De Novo Transcriptome Profiling for the Generation and Validation of Microsatellite Markers, Transcription Factors, and Database Development for Andrographis paniculata.

Andrographis paniculata belongs to the family Acanthaceae and is known for its medicinal properties owing to the presence of unique constituents belonging to the lactones, diterpenoids, diterpene glycosides, flavonoids, and flavonoid glycosides groups of chemicals. Andrographolide, a major therapeutic constituent of A. paniculata, is extracted primarily from the leaves of this plant and exhibits antimicrobial and anti-inflammatory activities. Using 454 GS-FLX pyrosequencing, we have generated a whole transcriptome profile of entire leaves of A. paniculata. A total of 22,402 high-quality transcripts were generated, with an average transcript length and N50 of 884 bp and 1007 bp, respectively. Functional annotation revealed that 19,264 (86%) of the total transcripts showed significant similarity with the NCBI-Nr database and were successfully annotated. Out of the 19,264 BLAST hits, 17,623 transcripts were assigned GO terms and distributed into three major functional categories: molecular function (44.62%), biological processes (29.19%), and cellular component (26.18%) based on BLAST2GO. Transcription factor analysis showed 6669 transcripts, belonging to 57 different transcription factor families. Fifteen TF genes that belong to the NAC, MYB, and bHLH TF categories were validated by RT PCR amplification. In silico analysis of gene families involved in the synthesis of biochemical compounds having medicinal values, such as cytochrome p450, protein kinases, heat shock proteins, and transporters, was completed and a total of 102 different transcripts encoding enzymes involved in the biosynthesis of terpenoids were predicted. Out of these, 33 transcripts belonged to terpenoid backbone biosynthesis. This study also identified 4254 EST-SSRs from 3661 transcripts, representing 16.34% of the total transcripts. Fifty-three novel EST-SSR markers generated from our EST dataset were used to assess the genetic diversity among eighteen A. paniculata accessions. The genetic diversity analysis revealed two distinct sub-clusters and all accessions based on the genetic similarity index were distinct from each other. A database based on EST transcripts, EST-SSR markers, and transcription factors has been developed using data generated from the present study combined with available transcriptomic resources from a public database using Meta transcriptome analysis to make genomic resources available in one place to the researchers working on this medicinal plant.

Comparative analysis of chromosome 2A molecular organization in diploid and hexaploid wheat.

Diploid A genome wheat species harbor immense genetic variability which has been targeted and proven useful in wheat improvement. Development and deployment of sequence-based markers has opened avenues for comparative analysis, gene transfer and marker assisted selection (MAS) using high throughput cost effective genotyping techniques. Chromosome 2A of wheat is known to harbor several economically important genes. The present study aimed at identification of genic sequences corresponding to full length cDNAs and mining of SSRs and ISBPs from 2A draft sequence assembly of hexaploid wheat cv. Chinese Spring for marker development. In total, 1029 primer pairs including 478 gene derived, 501 SSRs and 50 ISBPs were amplified in diploid A genome species Triticum monococcum and T. boeoticum identifying 221 polymorphic loci. Out of these, 119 markers were mapped onto a pre-existing chromosome 2A genetic map consisting of 42 mapped markers. The enriched genetic map constituted 161 mapped markers with final map length of 549.6 cM. Further, 2A genetic map of T. monococcum was anchored to the physical map of 2A of cv. Chinese Spring which revealed several rearrangements between the two species. The present study generated a highly saturated genetic map of 2A and physical anchoring of genetically mapped markers revealed a complex genetic architecture of chromosome 2A that needs to be investigated further.

Comparative RNA sequencing based transcriptome profiling of regular bearing and alternate bearing mango (Mangifera indica L.) varieties reveals novel insights into the regulatory mechanisms underlying alternate bearing.

OBJECTIVE: This study is to understand a comprehensive perspective on the molecular mechanisms underlying alternate bearing in mango (Mangifera indica L.) via transcriptome wide gene expression profiling of both regular and irregular mango varieties. RESULTS: Transcriptome data of regular (Neelam) and irregular (Dashehari) mango varieties revealed a total of 42,397 genes. Out of that 12,557 significantly differentially expressed genes were identified, of which 6453 were found to be up-regulated and 6104 were found to be down-regulated genes. Further, many of the common unigenes which were involved in hormonal regulation, metabolic processes, oxidative stress, ion homeostasis, alternate bearing etc. showed significant differences between these two different bearing habit varieties. Pathway analysis showed the highest numbers of differentially expressed genes were related with the metabolic processes (523). A total of 26 alternate bearing genes were identified and principally three genes viz; SPL-like gene (GBVX01015803.1), Rumani GA-20-oxidase-like gene (GBVX01019650.1) and LOC103420644 (GBVX01016070.1) were significantly differentially expressed (at log2FC and pval less than 0.05) while, only single gene (gbGBVW01004309.1) related with flowering was found to be differentially expressed. A total of 15 differentially expressed genes from three important pathways viz; alternate bearing, carbohydrate metabolism and hormone synthesis were validated using Real time PCR and results were at par with in silico analysis. CONCLUSIONS: Deciphering the differentially expressed genes (DEGs) and potential candidate genes associated with alternate bearing, hormone and carbohydrate metabolism pathways will help for illustrating the molecular mechanisms underlying the bearing tendencies in mango.

Comparative transcriptome analyses provide novel insights into the differential response of Pigeonpea (Cajanus cajan L.) and its wild relative (Cajanus platycarpus (Benth.) Maesen) to herbivory by Helicoverpa armigera (Hübner).

KEY MESSAGE: Deeper insights into the resistance response of Cajanus platycarpus were obtained based on comparative transcriptomics under Helicoverpa armigera infestation. Devastation by pod borer, Helicoverpa armigera is one of the major factors for stagnated productivity in Pigeonpea. Despite possessing a multitude of desirable traits including pod borer resistance, wild relatives of Cajanus spp. have remained under-utilized due to linkage drag and cross-incompatibility. Discovery and deployment of genes from them can provide means to tackle key pests like H. armigera. Transcriptomic differences between Cajanus platycarpus and Cajanus cajan during different time points (0, 18, 38, 96 h) of pod borer infestation were elucidated in this study. For the first ever time, we demonstrated captivating variations in their response; C. platycarpus apparently being reasonably agile with effectual transcriptomic reprogramming to deter the insect. Deeper insights into the differential response were obtained by identification of significant GO-terms related to herbivory followed by combined KEGG and ontology analyses. C. platycarpus portrayed a multilevel response with cardinal involvement of SAR, redox homeostasis and reconfiguration of primary metabolites leading to a comprehensive defense response. The credibility of RNA-seq analyses was ascertained by transient expression of selected putative insect resistance genes from C. platycarpus viz., chitinase (CHI4), Alpha-amylase/subtilisin inhibitor (IAAS) and Flavonoid 3_5 hydroxylase (C75A1) in Nicotiana benthamiana followed by efficacy analysis against H. armigera. qPCR validated results of the study provided innovative insights and useful leads for development of durable pod borer resistance.

De novo transcriptome sequencing facilitates genomic resource generation in Tinospora cordifolia.

Tinospora cordifolia is known for its medicinal properties owing to the presence of useful constituents such as terpenes, glycosides, steroids, alkaloids, and flavonoids belonging to secondary metabolism origin. However, there is little information available pertaining to critical genomic elements (ESTs, molecular markers) necessary for judicious exploitation of its germplasm. We employed 454 GS-FLX pyrosequencing of entire transcripts and altogether ∼25 K assembled transcripts or Expressed sequence tags (ESTs) were identified. As the interest in T. cordifolia is primarily due to its secondary metabolite constituents, the ESTs pertaining to terpenoids biosynthetic pathway were identified in the present study. Additionally, several ESTs were assigned to different transcription factor families. To validate our transcripts dataset, the novel EST-SSR markers were generated to assess the genetic diversity among germplasm of T. cordifolia. These EST-SSR markers were found to be polymorphic and the dendrogram based on dice similarity index revealed three distinct clustering of accessions. The present study demonstrates effectiveness in using both NEWBLER and MIRA sequence read assembler software for enriching transcript-dataset and thus enables better exploitation of EST resources for mining candidate genes and designing molecular markers.

Mitochondria in biology and medicine - 2023.

Mitochondria are an indispensable part of the cell that plays a crucial role in regulating various signaling pathways, energy metabolism, cell differentiation, proliferation, and cell death. Since mitochondria have their own genetic material, they differ from their nuclear counterparts, and dysregulation is responsible for a broad spectrum of diseases. Mitochondrial dysfunction is associated with several disorders, including neuro-muscular disorders, cancer, and premature aging, among others. The intricacy of the field is due to the cross-talk between nuclear and mitochondrial genes, which has also improved our knowledge of mitochondrial functions and their pathogenesis. Therefore, interdisciplinary research and communication are crucial for mitochondrial biology and medicine due to the challenges they pose for diagnosis and treatment. The ninth annual conference of the Society for Mitochondria Research and Medicine (SMRM)- India, titled "Mitochondria in Biology and Medicine" was organized at the Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India, on June 21-23, 2023. The latest advancements in the field of mitochondrial biology and medicine were discussed at the conference. In this article, we summarize the entire event for the benefit of researchers working in the field of mitochondrial biology and medicine.

Delineation of novel genomic loci and putative candidate genes associated with seed iron and zinc content in lentil (Lens culinaris Medik.).

The use of molecular breeding approaches for development of lentil genotypes biofortified with essential micro-nutrients such as iron and zinc, could serve as a promising solution to address the problem of global malnutrition. Thus, genome-wide association study (GWAS) strategy was adopted in this study to identify the genomic regions associated with seed iron and zinc content in lentil. A panel of 95 diverse lentil genotypes, grown across three different geographical locations and evaluated for seed iron and zinc content, exhibited a wide range of variation. Genotyping-by-sequencing (GBS) analysis of the panel identified 33,745 significant single nucleotide polymorphisms (SNPs) that were distributed across all the 7 lentil chromosomes. Association mapping revealed 23 SNPs associated with seed iron content that were distributed across all the chromosomes except chromosome 3. Similarly, 14 SNPs associated with seed zinc content were also identified that were distributed across chromosomes 1, 2, 4, 5 and 6. Further, 80 genes were identified in the proximity of iron associated markers and 36 genes were identified in the proximity of zinc associated markers. Functional annotation of these genes revealed their putative involvement in iron and zinc metabolism. For seed iron content, two highly significant SNPs were found to be located within two putative candidate genes namely iron-sulfur cluster assembly (ISCA) and flavin binding monooxygenase (FMO) respectively. For zinc content, a highly significant SNP was detected in a gene encoding UPF0678 fatty acid-binding protein. Expression analysis of these genes and their putative interacting partners suggests their involvement in iron and zinc metabolism in lentil. Overall, in this study we have identified markers, putative candidate genes and predicted putative interacting protein partners significantly associated with iron and zinc metabolism that could be utilized in future breeding studies of lentil for nutrient biofortification.

An Immobilized Form of a Blend of Essential Oils Improves the Density of Beneficial Bacteria, in Addition to Suppressing Pathogens in the Gut and Also Improves the Performance of Chicken Breeding.

Antimicrobial growth promoters (AGP) are used in chicken production to suppress pathogens in the gut and improve performance, but such products tend to suppress beneficial bacteria while favoring the development and spread of antimicrobial resistance. A green alternative to AGP with the ability to suppress pathogens, but with an additional ability to spare beneficial gut bacteria and improve breeding performance is urgently required. We investigated the effect of supplementation of a blend of select essential oils (cinnamon oil, carvacrol, and thyme oil, henceforth referred to as EO; at two doses: 200 g/t and 400 g/t feed) exhibiting an ability to spare Lactobacillus while exhibiting strong E. coli inhibition ability under in vitro tests and immobilized in a sunflower oil and calcium alginate matrix, to broiler chickens and compared the effects with those of a probiotic yeast (Y), an AGP virginiamycin (V), and a negative control (C). qPCR analysis of metagenomic DNA from the gut content of experimental chickens indicated a significantly (p < 0.05) lower density of E. coli in the EO groups as compared to other groups. Amplicon sequence data of the gut microbiome indicated that all the additives had specific significant effects (DESeq2) on the gut microbiome, such as enrichment of uncultured Clostridia in the V and Y groups and uncultured Ruminococcaceae in the EO groups, as compared to the control. LEfSe analysis of the sequence data indicated a high abundance of beneficial bacteria Ruminococcaceae in the EO groups, Faecalibacterium in the Y group, and Blautia in the V group. Supplementation of the immobilized EO at the dose rate of 400 g/ton feed improved body weight gain (by 64 g/bird), feed efficiency (by 5 points), and cellular immunity (skin thickness response to phytoheamagglutinin lectin from Phaseolus vulgaris by 58%) significantly (p < 0.05), whereas neither yeast nor virginiamycin showed a significant effect on performance parameters. Expression of genes associated with gut barrier and immunity function such as CLAUDIN1, IL6, IFNG, TLR2A, and NOD1 were significantly higher in the EO groups. This study showed that the encapsulated EO mixture can improve the density of beneficial microbes in the gut significantly, with concomitant suppression of potential pathogens such as E.coli and improved performance and immunity, and hence, has a high potential to be used as an effective alternative to AGP in poultry.

Amaranth Genomic Resource Database: an integrated database resource of Amaranth genes and genomics.

Amaranth (Amaranthus L.) is native to Mexico and North America, where it was cultivated thousands of years ago, but now amaranth is grown worldwide. Amaranth is one of the most promising food crops with high nutritional value and belongs to the family Amaranthaceae. The high-quality genome assembly of cultivated amaranth species (A. hypochondriacus, A. cruentus) and wild/weedy species (A. tuberculatus, A. hybridus, and A. palmeri) has already been reported; therefore, we developed an Amaranth Genomic Resource Database (AGRDB) to provide access to all the genomic information such as genes, SSRs, SNPs, TFs, miRNAs, and transporters in one place. The AGRDB database contains functionally annotated gene information with their sequence details, genic as well as genomic SSRs with their three sets of primers, transcription factors classified into different families with their sequence information and annotation details, putative miRNAs with their family, sequences, and targeted gene details, transporter genes with their superfamily, trans-membrane domain details, and details of genic as well as nongenic SNPs with 3' and 5' flanking sequence information of five amaranth species. A database search can be performed using the gene ID, sequence ID, sequence motif, motif repeat, family name, annotation keyword, scaffold or chromosome numbers, etc. This resource also includes some useful tools, including JBrowse for the visualization of genes, SSRs, SNPs, and TFs on the respective amaranth genomes and BLAST search to perform a BLAST search of the user's query sequence against the amaranth genome as well as protein sequences. The AGRDB database will serve as a potential platform for genetic improvement and characterization of this futuristic crop. The AGRDB database will be accessible via the link: http://www.nbpgr.ernet.in:8080/AmaranthGRD/.

High resolution mapping of QTLs for fruit color and firmness in Amrapali/Sensation mango hybrids.

Introduction: Mango (Mangifera indica L.), acclaimed as the 'king of fruits' in the tropical world, has historical, religious, and economic values. It is grown commercially in more than 100 countries, and fresh mango world trade accounts for ~3,200 million US dollars for the year 2020. Mango is widely cultivated in sub-tropical and tropical regions of the world, with India, China, and Thailand being the top three producers. Mango fruit is adored for its taste, color, flavor, and aroma. Fruit color and firmness are important fruit quality traits for consumer acceptance, but their genetics is poorly understood. Methods: For mapping of fruit color and firmness, mango varieties Amrapali and Sensation, having contrasting fruit quality traits, were crossed for the development of a mapping population. Ninety-two bi-parental progenies obtained from this cross were used for the construction of a high-density linkage map and identification of QTLs. Genotyping was carried out using an 80K SNP chip array. Results and discussion: Initially, we constructed two high-density linkage maps based on the segregation of female and male parents. A female map with 3,213 SNPs and male map with 1,781 SNPs were distributed on 20 linkages groups covering map lengths of 2,844.39 and 2,684.22cM, respectively. Finally, the integrated map was constructed comprised of 4,361 SNP markers distributed on 20 linkage groups, which consisted of the chromosome haploid number in Mangifera indica (n =20). The integrated genetic map covered the entire genome of Mangifera indica cv. Dashehari, with a total genetic distance of 2,982.75 cM and an average distance between markers of 0.68 cM. The length of LGs varied from 85.78 to 218.28 cM, with a mean size of 149.14 cM. Phenotyping for fruit color and firmness traits was done for two consecutive seasons. We identified important consistent QTLs for 12 out of 20 traits, with integrated genetic linkages having significant LOD scores in at least one season. Important consistent QTLs for fruit peel color are located at Chr 3 and 18, and firmness on Chr 11 and 20. The QTLs mapped in this study would be useful in the marker-assisted breeding of mango for improved efficiency.

The first draft of the pigeonpea genome sequence.

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety 'Asha'. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550 bp and >10-fold genome coverage, resulting in 510,809,477 bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable 'Arhar' simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement.

TinoTranscriptDB: A Database of Transcripts and Microsatellite Markers of Tinospora cordifolia, an Important Medicinal Plant.

Tinospora cordifolia, commonly known as "Giloe" in India, is a shrub belonging to the family Menispermaceae. It is an important medicinal plant known for its antipyretic, anti-inflammatory, antispasmodic, and antidiabetic properties and is used in the treatment of jaundice, gout, and rheumatism. Despite its economic importance, the limited information related to its genomic resources prohibits its judicious exploitation through molecular breeding or biotechnological approaches. In this study, we generated a meta-transcriptome assembly of 43,090 non-redundant transcripts by merging the RNASeq data obtained from Roche 454 GS-FLX, and Illumina platforms, and report the first transcriptome-based database for simple sequence repeats and transcription factors ("TinoTranscriptDB" (Tinospora cordifolia Transcriptome Database)). We annotated 26,716 (62%) of the total transcripts successfully from National Center for Biotechnology Information non-redundant protein (NCBI-NR), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-Prot, and Pfam databases. This database contains information of 2620 perfect simple sequence repeats (P-SSRs) with a relative abundance of 340.12 (loci/Mb), and relative density of 6309.29 (bp/Mb). Excluding mono-nucleotides, the most abundant SSR motifs were tri-nucleotides (54.31%), followed by di-nucleotides (37.51%), tetra-nucleotides (4.54%), penta-nucleotides (3.16%) and hexa-nucleotides (0.45%). Additionally, we also identified 4,311 transcription factors (TFs) and categorized them into 55 sub-families. This database is expected to fill the gap in genomic resource availability in T. cordifolia and thus accelerate molecular breeding and related functional and other applied studies aimed towards genetic improvements of T. cordifolia and related species.

Utility of Triti-Map for bulk-segregated mapping of causal genes and regulatory elements in Triticeae.

Triticeae species, including wheat, barley, and rye, are critical for global food security. Mapping agronomically important genes is crucial for elucidating molecular mechanisms and improving crops. However, Triticeae includes many wild relatives with desirable agronomic traits, and frequent introgressions occurred during Triticeae evolution and domestication. Thus, Triticeae genomes are generally large and complex, making the localization of genes or functional elements that control agronomic traits challenging. Here, we developed Triti-Map, which contains a suite of user-friendly computational packages specifically designed and optimized to overcome the obstacles of gene mapping in Triticeae, as well as a web interface integrating multi-omics data from Triticeae for the efficient mining of genes or functional elements that control particular traits. The Triti-Map pipeline accepts both DNA and RNA bulk-segregated sequencing data as well as traditional QTL data as inputs for locating genes and elucidating their functions. We illustrate the usage of Triti-Map with a combination of bulk-segregated ChIP-seq data to detect a wheat disease-resistance gene with its promoter sequence that is absent from the reference genome and clarify its evolutionary process. We hope that Triti-Map will facilitate gene isolation and accelerate Triticeae breeding.

A 62K genic-SNP chip array for genetic studies and breeding applications in pigeonpea (Cajanus cajan L. Millsp.).

Pigeonpea is the second most important pulse legume crop for food and nutritional security of South Asia that requires accelerated breeding using high throughput genomic tools. Single nucleotide polymorphisms (SNPs) are highly suitable markers for this purpose because of their bi-allelic nature, reproducibility and high abundance in the genome. Here we report on development and use of a pigeonpea 62 K SNP chip array 'CcSNPnks' for Affymetrix GeneTitan® platform. The array was designed after filtering 645,662 genic-SNPs identified by re-sequencing of 45 diverse genotypes and has 62,053 SNPs from 9629 genes belonging to five different categories, including 4314 single-copy genes unique to pigeonpea, 4328 single-copy genes conserved between soybean and pigeonpea, 156 homologs of agronomically important cloned genes, 746 disease resistance and defense response genes and 85 multi-copy genes of pigeonpea. This fully genic chip has 28.94% exonic, 33.04% intronic, 27.56% 5'UTR and 10.46% 3'UTR SNPs and incorporates multiple SNPs per gene allowing gene haplotype network analysis. It was used successfully for the analysis of genetic diversity and population structure of 95 pigeonpea varieties and high resolution mapping of 11 yield related QTLs for number of branches, pod bearing length and number of seeds per pod in a biparental RIL population. As an accurate high-density genotyping tool, 'CcSNPnks' chip array will be useful for high resolution fingerprinting, QTL mapping and genome wide as well as gene-based association studies in pigeonpea.

Allelic sequence variation in the Sub1A, Sub1B and Sub1C genes among diverse rice cultivars and its association with submergence tolerance.

Erratic rainfall leading to flash flooding causes huge yield losses in lowland rice. The traditional varieties and landraces of rice possess variable levels of tolerance to submergence stress, but gene discovery and utilization of these resources has been limited to the Sub1A-1 allele from variety FR13A. Therefore, we analysed the allelic sequence variation in three Sub1 genes in a panel of 179 rice genotypes and its association with submergence tolerance. Population structure and diversity analysis based on a 36-plex genome wide genic-SNP assay grouped these genotypes into two major categories representing Indica and Japonica cultivar groups with further sub-groupings into Indica, Aus, Deepwater and Aromatic-Japonica cultivars. Targetted re-sequencing of the Sub1A, Sub1B and Sub1C genes identfied 7, 7 and 38 SNPs making 8, 9 and 67 SNP haplotypes, respectively. Haplotype networks and phylogenic analysis revealed evolution of Sub1B and Sub1A genes by tandem duplication and divergence of the ancestral Sub1C gene in that order. The alleles of Sub1 genes in tolerant reference variety FR13A seem to have evolved most recently. However, no consistent association could be found between the Sub1 allelic variation and submergence tolerance probably due to low minor allele frequencies and presence of exceptions to the known Sub1A-1 association in the genotype panel. We identified 18 cultivars with non-Sub1A-1 source of submergence tolerance which after further mapping and validation in bi-parental populations will be useful for development of superior flood tolerant rice cultivars.

Assembly and Annotation of the Nuclear and Organellar Genomes of a Dwarf Coconut (Chowghat Green Dwarf) Possessing Enhanced Disease Resistance.

Coconut (Cocos nucifera L.), an important source of vegetable oil, nutraceuticals, functional foods, and housing materials, provides raw materials for a repertoire of industries engaged in the manufacture of cosmetics, soaps, detergents, paints, varnishes, and emulsifiers, among other products. The palm plays a vital role in maintaining and promoting the sustainability of farming systems of the fragile ecosystems of islands and coastal regions of the tropics. In this study, we present the genome of a dwarf coconut variety "Chowghat Green Dwarf" (CGD) from India, possessing enhanced resistance to root (wilt) disease. Utilizing short reads from the Illumina HiSeq 4000 platform and long reads from the Pacific Biosciences RSII platform, we have assembled the draft genome assembly of 1.93 Gb. The genome is distributed over 26,855 scaffolds, with ∼81.56% of the assembled genome present in scaffolds of lengths longer than 50 kb. About 77.29% of the genome was composed of transposable elements and repeats. Gene prediction yielded 51,953 genes, which upon stringent filtering, based on Annotation Edit Distance, resulted in 13,707 genes, which coded for 11,181 proteins. Among these, we gathered transcript level evidence for a total of 6828 predicted genes based on the RNA-Seq data from different coconut tissues, since they presented assembled transcripts within the genome annotation coordinates. A total of 112 nucleotide-binding and leucine-rich repeat loci, belonging to six classes, were detected. We have also undertaken the assembly and annotation of the CGD chloroplast and mitochondrial genomes. The availability of the dwarf coconut genome shall prove invaluable for deducing the origin of dwarf coconut cultivars, dissection of genes controlling plant habit and fruit color, and accelerated breeding for improved agronomic traits.

Detection of extended-spectrum beta-lactamase-producing E. coli and Klebsiella spp. in effluents of different hospitals sewage in Biratnagar, Nepal.

OBJECTIVE: This study was aimed to determine prevalence and resistance pattern like multidrug resistant (MDR) or ESBL nature of E. coli and Klebsiella spp. from various sewage drain samples with an idea to deliver baseline information that could be utilized for defining guidelines for the treatment of hospital sewages. RESULTS: Of 10 sewage samples analyzed, 7 (70%) contained E. coli while 6 (60%) contained Klebsiella. Except one sample, all positive samples contained both E. coli and Klebsiella spp. E. coli isolates were resistant to ampicillin, amoxicillin, cefoxitin, cefuroxime, and cefpodoxime; while 85.7% were resistant to amoxicillin/clavulanate, ceftazidime, cefotaxime and ceftriaxone. 71.4%, 57.1%, 42.9%, and 28.6% were resistant to aztreonam, trimethoprim/sulfamethoxazole, nitrofurantoin, and gentamicin. Most were sensitive to chloramphenicol, ofloxacin, ciprofloxacin, and azithromycin. 85.7% and 57.1% of E. coli were MDR and ESBL isolates, respectively. Klebsiella were resistant to ampicillin, amoxicillin, and amoxicillin/clavulanate. 83.4% of Klebsiella were resistant to cefoxitin. 66.7% of strains were resistant to cefuroxime, ceftazidime, cefotaxime, ceftriaxone, and cefpodoxime. Klebsiella showed 50% resistant to aztreonam and trimethoprim/sulfamethoxazole, while 33.3% were resistant to chloramphenicol, nitrofurantoin, ofloxacin, and ciprofloxacin. Klebsiella were sensitive to azithromycin and gentamicin. 66.7% and 33.3% of Klebsiella were MDR and ESBL isolates, respectively.

Solution-Processed Organic Field-Effect Transistors with High Performance and Stability on Paper Substrates.

High-performance operationally stable organic field-effect transistors were successfully fabricated on a PowerCoat HD 230 paper substrate with a TIPS-pentacene:polystyrene blend as the active layer and poly(4-vinylphenol)/HfO2 as the hybrid gate dielectric. The fabricated devices exhibited excellent p-channel characteristics with a maximum and av field effect mobility of 0.44 and 0.22(±0.11) cm2 V-1 s-1, respectively, av threshold voltage of 0.021(±0.63) V, and current on-off ratio of ∼105 while operating at -10 V. These devices exhibited remarkable stability under effects of gate bias stress and large number of repeated transfer scans with negligible performance spread. In addition, these devices displayed very stable electrical characteristics after long exposure periods to humidity and an excellent shelf life of more than 6 months in ambient environment. Thermal stress at high temperatures however deteriorates the device characteristics because of the generation and propagation of cracks in the active semiconductor crystals. Furthermore, novel paper-based phototransistors have been demonstrated with these devices.

Homeologue Specific Gene Expression Analysis of Two Vital Carbon Metabolizing Enzymes-Citrate Synthase and NADP-Isocitrate Dehydrogenase-from Wheat (Triticum aestivum L.) Under Nitrogen Stress : Homeologue Specific gene expression of CS and NADP-ICDH.

Citrate synthase (CS) and NADP-dependent isocitrate dehydrogenase (NADP-ICDH) have been considered as candidate enzymes to provide carbon skeletons for nitrogen assimilation, i.e., production of 2-oxoglutarate required by the glutamine synthetase/glutamate synthase cycle. The CS and NADP-ICDH cDNAs were encoded for polypeptides of 402 and 480 amino acids with an estimated molecular weight of 53.01 and 45 kDa and an isoelectric point of 9.08 and 5.98, respectively. Phylogenetic analysis of these proteins in wheat across kingdoms confirmed the close relationship with Aegilops tauschii and Hordeum vulgare. Further, their amino acid sequences were demonstrated to have some conserved motifs such as Mg2+ or Mn2 binding site, catalytic sites, NADP binding sites, and active sites. In-silico-identified genomic sequences for the three homeologues A, B, and Dof CS and NADP-ICDH were found to be located on long arm of chromosomes 5 and 3, and sequence analysis also revealed that the three homeologues consisted of 13 and 15 exons, respectively. The total expression analysis indicated that both genes are ubiquitously expressed in shoot and root tissues under chronic as well as transient nitrogen stress. However, they are differentially and contrastingly expressed but almost in a coordinated manner in both the tissues. Under chronic as well as transient stress, both the genes in shoot tissue showed downregulation, lowest at 6 h of transient stress. However, in the root tissue, trend was found opposite except with exceptions. Moreover, all the three homeologues of both the genes were transcribed differentially, and the ratio of the individual homeologues transcripts to total homeologues transcripts also varied with the tissue, i.e., shoots or roots, as well as with nitrogen stress treatments. Thus, cDNA as well as genomic sequence information, apparent expression at different time point of nitrogen stress, and coordination between these enzymes would be ultimately linked to nitrate assimilation and nitrogen use efficiency in wheat.

Comparative Genomics Reveals the High Copy Number Variation of a Retro Transposon in Different Magnaporthe Isolates.

Magnaporthe oryzae is one of the fungal pathogens of rice which results in heavy yield losses worldwide. Understanding the genomic structure of M. oryzae is essential for appropriate deployment of the blast resistance in rice crop improvement programs. In this study we sequenced two M. oryzae isolates, RML-29 (avirulent) and RP-2421 (highly virulent) and performed comparative study along with three publically available genomes of 70-15, P131, and Y34. We identified several candidate effectors (>600) and isolate specific sequences from RML-29 and RP-2421, while a core set of 10013 single copy orthologs were found among the isolates. Pan-genome analysis showed extensive presence and absence variations (PAVs). We identified isolate-specific genes across 12 isolates using the pan-genome information. Repeat analysis was separately performed for each of the 15 isolates. This analysis revealed ∼25 times higher copy number of short interspersed nuclear elements (SINE) in virulent than avirulent isolate. We conclude that the extensive PAVs and occurrence of SINE throughout the genome could be one of the major mechanisms by which pathogenic variability is emerging in M. oryzae isolates. The knowledge gained in this comparative genome study can provide understandings about the fungal genome variations in different hosts and environmental conditions, and it will provide resources to effectively manage this important disease of rice.