De novo assembly and characterization of the draft genome of the cashew (Anacardium occidentale L.).

Cashew is the second most important tree nut crop in the global market. Cashew is a diploid and heterozygous species closely related to the mango and pistachio. Its improvement by conventional breeding is slow due to the long juvenile phase. Despite the economic importance, very little genomics/transcriptomics information is available for cashew. In this study, the Oxford nanopore reads and Illumina reads were used for de novo assembly of the cashew genome. The hybrid assembly yielded a 356.6 Mb genome corresponding to 85% of the estimated genome size (419 Mb). The BUSCO analysis showed 91.8% of genome completeness. Transcriptome mapping showed 92.75% transcripts aligned with the assembled genome. Gene predictions resulted in the identification of 31,263 genes coding for a total of 35,000 gene isoforms. About 46% (165 Mb) of the cashew genome comprised of repetitive sequences. Phylogenetic analyses of the cashew with nine species showed that it was closely related to Mangifera indica. Analysis of cashew genome revealed 3104 putative R-genes. The first draft assembly of the genome, transcriptome and R gene information generated in this study would be the foundation for understanding the molecular basis of economic traits and genomics-assisted breeding in cashew.

Insights on Genetic Diversity, Population Structure, and Linkage Disequilibrium in Globally Diverse Coconut Accessions Using Genotyping-by-Sequencing.

Genotyping-by-sequencing (GBS) has emerged as a cost-effective approach for genome-wide discovery of single-nucleotide polymorphism (SNP) markers and high-throughput genotyping. In this study, 96 coconut palms, representing 16 accessions from globally diverse origins, were genotyped using the GBS strategy. A total of 10,835 high-quality SNPs, which were identified after stringent filtering, were utilized to assess genetic diversity, population structure, and linkage disequilibrium (LD) analyses. The polymorphism information content (PIC) values of SNPs ranged from 0.1 to 0.4, with a large proportion of SNPs (8633 nos.; 79.7%) having a higher PIC in the range of 0.3-0.4. The genetic diversity analysis revealed the existence of a high level of variation in coconut accessions, with an average expected heterozygosity (He) value of 0.43. Unweighted neighbor-joining phylogenetic tree and Bayesian-based model population structure grouped coconut genotypes into four main clusters. The accessions are generally clustered based on their height (tall or dwarf), with a few accession clusterings based on geographical origins. Investigation of LD pattern in coconut indicated a relatively rapid LD decay with a short range (9 kb). The results obtained in this study will contribute to enhancing the capacity of coconut researchers to utilize genetic diversity for further genetic improvement. In addition, it would open up possibilities for performing genomic studies such as genome-wide association studies and genomic selection to accelerate the efficiency and speed of coconut genetic improvement.

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.

Facile coconut inflorescence sap mediated synthesis of silver nanoparticles and its diverse antimicrobial and cytotoxic properties.

Green synthesis of nanoparticles (NPs) involves the use of diverse extracts of biological origin as substrates to synthesize NPs and can overcome the hazards associated with chemical methods. Coconut inflorescence sap, which is unfermented phloem sap obtained by tapping of coconut inflorescence, is a rich source of sugars and secondary metabolites. In this study, coconut inflorescence sap was used to synthesize silver NPs (AgNPs). We have initially undertaken metabolomic profiling of coconut inflorescence sap from West Coast Tall cultivar to delineate its individual components. It was found to comprise of 64% secondary metabolites, 9% sugars, 12% lipids/fats and 9% peptides in positive mode, whereas in the negative mode, it was 33, 20, 9 and 11%, respectively. The concentration of silver nitrate, inflorescence sap and incubation temperature for the synthesis of AgNPs were optimized. Incubating the reaction mixture at 40 °C was found to enhance AgNP synthesis. The AgNPs synthesized were characterized using UV-visible (UV-Vis) spectrophotometry, X-Ray Diffraction (XRD), Fourier Transform Infrared spectroscopy (FTIR) and Transmission Electron Microscopy (TEM). The particles were crystalline in nature and the bulk of the particles were spherical with smooth (thin) shell and poly-dispersed with a diameter ranging from 10 nm to 30 nm. Antimicrobial property of AgNPs was tested in tissue culture of arecanut (Areca catechu L.) where bacterial contamination (Bacillus pumilus) was a frequent occurrence. A significant reduction in the contamination was observed when plantlets were treated with aqueous solutions of AgNPs. Notably, treatment with AgNPs did not affect the growth and development of the arecanut plantlets. Antimicrobial properties of AgNPs synthesized from inflorescence sap were also evaluated in human pathogenic bacteria viz., Escherichia coli ATCC 25922; Salmonella Typhimurium ATCC 14028 and Vibrio parahaemolyticus AQ4037. The antibacterial action was confirmed by determining the production of reactive oxygen species (ROS) and protein leakage studies. Cytotoxicity of AgNPs was quantified in HeLa cells. The viability (%) of HeLa cells declined significantly at 10 mg L-1 concentration of AgNP and complete mortality was observed at a concentration of 60 mg L-1. The study concludes that unfermented inflorescence sap, with above neutral pH, serves as an excellent reducing agent to synthesize AgNPs from Ag+.

Transcriptome sequencing and de novo assembly in arecanut, Areca catechu L elucidates the secondary metabolite pathway genes.

Areca catechu L. belongs to the Arecaceae family which comprises many economically important palms. The palm is a source of alkaloids and carotenoids. The lack of ample genetic information in public databases has been a constraint for the genetic improvement of arecanut. To gain molecular insight into the palm, high throughput RNA sequencing and de novo assembly of arecanut leaf transcriptome was undertaken in the present study. A total 56,321,907 paired end reads of 101 bp length consisting of 11.343 Gb nucleotides were generated. De novo assembly resulted in 48,783 good quality transcripts, of which 67% of transcripts could be annotated against NCBI non - redundant database. The Gene Ontology (GO) analysis with UniProt database identified 9222 biological process, 11268 molecular function and 7574 cellular components GO terms. Large scale expression profiling through Fragments per Kilobase per Million mapped reads (FPKM) showed major genes involved in different metabolic pathways of the plant. Metabolic pathway analysis of the assembled transcripts identified 124 plant related pathways. The transcripts related to carotenoid and alkaloid biosynthetic pathways had more number of reads and FPKM values suggesting higher expression of these genes. The arecanut transcript sequences generated in the study showed high similarity with coconut, oil palm and date palm sequences retrieved from public domains. We also identified 6853 genic SSR regions in the arecanut. The possible primers were designed for SSR detection and this would simplify the future efforts in genetic characterization of arecanut.

De novo assembly and characterization of global transcriptome of coconut palm (Cocos nucifera L.) embryogenic calli using Illumina paired-end sequencing.

Production and supply of quality planting material is significant to coconut cultivation but is one of the major constraints in coconut productivity. Rapid multiplication of coconut through in vitro techniques, therefore, is of paramount importance. Although somatic embryogenesis in coconut is a promising technique that will allow for the mass production of high quality palms, coconut is highly recalcitrant to in vitro culture. In order to overcome the bottlenecks in coconut somatic embryogenesis and to develop a repeatable protocol, it is imperative to understand, identify, and characterize molecular events involved in coconut somatic embryogenesis pathway. Transcriptome analysis (RNA-Seq) of coconut embryogenic calli, derived from plumular explants of West Coast Tall cultivar, was undertaken on an Illumina HiSeq 2000 platform. After de novo transcriptome assembly and functional annotation, we have obtained 40,367 transcripts which showed significant BLASTx matches with similarity greater than 40 % and E value of ≤10(-5). Fourteen genes known to be involved in somatic embryogenesis were identified. Quantitative real-time PCR (qRT-PCR) analyses of these 14 genes were carried in six developmental stages. The result showed that CLV was upregulated in the initial stage of callogenesis. Transcripts GLP, GST, PKL, WUS, and WRKY were expressed more in somatic embryo stage. The expression of SERK, MAPK, AP2, SAUR, ECP, AGP, LEA, and ANT were higher in the embryogenic callus stage compared to initial culture and somatic embryo stages. This study provides the first insights into the gene expression patterns during somatic embryogenesis in coconut.

Genetic relationship and diversity among coconut (Cocos nucifera L.) accessions revealed through SCoT analysis.

Coconut (Cocos nucifera L.) is one of the important palms grown both as a homestead and plantation crop in countries and most island territories of tropical regions. Different DNA-based marker systems have been utilized to assess the extent of genetic diversity in coconut. Advances in genomics research have resulted in the development of novel gene-targeted markers. In the present study, we have used a simple and novel marker system, start codon targeted polymorphism (SCoT), for its evaluation as a potential marker system in coconut. SCoT markers were utilized for assessment of genetic diversity in 23 coconut accessions (10 talls and 13 dwarfs), representing different geographical regions. Out of 25 SCoT primers screened, 15 primers were selected for this study based on their consistent amplification patterns. A total of 102 scorable bands were produced by the 15 primers, 88 % of which were polymorphic. The scored data were used to construct a similarity matrix. The similarity coefficient values ranged between 0.37 and 0.91. These coefficients were utilized to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA). The extent of genetic diversity observed based on SCoT analysis of coconut accessions was comparable to earlier findings using other marker systems. Tall and dwarf coconut accessions were clearly demarcated, and in general, coconut accessions from the same geographical region clustered together. The results indicate the potential of SCoT markers to be utilized as molecular markers to detect DNA polymorphism in coconut accessions.

Accumulation of phenylpropanoid derivatives in chitosan-induced cell suspension culture of Cocos nucifera.

Chitosan-induced elicitation responses of dark-incubated Cocos nucifera (coconut) endosperm cell suspension cultures led to the rapid formation of phenylpropanoid derivatives, which essentially mimics the defense-induced biochemical changes in coconut palm as observed under in vivo conditions. An enhanced accumulation of p-hydroxybenzoic acid as the major wall-bound phenolics was evident. This was followed by p-coumaric acid and ferulic acid. Along with enhanced peroxidases activities in elicited lines, the increase in activities of the early phenylpropanoid pathway enzymes such as, phenylalanine ammonia lyase (PAL), p-coumaroyl-CoA ligase (4CL) and p-hydroxybenzaldehyde dehydrogenase (HBD) in elicited cell cultures were also observed. Furthermore, supplementation of specific inhibitors of PAL, C4H and 4CL in elicited cell cultures led to suppressed accumulation of p-hydroxybenzoic acid, which opens up interesting questions regarding the probable route of the biosynthesis of this phenolic acid in C. nucifera.

A database for coconut crop improvement.

UNLABELLED: Coconut crop improvement requires a number of biotechnology and bioinformatics tools. A database containing information on CG (coconut germplasm), CCI (coconut cultivar identification), CD (coconut disease), MIFSPC (microbial information systems in plantation crops) and VO (vegetable oils) is described. The database was developed using MySQL and PostgreSQL running in Linux operating system. The database interface is developed in PHP, HTML and JAVA. AVAILABILITY: http://www.bioinfcpcri.org.