Ishtiaq A. Khan

Variations in intergenic spacer rpl20-rps12 of mango (Mangifera indica) chloroplast DNA: implications for cultivar identification and phylogenetic analysis

We characterized rpl20-rps12 and atp-rbcL intergenic spacers of chloroplast DNA from 19 mango cultivars to analyze suitability of these regions for assessing intraspecific variation. Analysis of the atp-rbcL region showed no variation, while rpl20-rps12 spacer sequences revealed both intravarietal and inter-subspecific polymorphisms. The intravarietal SNP was at position 360 of rpl20-rps12 spacer [SNP type C(G)]. The first inter-subspecific genotype detected was a C-A variation at position 755 in 16 out of 19 mango cultivars tested. The second inter-subspecific genotype was an insertion of C at position 714 of the rpl20-rps12 spacer, which was detected only in the ‘Langra’ cultivar. Phylogenetic tree construction based on the rpl20-rps12 region grouped ‘Shane-e-khuda,’ ‘Sonara,’ ‘Padri,’ ‘Neelum,’ ‘Gulab Khas,’ ‘Dusehri,’ ‘Chonsa,’ and ‘Cari Saloli’ cultivars together in one group; ‘Anwar Ratol,’ ‘Zafran,’ ‘Tota Pari,’ ‘Safada,’ ‘Kalapahar,’ and ‘Almas’ cultivars in a second group; and ‘Bangan Palli,’ ‘Lohatia,’ ‘Kesar,’ and ‘Bombay Alphonso’ cultivars in a third group. This study supports the applicability of the rpl20-rps12 intergenic region of cpDNA for mango cultivar identification and phylogenetics.

Characterisation of hydrazides and hydrazine derivatives as novel aspartic protease inhibitors

Virtual screening of an in-house virtual library of synthetic compounds using FlexX, followed by enzyme inhibition, identified hydrazide and hydrazine derivatives as novel aspartic protease inhibitors. These compounds inhibited human cathepsin D and Plasmodium falciparum plasmepsin-II with low micromolar concentrations (IC50 = 1-2.5 μM). Modelling studies with plasmepsin-II predicted binding of ligands at the centre of the extended substrate-binding cleft, where hydrazide/hydrazine parts of the inhibitors acted as the transition state mimic by forming electrostatic interactions with catalytic aspartates.

Current trends in chloroplast genome research

Chloroplast is an important cellular organelle of autotrophs which has an independent, circular, doublestranded DNA molecule termed as chloroplast genome. The chloroplast DNA (cpDNA) contains essential genes for its maintenance and operation. Several components of the photosystems and proteins involved in biosynthetic pathways are also encoded by the chloroplast genome. Exploring the genetic repository of this organelle is vital due to its conserved nature, small size, persistent gene organization and promising ability for transgenic expression. Therefore, cpDNA sequence information has been instrumental in phylogenetic studies and molecular taxonomy of plants. Chloroplast genome sequencing efforts have being initiated with conventional cloning and chain-termination sequencing technologies. Dedicated databases such as CGDB and GOBASE among others have been established as more and more complete cpDNA sequences are being reported. Presently, elegant molecular biology techniques including shotgun sequencing, rolling circle amplification (RCA), Amplification, Sequencing and Annotation of Plasteome (ASAP) and Next generation sequencing are being used to accelerate data output. Owing to many fold increase in submission of cpDNA sequences in nucleotide databases, challenges of in-depth data analysis stimulated the emergence of devoted annotation, assembling and phylogenetic software. Recently, reported bioinformatics software for chloroplast genome studies comprise of DOGMA for annotation, SCAN-SE, ARAGON and PREP suit for RNA analyses and CG viewer for circular map construction/comparative analysis. Faster algorithms for gene-order based phylogenetic reconstruction and bootstrap analysis have attracted the attention of research community. Current trends in sequencing strategies and bioinformatics with reference to chloroplast genomes hold great potential to illuminate more hidden corners of this ancient cell organelle.

Genome characterization of a novel Burkholderia cepacia complex genomovar isolated from dieback affected mango orchards

We characterized the genome of the antibiotic resistant, caseinolytic and non-hemolytic Burkholderia sp. strain TJI49, isolated from mango trees (Mangifera indica L.) with dieback disease. This isolate produced severe disease symptoms on the indicator plants. Next generation DNA sequencing and short-read assembly generated the 60X deep 7,631,934 nucleotide draft genome of Burkholderia sp. TJI49 which comprised three chromosomes and at least one mega plasmid. Genome annotation studies revealed a total 8,992 genes, out of which 8,940 were protein coding genes. Comparative genomics and phylogenetics identified Burkholderia sp. TJI49 as a distinct species of Burkholderia cepacia complex (BCC), closely related to B.multivorans ATCC17616. Genome-wide sequence alignment of this isolate with replicons of BCC members showed conservation of core function genes but considerable variations in accessory genes. Subsystem-based gene annotation identified the active presence of wide spread colonization island and type VI secretion system in Burkholderia sp. TJI49. Sequence comparisons revealed (a) 28 novel ORFs that have no database matches and (b) 23 ORFs with orthologues in species other than Burkholderia, indicating horizontal gene transfer events. Fold recognition of novel ORFs identified genes encoding pertactin autotransporter-like proteins (a constituent of type V secretion system) and Hap adhesion-like proteins (involved in cell–cell adhesion) in the genome of Burkholderia sp. TJI49. The genomic characterization of this isolate provided additional information related to the ‘pan-genome’ of Burkholderia species.

Estimating the mutational load for cardiovascular diseases in Pakistani population

The deleterious genetic variants contributing to certain diseases may differ in terms of number and allele frequency from population to population depending on their evolutionary background. Here, we prioritize the deleterious variants from Pakistani population in manually curated gene list already reported to be associated with common, Mendelian, and congenital cardiovascular diseases (CVDs) using the genome/exome sequencing data of Pakistani individuals publically available in 1000 Genomes Project (PJL), and Exome Aggregation Consortium (ExAC) South Asia. By applying a set of tools such as Combined Annotation Dependent Depletion (CADD), ANNOVAR, and Variant Effect Predictor (VEP), we highlighted 561 potentially detrimental variants from PJL data, and 7374 variants from ExAC South Asian data. Likewise, filtration from ClinVar for CVDs revealed 03 pathogenic and 02 likely pathogenic variants from PJL and 112 pathogenic and 42 likely pathogenic variants from ExAC South Asians. The comparison of derived allele frequencies (DAF) revealed many of these prioritized variants having two fold and higher DAF in Pakistani individuals than in other populations. The highest number of deleterious variants contributing to common CVDs in descending order includes hypertension, atherosclerosis, heart failure, aneurysm, and coronary heart disease, and for Mendelian and congenital CVDs cardiomyopathies, cardiac arrhythmias, and atrioventricular septal defects.

Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard

Abstract We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches.

Rare genetic mutations in Pakistani patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a leading cause of heart failure, and heart transplantation globally. There is enlargement of left ventricle of the heart impairing the systolic function in this disorder. The involvement of genetic factors in the pathogenesis of DCM has been reported in up to 50% of the cases. However, due to the complexity and heterogeneity of the disease, the complete pathophysiology remains unclear. In this study, whole exomes of five unrelated patients of idiopathic DCM were sequenced to an average depth of 100× using Illumina HiSeq4000 system. The analysis of the data with in silico tools SIFT, Polyphen2, and CADD showed 494 rare (AF < 1.0%) missense SNVs predicted as deleterious. Detrimental variants in genes highly expressed in cardiac tissue included 3 rare allele frequency loss-of-function SNVs in C2orf40, MYOM3, and TMED4 genes, a homozygous frameshift insertion in RTKN2, and a splice site homozygous deletion in SLC6A6 in at least one of the patients. The stop-gained SNV rs143187236 of MYOM3 (myomesin 3) was found in perfect linkage disequilibrium (r2 = 1.0) with its neighboring missense SNV rs149105212 in two of the patients, representing the role of myomesin 3 in pathophysiology of DCM. Allele frequency comparison showed three variants rs375563861 (C2orf40), rs143187236 (MYOM3), and rs564181443 (RTKN2) having 3 fold or higher allele frequency in South Asians than in the global populations. The identified pathogenic variants can be used in risk assessment and precision therapy in DCM patients.