes of primary, secondary and tertiary gene pools. This indicates the effect of strong artificial and/or natural selection pressure on target SW locus during chickpea domestication. The proposed QTL-seq-driven integrated genome-wide strategy has potential to delineate major candidate gene(s) harbouring a robust trait regulatory QTL rapidly with optimal use of resources. This will further assist us to extrapolate the molecular mechanism underlying complex quantitative traits at a genome-wide scale leading to fast-paced marker-assisted genetic improvement in diverse crop plants, including chickpea.     

Metal ion mediated imprinting for electrochemical enantioselective sensing of L-histidine at trace level

Enantioselective trace level sensing of l-histidine (limit of detection, 1.980 ngm L(-1), S/N=3) was feasible with the use of a typical, reproducible, and rugged complex imprinted polymer-based pencil graphite electrode, in aqueous samples. In the present instance, the Cu(2+) ion-mediated imprinting of l-histidine in an molecularly imprinted polymer motif actually helped upbringing electrocatalytic activity to respond an enhanced differential pulse anodic stripping voltammetric oxidation peak of l-histidine, without any cross-reactivity and false-positive, in real samples. The proposed sensor could be considered suitable for the practical applications in biomarking histedinemia, a disease associated with L-histidine metabolic disorders, in clinical settings.