Exploring the functional profiles of odorant binding proteins crucial for sensing key odorants in the new leaves of coconut palms in Rhynchophorus ferrugineus.

The red palm weevil (RPW), Rhynchophorus ferrugineus (Curculionidae: Coleoptera) is a highly destructive global pest of coconut trees, with a preference for laying its eggs on new leaves. Females can identify where to lay eggs by using their sense of smell to detect specific odorants found in new leaves. In this study, we focused on the two odorants commonly found in new leaves by GC-MS: trans, trans-2,4-nonadienal and trans-2-nonenal. Our behavioral assays demonstrated a significant attraction of females to both of these odorants, with their electrophysiological responses being dose-dependent. Furthermore, we examined the expression patterns induced by these odorants in eleven RferOBP genes. Among them, RferOBP3 and RferOBP1768 exhibited the most significant and simultaneous upregulation. To further understand the role of these two genes, we conducted experiments with females injected with OBP-dsRNA. This resulted in a significant decrease in the expression of RferOBP3 and RferOBP1768, as well as impaired the perception of the two odorants. A fluorescence competitive binding assay also showed that both RferOBPs strongly bound to the odorants. Additionally, sequence analysis revealed that these two RferOBPs belong to the Minus-C family and possess four conserved cysteines. Molecular docking simulations showed strong interactions between these two RferOBPs and the odorant molecules. Overall, our findings highlight the crucial role of RferOBP3 and RferOBP1768 in the olfactory perception of the key odorants in coconut palm new leaves. This knowledge significantly improves our understanding of how RPW females locate sites for oviposition and lays the foundation for future research on the development of environmentally friendly pest attractants.

Variants in the mitochondrial genome sequence of Oryctes rhinoceros (Coleoptera: Scarabaeidae) infected with Oryctes rhinoceros nudivirus in oil palm and coconut plantations.

The CRB (coconut rhinoceros beetle) haplotype was classified into CRB-S and CRB-G, based on the presence of single nucleotide polymorphisms (SNPs) in the mitochondrial cox1 gene. Mitochondrial genomes (mitogenomes) are the most widely used genetic resources for molecular evolution, phylogenetics, and population genetics in relation to insects. This study presents the mitogenome CRB-G and CRB-S which were collected in Johor, Malaysia. The mitogenome of CRB-G collected from oil palm plantations in 2020 and 2021, and wild coconut palms in 2021 was 15,315 bp, 15,475 bp, and 17,275 bp, respectively. The CRB-S was discovered in coconut and oil palms in 2021, and its mitogenome was 15,484 bp and 17,142 bp, respectively. All the mitogenomes have 37 genes with more than 99% nucleotide sequence homology, except the CRB-G haplotype collected from oil palm in 2021 with 89.24% nucleotide sequence homology. The mitogenome of Johor CRBs was variable in the natural population due to its elevated mutation rate. Substitutions and indels in cox1, cox2, nad2 and atp6 genes were able to distinguish the Johor CRBs into two haplotypes. The mitogenome data generated in the present study may provide baseline information to study the infection and relationship between the two haplotypes of Johor CRB and OrNV in the field. This study is the first report on the mitogenomes of mixed haplotypes of CRB in the field.

A Preliminary Survey of Transfer RNA Modifications and Modifying Enzymes of the Tropical Plant Cocos nucifera L.

The coconut (Cocos nucifera L.) is a commercial crop widely distributed among coastal tropical regions. It provides millions of farmers with food, fuel, cosmetics, folk medicine, and building materials. Among these, oil and palm sugar are representative extracts. However, this unique living species of Cocos has only been preliminarily studied at molecular levels. Benefiting from the genomic sequence data published in 2017 and 2021, we investigated the transfer RNA (tRNA) modifications and modifying enzymes of the coconut in this survey. An extraction method for the tRNA pool from coconut flesh was built. In total, 33 species of modified nucleosides and 66 homologous genes of modifying enzymes were confirmed using a nucleoside analysis using high-performance liquid chromatography combined with high-resolution mass spectrometry (HPLC-HRMS) and homologous protein sequence alignment. The positions of tRNA modifications, including pseudouridines, were preliminarily mapped using a oligonucleotide analysis, and the features of their modifying enzymes were summarized. Interestingly, we found that the gene encoding the modifying enzyme of 2'-O-ribosyladenosine at the 64th position of tRNA (Ar(p)64) was uniquely overexpressed under high-salinity stress. In contrast, most other tRNA-modifying enzymes were downregulated with mining transcriptomic sequencing data. According to previous physiological studies of Ar(p)64, the coconut appears to enhance the quality control of the translation process when subjected to high-salinity stress. We hope this survey can help advance research on tRNA modification and scientific studies of the coconut, as well as thinking of the safety and nutritional value of naturally modified nucleosides.

Whole genome sequence analysis and in-vitro probiotic characterization of Bacillus velezensis FCW2 MCC4686 from spontaneously fermented coconut water.

In this study, the probiotic potential of B. velezensis FCW2, isolated from naturally fermented coconut water, was investigated by in vitro and genomic characterization. Our findings highlight key features of the bacterium which includes, antibacterial activity, high adhesive potential, aggregation capacity, production of nutrient secondary metabolites. In vivo safety assessment revealed no adverse effects on zebrafish. WGS data of B. velezensis FCW2 revealed a complete circular genome of 4,147,426 nucleotides and a GC content of 45.87%. We have identified 4059 coding sequence (CDS) genes that encode proteins involved in stress resistance, adhesion and micronutrient production. The genes responsible for producing secondary metabolites, exopolysaccharides, and other beneficial nutrients were identified. The KEGG and COG databases revealed that genes mainly involved amino acid metabolism, carbohydrate utilization, vitamin and cofactor metabolism, and biological adhesion. These findings suggest that B. velezensis FCW2 could be a putative probiotic in the development of fermented foods.

Genomic and metabolic properties of Staphylococcus gallinarum FCW1 MCC4687 isolated from naturally fermented coconut water towards GRAS assessment.

Staphylococcus gallinarum FCW1 was isolated from naturally fermented coconut-water and identified by biochemical and molecular methods. Probiotic characterization and safety assessment were conducted through a series of in vitro tests. A high survival rate was observed when the strain was tested for resistance to bile, lysozyme, simulated gastric and intestinal fluid, phenol, and different temperature and salt concentrations. The strain showed antagonism against some pathogens, was susceptible to all antibiotics tested except penicillin, and showed no hemolytic and DNase activity. Hydrophobicity, autoaggregation, biofilm formation, and antioxidation tests indicated that the strain possessed a high adhesive and antioxidant ability. Enzymatic activity was used to evaluate the metabolic capacities of the strain. In-vivo experiment on zebrafish was performed to check its safety status. The whole-genome sequencing indicated that the genome contained 2,880,305 bp with a GC content of 33.23%. The genome annotation confirmed the presence of probiotic-associated genes and genes for oxalate degradation, sulfate reduction, acetate metabolism, and ammonium transport in the FCW1 strain, adding to the theory that this strain may be helpful in treating kidney stones. This study revealed that the strain FCW1 might be an excellent potential probiotic in developing fermented coconut beverages and treating and preventing kidney stone disease.

Functional Characterization of Lactobacillus plantarum Isolated from Cow Milk and the Development of Fermented Coconut and Carrot Juice Mixed Beverage.

Screening probiotics are crucial for assessing their safety, security, and further production of functional foods for human health. The present study aimed to isolate and identify bacteria from raw cow's milk samples that exhibit health benefits upon consumption. We characterized the probiotic properties of Lactobacillus plantarum (also called Lactiplantibacillus plantarum) strains CMGC2 and CMJC7 isolated from cow milk by in vitro study. The strains exhibited tolerance to simulated gastric conditions and were further identified by 16S rRNA sequencing as Lactobacillus plantarum (L. plantarum) CMGC2 and CMJC7. Both isolates were evaluated in vitro for their probiotic attributes, viz. hydrophobicity, autoaggregation, co-aggregation, lysozyme tolerance, antibacterial activity, antibiotic susceptibility, hemolytic activity, and phenol tolerance. The isolates CMGC2 and CMJC7 showed excellent probiotic attributes; hence, both strains were selected to produce coconut and carrot juice mixed beverages (CCMB). The CCMB was evaluated for the pH, acid-production rate, and total viable bacterial counts. The results showed that the CCMB was an excellent medium for the growth of CMGC2 and CMJC7 as it supported adequate growth of organisms (8.93 CFU/mL and 8.68 CFU/mL, respectively) even after 48 h of incubation. In conclusion, CMGC2 and CMJC7 can be used to develop different beverages possessing nutritive and probiotic values, and these beverages can be used for producing unique products.

Universal, Rapid, and Visual Detection Methods for Phytoplasmas Associated with Coconut Lethal Yellowing Diseases Targeting 16S rRNA Gene Sequences.

Coconut lethal yellowing (LY) diseases caused by phytoplasmas are devastating diseases for coconut cultivation and seriously threaten the coconut industry around world. The phytoplasmas associated with the LY diseases belonged to six 16Sr groups containing 16SrI, 16SrIV, 16SrXI, 16SrXIV, 16SrXXII, and 16SrXXXII with comparatively higher variable levels. Conserved regions of the 16S rRNA genes of LY phytoplasmas belonging to the six 16Sr groups were obtained in the study. Based on the conserved region sequences of 16S rRNA genes, two sets of LAMP primers, Co-4 and Co-6, were designed and screened, and the rapid and visual detection methods universal for different groups LY phytoplasmas were established. The entire detection reactions of the universal detection methods could be completed with only 30 to 40 min of constant temperature amplification at 64°C, and the detection results were judged by the color changes of the reaction systems, which are convenient and quick. For the six groups of phytoplasmas, the estimated minimum detection limit range of the universal detection primers Co-4 and Co-6 were identical: 4.8 × 101 to 4.8 × 107 copies per 200 µl. The universal detection methods for the LY phytoplasmas established in the study are of great significance for the rapid diagnosis and identification and the efficient monitoring and early warning as well as the port inspection and quarantine of the LY phytoplasmas and their related diseases.

Physicochemical characterization and fatty acid profiles of testa oils from various coconut (Cocos nucifera L.) genotypes.

BACKGROUND: Cocos nucifera (L.) is an important plantation crop with immense but untapped nutraceutical potential. Despite its bioactive potential, the biochemical features of testa oils of various coconut genotypes are poorly understood. Hence, in this study, the physicochemical characteristics of testa oils extracted from six coconut genotypes - namely West Coast Tall (WCT), Federated Malay States Tall (FMST), Chowghat Orange Dwarf (COD), Malayan Yellow Dwarf (MYD), and two Dwarf × Dwarf (D × D hybrids) viz., Cameroon Red Dwarf (CRD) × Ganga Bondam Green Dwarf (GBGD) and MYD × Chowghat Green Dwarf (CGD) - were analyzed. RESULTS: The proportion of testa in the nuts (fruits) (1.29-3.42%), the proportion of oil in the testa (40.97-50.56%), and biochemical components in testa oils - namely proxidant elements Fe (34.17-62.48 ppm) and Cu (1.63-2.77 ppm), and the total phenolic content (6.84-8.67 mg GAE/100 g), and phytosterol content (54.66-137.73 mg CE/100 g) varied depending on the coconut genotypes. The saturated fatty acid content of testa oils (67.75 to 78.78%) was lower in comparison with that of coconut kernel oils. Similarly, the lauric acid (26.66-32.04%), myristic (18.31-19.60%), and palmitic acid (13.43-15.71%,) content of testa oils varied significantly in comparison with the coconut kernel oils (32-51%, 17-21% and 6.9-14%, respectively). Liquid chromatography-mass spectrometry (LC-MS) analysis revealed the presence of 18 phenolic acids in coconut testa oil. Multivariate analysis revealed the biochemical attributes that defined the principal components loadings. Hierarchical clustering analysis of the genotypes showed two distinct clusters. CONCLUSION: This study reveals the genotypic variations in the nutritionally important biochemical components of coconut testa oils. The relatively high concentration of polyunsaturated fatty acids (PUFA) and polyphenol content in testa oils warrant further investigation to explore their nutraceutical potential. © 2022 Society of Chemical Industry.

A new species of planthopper in the genus Bothriocera (Hemiptera: Auchenorrhyncha: Cixiidae) from coconut palm (Cocos nucifera) in Jamaica.

Recent survey work in Jamaica on palm-associated planthoppers seeks to identify putative vectors of the lethal yellowing phytoplasma. Herein, a new species of planthopper, Bothriocera harthi sp. n., is described from coconut palm. Molecular data for the cytochrome c oxidase subunit I (COI), 18S rRNA gene, histone 3 (H3) gene, and 28S rRNA gene is provided to support placement of the novel taxon in Bothriocera. These findings are important because it provides novel data to help better understand the diversity and evolution of this unique group of planthoppers.

Development and characterization of non-coding RNA-derived simple sequence repeat markers in coconut (Cocos nucifera L.).

Non-coding RNA (ncRNA)-based SSR markers are highly useful in molecular breeding as ncRNAs play a significant role in gene regulation. In the present study, for the first time in coconut, we have identified 597 ncRNA-derived SSR markers, including 509 long non-coding RNASSRs (lncRNASSRs) and 88 micro RNASSRs (miRNASSRs). Of these, 20 primers (10 each from lncRNA-SSR and miRNA-SSR) were selected, screened on 6 coconut accessions, and 50% produced polymorphic fragments. These 10 polymorphic primers were used for genotyping 96 palms of 16 coconut accessions, comprising eight tall and dwarf accessions each. The number of alleles ranged from 2 to 9 per SSR marker, with an average of 4.6 alleles per locus. The average heterozygosity and Shannon index were 0.5 and 1.1, respectively, suggesting that ncRNA-SSRs show high polymorphism level. Distance-based cluster analyses revealed that all the tall and dwarf accessions were differentiated and grouped in different clusters. The study demonstrates the usefulness of ncRNA-based SSR markers for assessing genetic diversity and genetic improvement in coconut.

Reference-aided full-length transcript assembly, cDNA cloning, and molecular characterization of coronatine-insensitive 1b (COI1b) gene in coconut (Cocos nucifera L.).

BACKGROUND: In the Philippines, 26% of the total agricultural land is devoted to coconut production making coconut one of the most valuable industrial crop in the country. However, the country's multimillion-dollar coconut industry is threatened by the outbreak of coconut scale insect (CSI) and other re-emerging insect pests promoting national research institutes to work jointly on developing new tolerant coconut varieties. Here, we report the cloning and characterization of coronatine-insensitive 1 (COI1) gene, one of the candidate insect defense genes, using 'Catigan Green Dwarf' (CATD) genome sequence assembly as reference. METHODS AND RESULTS: Two (2) splicing variants were identified and annotated-CnCOI1b-1 and CnCOI1b-2. The full-length cDNA of CnCOI1b-1 was 7919 bp with an ORF of 1176 bp encoding for a deduced protein of 391 amino acids while CnCOI1b-2 has 2360 bp full-length cDNA with an ORF of 1743 bp encoding a deduced protein of 580 amino acids. The 3D structural model for the two (2) isoforms were generated through homology modelling. Functional analysis revealed that both isoforms are involved in various physiological and developmental plant processes including defense response of plants to insects and pathogens. Phylogenetic analysis confirms high degree of COI1 protein conservation during evolution, especially among monocot species. Differential gene expression via qRT-PCR analysis revealed a seven-fold increase of COI1 gene expression in coconut post introduction of CSI relative to base levels. CONCLUSION: This study provided the groundwork for further research on the actual role of COI1 in coconut in response to insect damage. The findings of this study are also vital to facilitate the development of improved insect-resistant coconut varieties for vibrant coconut industry.

Genome-wide diversity analysis to infer population structure and linkage disequilibrium among Colombian coconut germplasm.

Genetic diversity and relatedness of accessions for coconut growing in Colombia was unknown until this study. Here we develop single nucleotide polymorphisms (SNPs) along the coconut genome based on Genotyping by Sequencing (GBS) with the goal of analyze the genetic diversity, population structure, and linkage disequilibrium (LD) of a diverse coconut panel consisting of 112 coconut accessions from the Atlantic and Pacific coasts of Colombia. A comprehensive catalog of approximately 40,000 SNPs with a minor allele frequency (MAF) of > 0.05 is presented. A total of 40,614 SNPs were found but only 19,414 anchored to chromosomes. Of these, 10,338 and 4606 were exclusive to the Atlantic and Pacific gene pools, respectively, and 3432 SNPs could differentiate both gene pools. A filtered subset of unlinked and anchored SNPs (1271) showed a population structure at K = 4, separating accessions from the Pacific and Atlantic coasts that can also be distinguished by palm height, as found in previous studies. The Pacific groups had a slow LD decay, low Fixation Index (Fst) and low nucleotide diversity (π), while the Atlantic group had slightly higher genetic diversity and faster LD decay. Genome-wide diversity analyses are of importance to promote germplasm conservation and breeding programs aimed at developing new cultivars better adapted to the region.

Transcriptome analysis reveals key defense-related genes upon SA induction in Cocos nucifera L.

BACKGROUND: Salicylic acid (SA) is an important regulator of genes involved in plant defense and pathogen-triggered systemic acquired resistance (SAR). Coconut is an important crop affected by several pathogens. Reported evidence suggests SA involvement in defense responses, including SAR in coconut. OBJECTIVE: To identified differentially expressed genes in leaf and root tissues of coconut plantlets, as a result of SA, that might be involved in coconut defense responses. METHODS: Comparative transcriptomic analysis by RNA-Seq of leaf and root tissues from in vitro coconut plantlets unexposed and exposed to SA 2.5 mM for 48 h. And in silico validation of gene expression by qRT-PCR. RESULTS: We identified 4615 and 3940 differentially expressed unigenes (DEUs) in leaf and root tissues respectively. Our GO analysis showed functional categories related to the induction of defense responses, such as "systemic acquired resistance" and highly enriched hormone categories, such as abscisic acid. The most abundant KEGG pathway in our results was "Biosynthesis of antibiotics". Our findings support that exogenous application of SA to plantlets induced the activation of PRs, RGAs, ICS2, NLTP2, PER4, TRXM and some WRKYs mediated by NPR1-dependent pathways. Also, we found DEUs, such as BZR1, HSL1, and WHY2 that support that SA could regulate defense-related genes through NPR1-independent pathways. CONCLUSION: The present study of massive data analysis carried out on coconut plantlets exposed to SA, generates valuable information that increases our understanding of defense molecular mechanisms in coconut and open new venues for research for the improvement of management of coconut diseases.

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.

High-quality reference genome sequences of two coconut cultivars provide insights into evolution of monocot chromosomes and differentiation of fiber content and plant height.

BACKGROUND: Coconut is an important tropical oil and fruit crop whose evolutionary position renders it a fantastic species for the investigation of the evolution of monocot chromosomes and the subsequent differentiation of ancient plants. RESULTS: Here, we report the assembly and annotation of reference-grade genomes of Cn. tall and Cn. dwarf, whose genome sizes are 2.40 Gb and 2.39 Gb, respectively. The comparative analysis reveals that the two coconut subspecies diverge about 2-8 Mya while the conserved Arecaceae-specific whole-genome duplication (ω WGD) occurs approximately 47-53 Mya. It additionally allows us to reconstruct the ancestral karyotypes of the ten ancient monocot chromosomes and the evolutionary trajectories of the 16 modern coconut chromosomes. Fiber synthesis genes in Cn. tall, related to lignin and cellulose synthesis, are found at a higher copy number and expression level than dwarf coconuts. Integrated multi-omics analysis reveals that the difference in coconut plant height is the result of altered gibberellin metabolism, with both the GA20ox copy number and a single-nucleotide change in the promoter together leading to the difference in plant height between Cn. tall and Cn. dwarf. CONCLUSION: We provide high-quality coconut genomes and reveal the genetic basis of trait differences between two coconuts through multi-omics analysis. We also reveal that the selection of plant height has been targeted for the same gene for millions of years, not only in natural selection of ancient plant as illustrated in coconut, but also for artificial selection in cultivated crops such as rice and maize.

Kopyor versus macapuno coconuts: are these two edible mutants of Southeast Asia the same?

MAIN CONCLUSION: Kopyor and macapuno are two coconut mutants from Southeast Asia that are often described erroneously or interchangeably mismatched due to a lack of research, so future studies are encouraged. Coconut (Cocos nucifera L.; Arecaceae), a widely distributed plant with popular culinary applications, especially of the endosperm, has several nutritional and medicinal benefits. Two coconut mutants are widely recognized in Southeast Asia, namely kopyor and macapuno, specifically in Indonesia and Philippines, respectively. Kopyor coconut is known for its brittle solid endosperm while macapuno coconut is known for its gelatinous solid endosperm. Both mutant types have many other synonyms in other countries. Over many decades, the biology of macapuno coconut, including endosperm anatomy, histology, cytology, physiology, and genetics have been described, while kopyor coconut is still understudied. However, some literature and websites erroneously describe kopyor as macapuno coconut, or consider them interchangeably, which is an unintentional consequence of insufficient scientific research on these coconut mutants. Additionally, in Indonesia, there is another local mutant in Banten called wax coconut ("kelapa lilin") that some researchers claim as the actual Indonesian macapuno coconut due to its strong resemblance to kopyor coconut. Unfortunately, wax coconut is not only understudied, it is rarely documented. Additional evidence of their differences, in terms of morphological, biochemical and genetic characteristics, is needed. Moreover, clear documentation will also be needed for a better comparison. Understanding the differences between kopyor and macapuno coconuts will not only help to further clarify their scientific description in the literature, but will also guide locals, researchers, and industries to characterize similar mutants, if found in specific regions, for future study and bioprospecting.

Anther Culture in Coconut (Cocos nucifera L.).

Doubled haploids have a high impact on the improvement of heterozygous crops through hybridization. Anther culture is a doubled haploid technique for producing homozygous lines. In coconut, a tree species reported to be recalcitrant for tissue culture, a successful doubled haploid protocol was established through anther culture. All the factors affecting androgenesis induction have been optimized. In this chapter, a stepwise protocol, from doubled haploid induction including palm selection, anther isolation, pretreatment, and culture initiation, up to plant regeneration and thereafter acclimatization of the regenerated plants, is described. Furthermore, the protocol for testing the anther-derived plants for the ploidy level is also presented.

Coconut genome assembly enables evolutionary analysis of palms and highlights signaling pathways involved in salt tolerance.

Coconut (Cocos nucifera) is the emblematic palm of tropical coastal areas all around the globe. It provides vital resources to millions of farmers. In an effort to better understand its evolutionary history and to develop genomic tools for its improvement, a sequence draft was recently released. Here, we present a dense linkage map (8402 SNPs) aiming to assemble the large genome of coconut (2.42 Gbp, 2n = 32) into 16 pseudomolecules. As a result, 47% of the sequences (representing 77% of the genes) were assigned to 16 linkage groups and ordered. We observed segregation distortion in chromosome Cn15, which is a signature of strong selection among pollen grains, favouring the maternal allele. Comparing our results with the genome of the oil palm Elaeis guineensis allowed us to identify major events in the evolutionary history of palms. We find that coconut underwent a massive transposable element invasion in the last million years, which could be related to the fluctuations of sea level during the glaciations at Pleistocene that would have triggered a population bottleneck. Finally, to better understand the facultative halophyte trait of coconut, we conducted an RNA-seq experiment on leaves to identify key players of signaling pathways involved in salt stress response. Altogether, our findings represent a valuable resource for the coconut breeding community.