In vitro Adoption and Propagation of High Pathogenic Avian Influenza (HPAI) Virus Subtype H5N1 in non-avian Host System

The paradigmatic, fatal and devastating ailment called avian influenza or bird flu is a highly contagious viral disease caused by type A influenza virus, It primarily affects the respiratory, digestive and/or nervous system of chickens, turkeys, guinea fowls and other avian species and less commonly pigs and other species of mammals including human. In India, The first pandemic outbreak of Avian Influenza was reported during 2006. In this study, we selected an isolate of high pathogenic avian influenza (A/Ck/Jalgaon/India/12419/2006) H5N1 virus and propagated in chicken embryo fibroblast. Later this virus was adopted and propagated in Madin-Darby canine kidney cells (MDCK) and Vero cells. Infected non-avian cells with an avian virus shown cytopathic effects like rounding, cytoplasmic elongation, syncytia formation and later stages fluffing from the attached surface. The harvested virus suspension shown increased haemagglutination titre (HA) than viral suspension from chicken embryo fibroblast culture and the presence of virus was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). The obtained result reveals that virus had capacity to adopt for the invitro culture and propagate in non avian host cells with higher titre. This infers the chance of virus to cross the host barrier and probable chance of infection in human being

Is Endophytic Colonization of Host Plants a Method of Alleviating Drought Stress?: Conceptualizing the Hidden World of Endophytes

In the wake of changing climatic conditions, plants are frequently exposed to a wide range of biotic and abiotic stresses at various stages of their development, all of which negatively affect their growth, development, and productivity. Drought is one of the most devastating abiotic stresses for most cultivated crops, particularly in arid and semiarid environments. Conventional breeding and biotechnological approaches are used to generate drought-tolerant crop plants. However, these techniques are costly and time-consuming. Plant-colonizing microbes, notably, endophytic fungi, have received increasing attention in recent years since they can boost plant growth and yield and can strengthen plant responses to abiotic stress. In this review, we describe these microorganisms and their relationship with host plants, summarize the current knowledge on how they “reprogram” the plants to promote their growth, productivity, and drought tolerance, and explain why they are promising agents in modern agriculture

Investigation of Compressive Properties of Wollastonite (CaSiO3) reinforced Acrylonitrile Butadiene Styrene composites

Volume 8 Issue 4 (April 201

The first set of EST resource for gene discovery and marker development in pigeonpea (Cajanus cajanL.)

Background Pigeonpea (Cajanus cajan (L.) Millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses [Fusarium wilt (FW), sterility mosaic disease (SMD), etc.] are serious challenges for sustainable crop production. Modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. Availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. With an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of FW- and SMD- responsive expressed sequence tags (ESTs). Results A total of 16 cDNA libraries were constructed from four pigeonpea genotypes that are resistant and susceptible to FW ('ICPL 20102' and 'ICP 2376') and SMD ('ICP 7035' and 'TTB 7') and a total of 9,888 (9,468 high quality) ESTs were generated and deposited in dbEST of GenBank under accession numbers GR463974 to GR473857 and GR958228 to GR958231. Clustering and assembly analyses of these ESTs resulted into 4,557 unique sequences (unigenes) including 697 contigs and 3,860 singletons. BLASTN analysis of 4,557 unigenes showed a significant identity with ESTs of different legumes (23.2-60.3%), rice (28.3%), Arabidopsis (33.7%) and poplar (35.4%). As expected, pigeonpea ESTs are more closely related to soybean (60.3%) and cowpea ESTs (43.6%) than other plant ESTs. Similarly, BLASTX similarity results showed that only 1,603 (35.1%) out of 4,557 total unigenes correspond to known proteins in the UniProt database (≤ 1E-08). Functional categorization of the annotated unigenes sequences showed that 153 (3.3%) genes were assigned to cellular component category, 132 (2.8%) to biological process, and 132 (2.8%) in molecular function. Further, 19 genes were identified differentially expressed between FW- responsive genotypes and 20 between SMD- responsive genotypes. Generated ESTs were compiled together with 908 ESTs available in public domain, at the time of analysis, and a set of 5,085 unigenes were defined that were used for identification of molecular markers in pigeonpea. For instance, 3,583 simple sequence repeat (SSR) motifs were identified in 1,365 unigenes and 383 primer pairs were designed. Assessment of a set of 84 primer pairs on 40 elite pigeonpea lines showed polymorphism with 15 (28.8%) markers with an average of four alleles per marker and an average polymorphic information content (PIC) value of 0.40. Similarly, in silico mining of 133 contigs with ≥ 5 sequences detected 102 single nucleotide polymorphisms (SNPs) in 37 contigs. As an example, a set of 10 contigs were used for confirming in silico predicted SNPs in a set of four genotypes using wet lab experiments. Occurrence of SNPs were confirmed for all the 6 contigs for which scorable and sequenceable amplicons were generated. PCR amplicons were not obtained in case of 4 contigs. Recognition sites for restriction enzymes were identified for 102 SNPs in 37 contigs that indicates possibility of assaying SNPs in 37 genes using cleaved amplified polymorphic sequences (CAPS) assay. Conclusion The pigeonpea EST dataset generated here provides a transcriptomic resource for gene discovery and development of functional markers associated with biotic stress resistance. Sequence analyses of this dataset have showed conservation of a considerable number of pigeonpea transcripts across legume and model plant species analysed as well as some putative pigeonpea specific genes. Validation of identified biotic stress responsive genes should provide candidate genes for allele mining as well as candidate markers for molecular breeding

Inheritance of sterility mosaic disease resistance to Bangalore and Patancheru isolates in pigeonpea (Cajanus cajan (L.) Millsp.)

Sterility mosaic disease (SMD), is an important biotic constraint in pigeonpea (Cajanus cajan (L.) Millsp.) in Indian subcontinent. It is caused by a virus and transmitted by eriophyid mites, Aceria cajani Channabasavanna. A comprehensive study of variability in the sterility mosaic pathogen revealed the occurrence of five different isolates in India. Amongst them, three distinct isolates have been characterised, viz., Bangalore, Patancheru and Coimbatore. Studies were conducted at Bangalore and Patancheru to determine the inheritance of resistance to Bangalore and Patancheru isolates of the SMD involving a resistant (ICP 7035) and susceptible (TTB 7) genotypes. Observations in parents, F indicated dominance of susceptibility over resistance. The disease reaction of the individual F 2 plant derived F 3 1 families for Patancheru isolate was controlled by two genes with dominance epistasis and for Bangalore isolate, absence of resistant plants indicate action of two or more genes in controlling resistance to SMD

DNA barcoding and surveillance sampling strategies for Culicoides biting midges (Diptera: Ceratopogonidae) in southern India

Background: Culicoides spp. biting midges transmit bluetongue virus (BTV), the aetiological agent of bluetongue (BT), an economically important disease of ruminants. In southern India, hyperendemic outbreaks of BT exert high cost to subsistence farmers in the region, impacting on sheep production. Effective Culicoides spp. monitoring methods coupled with accurate species identification can accelerate responses for minimising BT outbreaks. Here, we assessed the utility of sampling methods and DNA barcoding for detection and identification of Culicoides spp. in southern India, in order to provide an informed basis for future monitoring of their populations in the region. Methods: Culicoides spp. collected from Tamil Nadu and Karnataka were used to construct a framework for future morphological identification in surveillance, based on sequence comparison of the DNA barcode region of the mitochondrial cytochrome c oxidase I (COI) gene and achieving quality standards defined by the Barcode of Life initiative. Pairwise catches of Culicoides spp. were compared in diversity and abundance between green (570 nm) and ultraviolet (UV) (390 nm) light emitting diode (LED) suction traps at a single site in Chennai, Tamil Nadu over 20 nights of sampling in November 2013. Results: DNA barcode sequences of Culicoides spp. were mostly congruent both with existing DNA barcode data from other countries and with morphological identification of major vector species. However, sequence differences symptomatic of cryptic species diversity were present in some groups which require further investigation. While the diversity of species collected by the UV LED Center for Disease Control (CDC) trap did not significantly vary from that collected by the green LED CDC trap, the UV CDC significantly outperformed the green LED CDC trap with regard to the number of Culicoides individuals collected. Conclusions: Morphological identification of the majority of potential vector species of Culicoides spp. samples within southern India appears relatively robust; however, potential cryptic species diversity was present in some groups requiring further investigation. The UV LED CDC trap is recommended for surveillance of Culicoides in southern India

An intra-specific consensus genetic map of pigeonpea [Cajanus cajan (L.) Millspaugh] derived from six mapping populations

Pigeonpea (Cajanus cajan L.) is an important food legume crop of rainfed agriculture. Owing to exposure of the crop to a number of biotic and abiotic stresses, the crop productivity has remained stagnant for almost last five decades at ca. 750 kg/ha. The availability of a cytoplasmic male sterility (CMS) system has facilitated the development and release of hybrids which are expected to enhance the productivity of pigeonpea. Recent advances in genomics and molecular breeding such as marker-assisted selection (MAS) offer the possibility to accelerate hybrid breeding. Molecular markers and genetic maps are pre-requisites for deploying MAS in breeding. However, in the case of pigeonpea, only one inter- and two intra-specific genetic maps are available so far. Here, four new intra-specific genetic maps comprising 59–140 simple sequence repeat (SSR) loci with map lengths ranging from 586.9 to 881.6 cM have been constructed. Using these four genetic maps together with two recently published intra-specific genetic maps, a consensus map was constructed, comprising of 339 SSR loci spanning a distance of 1,059 cM. Furthermore, quantitative trait loci (QTL) analysis for fertility restoration (Rf) conducted in three mapping populations identified four major QTLs explaining phenotypic variances up to 24 %. To the best of our knowledge, this is the first report on construction of a consensus genetic map in pigeonpea and on the identification of QTLs for fertility restoration. The developed consensus genetic map should serve as a reference for developing new genetic maps as well as correlating with the physical map in pigeonpea to be developed in near future. The availability of more informative markers in the bins harbouring QTLs for sterility mosaic disease (SMD) and Rf will facilitate the selection of the most suitable markers for genetic analysis and molecular breeding applications in pigeonpea

The first set of EST resource for gene discoveryand marker development in pigeonpea(Cajanus cajan L.)

Pigeonpea (Cajanus cajan (L.) Millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses [Fusarium wilt (FW), sterility mosaic disease (SMD), etc.] are serious challenges for sustainable crop production. Modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. Availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. With an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of FW- and SMD- responsive expressed sequence tags (ESTs). Results: A total of 16 cDNA libraries were constructed from four pigeonpea genotypes that are resistant and susceptible to FW ('ICPL 20102' and 'ICP 2376') and SMD ('ICP 7035' and 'TTB 7') and a total of 9,888 (9,468 high quality) ESTs were generated and deposited in dbEST of GenBank under accession numbers GR463974 to GR473857 and GR958228 to GR958231. Clustering and assembly analyses of these ESTs resulted into 4,557 unique sequences (unigenes) including 697 contigs and 3,860 singletons. BLASTN analysis of 4,557 unigenes showed a significant identity with ESTs of different legumes (23.2-60.3%), rice (28.3%), Arabidopsis (33.7%) and poplar (35.4%). As expected, pigeonpea ESTs are more closely related to soybean (60.3%) and cowpea ESTs (43.6%) than other plant ESTs. Similarly, BLASTX similarity results showed that only 1,603 (35.1%) out of 4,557 total unigenes correspond to known proteins in the UniProt database (= 1E-08). Functional categorization of the annotated unigenes sequences showed that 153 (3.3%) genes were involved in cellular component category, 132 (2.8%) in biological process, and 132 (2.8%) in molecular function. Further, nineteen genes were identified differentially expressed between FW- responsive genotypes and 20 between SMD- responsive genotypes. Generated ESTs were compiled together with 908 ESTs available in public domain, at the time of analysis, and a set of 5,085 unigenes were defined that were used for identification of molecular markers in pigeonpea. For instance, 3,583 simple sequence repeat (SSR) motifs were identified in 1,365 unigenes and 383 primer pairs were designed. Assessment of a set of 84 primer pairs on 40 elite pigeonpea lines showed polymorphism with 15 (28.8%) markers with an average of four alleles per marker and an average polymorphic information content (PIC) value of 0.40. Similarly, in silico mining of 133 contigs with ⩾ 5 sequences detected 102 single nucleotide polymorphisms (SNPs) in 37 contigs. As an example, a set of 10 contigs were used for confirming in silico predicted SNPs in a set of four genotypes using wet lab experiments. While occurrence of SNPs were confirmed for all the 6 contigs for which scorable and sequenceable amplicons were generated. PCR amplicons were not obtained in case of 4 contigs. Recognition sites for restriction enzymes were identified for 102 SNPs in 37 contigs that indicates possibility of assaying SNPs in 37 genes using cleaved amplified polymorphic sequences (CAPS) assa

ICP 7035 – A Sterility Mosaic Resistant Vegetable and Grain Purpose Pigeonpea Variety

ICP 7035 is a medium duration, non-determinate pigeonpea landrace collected in 1973 from Bedaghat (near Jabalpur), Madhya Pradesh, India. Plants mature in 170-200 days (in south-central regions of India) and, at this stage, reach an average height of 120-140 cm. Each plant produced around 100 pods and each pod contained 5 seeds, which are nutritionally rich and contain high percentages of digestible carbohydrates, vitamins and micronutrients. The large seeds (9-11 mm diameter) had purple seed coats and green cotyledons, and are suitable for consumption as vegetable. The fresh seed contains 8.6% protein, 12% fibre, and 45.7% carbohydrate and starch. The pinkish-purple colour of the pod and seed coat is due to high anthocyanin contents. While the normal sugar level in most pigeon pea cultivars is approximately 5%, the sugar content in ICP 7035 seeds is 8.8%. Decorticated dried split seeds measure 5-6 mm in diameter and 100 dried seeds weigh 19.2 g. The seed contains 19.6% protein, 27.4% dietary fibre, 33% starch and 67% carbohydrate, and has high amounts of copper, calcium, magnesium and phosphorous. Resistance to Pigeonpea sterility mosaic virus in ICP 7035 has a positive impact on yield as a result of negligible crop loss in endemic areas. In the absence of the disease, the yield of ICP 7035 is on a par with the yields of local cultivars. Recently, provisional approval was given for the release of this cultivar in SMD endemic areas of southern Karnataka

Sequencing Analysis of Genetic Loci for Resistance for Late Leaf Spot and Rust in Peanut (Arachis hypogaea L.)

The aim of this study was to identify candidate resistance genes for late leaf spot (LLS) and rust diseases in peanut (Arachis hypogaea L.). We used a double-digest restriction-site associated DNA sequencing (ddRAD-Seq) technique based on next-generation sequencing (NGS) for genotyping analysis across the recombinant inbred lines (RILs) derived from a cross between a susceptible line, TAG 24, and a resistant line, GPBD 4. A total of 171 SNPs from the ddRAD-Seq together with 282 markers published in the previous studies were mapped on a genetic map covering 1510.1 cM. Subsequent quantitative trait locus (QTL) analysis revealed major genetic loci for LLS and rust resistance on chromosomes A02 and A03, respectively. Heterogeneous inbred family-derived near isogenic lines and the pedigree of the resistant gene donor, A. cardenasii Krapov. & W.C. Greg., including the resistant derivatives of ICGV 86855 and VG 9514 as well as GPBD 4, were employed for whole-genome resequencing analysis. The results indicated the QTL candidates for LLS and rust resistance were located in 1.4- and 2.7-Mb genome regions on A02 and A03, respectively. In these regions, four and six resistance-related genes with deleterious mutations were selected as candidates for LLS and rust resistance, respectively. These delimited genomic regions may be beneficial in breeding programs aimed at improving disease resistance and enhancing peanut productivity