Dhiraj Singh

CRISPR/Cas9: a promising way to exploit genetic variation in plants

Creation of variation in existing gene pool of crop plants is the foremost requirement in crop improvement programmes. Genome editing is a tool to produce knock out of target genes either by introduction of insertion or by deletion that disrupts the function of a specific gene. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system is the most recent addition to the toolbox of sequence-specific nucleases that includes ZFNs and TALENs. The CRISPR/Cas9 system allows targeted cleavage of genomic DNA guided by a small noncoding RNA, resulting in gene modifications by both non-homologous end joining and homology-directed repair mechanisms. Here, we present an overview of mechanisms of CRISPR, its potential roles in creating variation in germplasm and applications of this novel interference pathway in crop improvement. The availability of the CRISPR/Cas9 system holds promise in facilitating both forward and reverse genetics and will enhance research in crops that lack genetic resources.

Furan fatty acids: their role in plant systems

Furan fatty acids (F-acids) are heterocyclic fatty acids having a furan ring in their structure. Most existing studies on F-acids are related to either fish or other marine animals. Even though F-acids have been detected in many plant species, not much work has been done exclusively in plants of economic importance, especially oilseed crops. This review focuses mainly on the functions and roles of F-acids in plants. In plants, they are bound to phospholipids by substituting PUFA and function as free radical scavengers suggesting their role in defense against oxidative stress. Owing to their antioxidative property F-acids are highly unstable and their photooxidative products can contribute to the flavor of edible oils.

SSR marker variations in Brassica species provide insight into the origin and evolution of Brassica amphidiploids

BackgroundOilseed Brassica represents an important group of oilseed crops with a long history of evolution and cultivation. To understand the origin and evolution of Brassica amphidiploids, simple sequence repeat (SSR) markers were used to unravel genetic variations in three diploids and three amphidiploid Brassica species of U’s triangle along with Eruca sativa as an outlier.ResultsOf 124 Brassica-derived SSR loci assayed, 100% cross-transferability was obtained for B. juncea and three subspecies of B. rapa, while lowest cross-transferability (91.93%) was obtained for Eruca sativa. The average % age of cross-transferability across all the seven species was 98.15%. The number of alleles detected at each locus ranged from one to six with an average of 3.41 alleles per primer pair. Neighbor-Joining-based dendrogram divided all the 40 accessions into two main groups composed of B. juncea/B. nigra/B. rapa and B. carinata/B. napus/B. oleracea. C-genome of oilseed Brassica species remained relatively more conserved than A- and B-genome. A- genome present in B. juncea and B. napus seems distinct from each other and hence provides great opportunity for generating diversity through synthesizing amphidiploids from different sources of A- genome. B. juncea had least intra-specific distance indicating narrow genetic base. B. rapa appears to be more primitive species from which other two diploid species might have evolved.ConclusionThe SSR marker set developed in this study will assist in DNA fingerprinting of various Brassica species cultivars, evaluating the genetic diversity in Brassica germplasm, genome mapping and construction of linkage maps, gene tagging and various other genomics-related studies in Brassica species. Further, the evolutionary relationship established among various Brassica species would assist in formulating suitable breeding strategies for widening the genetic base of Brassica amphidiploids by exploiting the genetic diversity present in diploid progenitor gene pools.

A simple spectrophotometric method for estimating total glucosinolates in mustard de-oiled cake

ABSTRACT Glucosinolates are anti-nutritional factors present abundantly in the seed meal fraction of oilseed Brassica species. They are found in varying levels among different genotypes. Those genotypes containing less than 30 µmol/g are considered low/zero glucosinolate type and are preferred for edible purposes due to low pungency. Twenty two different genotypes were taken for the analysis of glucosinolates by spectrophotometry. A regression model was obtained using Ordinary Least Square technique which predicted a formula. Total glucosinolates (µmol/g) = 1.40 + 118.86 × A425, where A425 is the absorbance at 425 nm. The total glucosinolate content obtained by the prediction formula when compared with HPLC data showed a correlation coefficient of 0.942. This high correlation between the two data sets validated the developed methodology. This method also simplifies the estimation of total glucosinolates by excluding the use of HPLC or other sophisticated instruments.

Recent Perspective of Next Generation Sequencing: Applications in Molecular Plant Biology and Crop Improvement

Nearly after three decades of Sanger sequencing method dominating the field of nucleotide sequencing, the new age of novel sequencing techniques commenced with 454-pyrosequencing in the year 2005 and thus started the era of next generation sequencing techniques. Since then many other novel sequencing techniques with increased accuracy, simplicity and cost-effectiveness have come up and called next to next generation sequencing techniques or third generation sequencing techniques. The scientific knowledge generated from next generation sequencing techniques has transformed the field of structural and functional genomic studies in different crop plants. In this review, various next generation sequencing techniques are described, with their applications and future prospective.