Integrated analysis of the metabolome and transcriptome provides insights into anthocyanin biosynthesis of cashew apple.

The cashew apple remains an underutilized agricultural product despite its abundance as a by-product of cashew nut production. Anthocyanins are water-soluble pigments responsible for red, purple, and blue hues in plant tissues and have various health-promoting properties. To investigate the anthocyanin biosynthesis in cashew apples, fruits with varying peel colors from three cultivars were subjected to integrative analyses with metabolomics and transcriptomics. Through a UPLC-ESI-MS/MS-based targeted metabolomics analysis, a total of 26 distinct anthocyanin compounds were identified in the fruits of the three cashew cultivars. Subsequent quantification revealed that Pelargonidin-3-O-galactoside, Petunidin-3-O-arabinoside, and Cyanidin-3-O-galactoside were the primary contributors responsible for the red pigmentation in cashew apple peels. Following transcriptomic analysis showed that the expression levels of anthocyanin biosynthetic genes were predominantly higher in the red cashew apples as compared to the other two cultivars. Moreover, correlation analysis revealed that eight potential transcription factors implicated in the regulation of anthocyanin biosynthesis. Among these, four transcription factors exhibited positive correlations with both anthocyanin contents and anthocyanin biosynthetic gene expression, while the remaining four transcription factors displayed negative correlations. These findings provide a comprehensive understanding of the molecular basis of anthocyanin biosynthesis in cashew apple peels.

Metabolomic and transcriptomic analyses provide insights into metabolic networks during cashew fruit development and ripening.

Cashew nut is a popular food around the world. The high-resolution profiles and dynamics of metabolomes in cashew fruits are poorly understood till now. In this study, we analyzed the temporal metabolome of cashew nut via a non-targeted method based on UHPLC-Q-Exactive-MS, and analyzed that of cashew apple via a widely targeted method based on UHPLC-QTRAP-MS/MS (MRM). Furthermore, we performed integrative analyses of temporal metabolome and transcriptome data, characterized the accumulation of specific metabolites, and identified the transcriptional changes during cashew fruit development. Specifically, we found that phosphatidylinositol species were the predominant fractions in the unsaturated glycerophospholipids, and we identified a transcription factor that was the potential regulator of phosphatidylinositol biosynthesis. Analysis of cashew apple revealed metabolic genes and transcription factors involved in sugar biosynthesis. Taken together, our results provide insights into metabolic networks during cashew fruit development and generate a valuable resource for further cashew breeding studies.

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.

Transcriptome profiling of cashew apples (Anacardium occidentale) genotypes reveals specific genes linked to firmness and color during pseudofruit development.

KEY MESSAGE: We found 34 and 71 key genes potentially involved in flavonoid biosynthesis and cell wall disassembly, respectively, which could be associated with specific peel coloration and softening of each genotype. Cashew apple (Anacardium occidentale) has a great economic importance worldwide due to its high nutritional value, peculiar flavor and aroma. During ripening, the peduncle develops different peel color and becomes quickly fragile due to its oversoftening, impacting its consumers' acceptance. In view of this, the understanding about its transcriptional dynamics throughout ripening is imperative. In this study, we performed a transcriptome sequencing of two cashew apple genotypes (CCP 76 and BRS 265), presenting different firmness and color peel, in the immature and ripe stages. Comparative transcriptome analysis between immature and ripe cashew apple revealed 4374 and 3266 differentially expressed genes (DEGs) to CCP 76 and BRS 265 genotypes, respectively. These genes included 71 and 34 GDEs involved in the cell wall disassembly and flavonoid biosynthesis, respectively, which could be associated with firmness loss and anthocyanin accumulation during cashew apple development. Then, softer peduncle of CCP 76 could be justified by down-regulated EXP and up-regulation of genes involved in pectin degradation (PG, PL and PAE) and in cell wall biosynthesis. Moreover, genes related to flavonoid biosynthesis (PAL, C4H and CHS) could be associated with early high accumulation of anthocyanin in red-peel peduncle of BRS 265. Finally, expression patterns of the selected genes were tested by real-time quantitative PCR (qRT-PCR), and the qRT-PCR results were consistent with transcriptome data. The information generated in this work will provide insights into transcriptome responses to cashew apple ripening and hence, it will be helpful for cashew breeding programs aimed at developing genotypes with improved quality traits.

Population dynamics and infestation of Holopothrips fulvus Morgan (Thysanoptera: Phlaeothripidae) in dwarf cashew genotypes.

The objective of this study was to evaluate the Holopothrips fulvus Morgan (Thysanoptera: Phlaeothripidae) population dynamics and to identify dwarf cashew genotypes less infested by the pest in 2015 and 2016, under field conditions. H. fulvus population evaluations were carried out by monthly observations in the plants and using a score scale varying from 0 to 4. H. fulvus infestation occurred from October to December, and in the cashew genotypes CAP 112/8, CAP 121/1, CAP 131/2, CAP 145/2, CAP 145/7, CAP 128/2, CAP 120/4, CAP 123/6, CAP 130/1, and CAP 157/2 was dependent on the flowering period of the crop in 2015. In 2016, there was dependence in all evaluated genotypes between H. fulvus infestation and the cashew flowering period. In 2015, no significant differences were observed between the evaluated genotypes regarding H. fulvus infestation. In 2016, genotypes CAP 105/5, CAP 143/7, CAP 150/3, CAP 155/2, CAP 158/8, CAP 161/7, CAP 163/8, CAP 31, CAP 71, CAP 92, CAP 113, CAP 120, CAP 155, CAP 165, CAP 106/1, CAP 111/2, CAP 127/3, CAP 157/2, and BRS 226 were less infested. H. fulvus occurs from October to December and we could identify the dwarf cashew genotypes less infested by the pest.

Detection and quantification of cashew in commercial tea products using High Resolution Melting (HRM) analysis.

Tea, a popular aromatic infusion and food supplement, prepared from Camellia sinensis (L.) Kuntze leaves, is often subjected to adulteration with various undeclared inorganic and plant-derived materials. Cashew (Anacardium occidentale L.) nut husk is one of the most common plant tea adulterants. To date, there are limited DNA-based technologies for tea authentication and quantitative detection of adulterants. Herein, we used a universal plant DNA barcoding marker coupled with High Resolution Melting (Bar-HRM) analysis to authenticate tea products from cashew ground nut. Additionally, cashew-specific markers coupled with HRM technology were used to detect and quantify adulteration of tea with cashew DNA. This methodology can reliably detect admixtures as low as 1% v/v cashew in commercial tea products. Overall, our results demonstrate that the HRM technology is a strong molecular approach in tea authentication, capable of detecting very low adulterations in DNA admixtures. PRACTICAL APPLICATION: In this study, we established the use of high-resolution DNA-based technologies for the detection of cashew adulteration in tea, even in very low quantities. The technology could be applied to a greater range of plant-based tea adulterants. This work is expected to facilitate the traceability and authenticity of tea products and form the basis for the development of strategies against fraudulent practices.

Identification and in silico bioinformatics analysis of PR10 proteins in cashew nut.

Proteins from cashew nut can elicit mild to severe allergic reactions. Three allergenic proteins have already been identified, and it is expected that additional allergens are present in cashew nut. pathogenesis-related protein 10 (PR10) allergens from pollen have been found to elicit similar allergic reactions as those from nuts and seeds. Therefore, we investigated the presence of PR10 genes in cashew nut. Using RNA-seq analysis, we were able to identify several PR10-like transcripts in cashew nut and clone six putative PR10 genes. In addition, PR10 protein expression in raw cashew nuts was confirmed by immunoblotting and liquid chromatography-mass spectrometry (LC-MS/MS) analyses. An in silico allergenicity assessment suggested that all identified cashew PR10 proteins are potentially allergenic and may represent three different isoallergens.

Plastome Sequencing of Ten Nonmodel Crop Species Uncovers a Large Insertion of Mitochondrial DNA in Cashew.

In plant evolution, intracellular gene transfer (IGT) is a prevalent, ongoing process. While nuclear and mitochondrial genomes are known to integrate foreign DNA via IGT and horizontal gene transfer (HGT), plastid genomes (plastomes) have resisted foreign DNA incorporation and only recently has IGT been uncovered in the plastomes of a few land plants. In this study, we completed plastome sequences for l0 crop species and describe a number of structural features including variation in gene and intron content, inversions, and expansion and contraction of the inverted repeat (IR). We identified a putative in cinnamon ( J. Presl) and other sequenced Lauraceae and an apparent functional transfer of to the nucleus of quinoa ( Willd.). In the orchard tree cashew ( L.), we report the insertion of an ∼6.7-kb fragment of mitochondrial DNA into the plastome IR. BLASTn analyses returned high identity hits to mitogenome sequences including an intact open reading frame. Using three plastome markers for five species of , we generated a phylogeny to investigate the distribution and timing of the insertion. Four species share the insertion, suggesting that this event occurred <20 million yr ago in a single clade in the genus. Our study extends the observation of mitochondrial to plastome IGT to include long-lived tree species. While previous studies have suggested possible mechanisms facilitating IGT to the plastome, more examples of this phenomenon, along with more complete mitogenome sequences, will be required before a common, or variable, mechanism can be elucidated.

Purification and Characterization of Anacardium occidentale (Cashew) Allergens Ana o 1, Ana o 2, and Ana o 3.

In this study a fast and simple purification procedure for the three known allergens from cashew (7S globulin Ana o 1, 11S globulin Ana o 2, and 2S albumin Ana o 3) is described. The purified allergens are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, glycoprotein stain, and protein identification. The purified proteins still bind IgE, and this IgE binding varied between different pools of patient serum. Ana o 1 was found to be a glycoprotein. Ana o 3 has been studied more in detail to identify both the small and large subunits, both displaying microheterogeneity, and epitope mapping of Ana o 3 has been performed.

A modified acidic approach for DNA extraction from plant species containing high levels of secondary metabolites.

Purified genomic DNA can be difficult to obtain from some plant species because of the presence of impurities such as polysaccharides, which are often co-extracted with DNA. In this study, we developed a fast, simple, and low-cost protocol for extracting DNA from plants containing high levels of secondary metabolites. This protocol does not require the use of volatile toxic reagents such as mercaptoethanol, chloroform, or phenol and allows the extraction of high-quality DNA from wild and cultivated tropical species.

Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.

Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.

Rapid screening for potential epitopes reactive with a polycolonal antibody by solution-phase H/D exchange monitored by FT-ICR mass spectrometry.

The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.

Transferability and characterization of microssatellite loci in Anacardium humile A. St. Hil. (Anacardiaceae).

Microsatellite markers were transferred from the cashew, Anarcadium occidentale, to Anacardium humile (Anacardiaceae), a Neotropical shrub from the Brazilian savanna, that produces an edible nut and pseudo-fruit. We tested 14 microsatellite primers from A. occidentale on A. humile. Polymorphism of each microsatellite locus was analyzed based on 58 individuals from three populations. Twelve loci amplified successfully and presented 2 to 9 alleles; expected heterozygosity ranged from 0.056 to 0.869. These 12 microsatellite loci provide a new tool for the generation of fundamental population genetic data for devising conservation strategies for A. humile.

Transferability and characterization of simple sequence repeat markers from Anacardium occidentale to A. humile (Anacardiaceae).

Use of molecular markers can be limited by the high cost and extensive time required for their development. Transfer of simple sequence repeat (SSR) markers reduces the cost and time limitations and has allowed the use of these markers in a larger number of species. We tested 11 SSR markers previously developed for Anacardium occidentale on A. humile. The 11 loci were successfully amplified in A. humile. All loci were polymorphic and generated a mean of 5.4 alleles per locus. The observed heterozygosity was lower than the expected heterozygosity under Hardy-Weinberg equilibrium for most loci, with mean values of 0.463 and 0.696, respectively. The endogamy coefficients were positive and significant for seven loci. However, the combined probability of paternity exclusion was high, and the combined probability of genetic identity was low. None of the pairs of loci were in linkage disequilibrium. The informative power of these loci demonstrates that they are suitable for studies of diversity and genetic structure of natural populations of A. humile. In addition, the loci are suitable for estimating gene flow between populations, assessing species crossing preferences, and performing interspecific comparisons.

Isolation, characterization, and determination of 1-O-trans-cinnamoyl-beta-D-glucopyranose in the epidermis and flesh of developing cashew apple (Anacardium occidentale L.) and four of its genotypes.

1-O-trans-Cinnamoyl-beta-d-glucopyranose was purified from cashew apple (Anacardium occidentale L.) juice and unambiguously characterized. Absent at the immature green stage, its concentration bursts at the turning and even more at the mature ripe stage, reaching 6.2 mg/100 g of fresh weight. Whatever the considered cashew apple genotype, this cinnamoyl glucoside ester was preferentially concentrated in the epidermis, which was 4-5 times richer than flesh, reaching 85 mg/100 g of fresh weight for skin of the Brazilian clone EMBRAPA 50. Entire cashew apples contained from 6 to 20 mg of 1-cinnamoylglucose/100 g, a concentration similar to that of red strawberry receptacle. Accumulation of such amounts in this false fruit remains to be explained.

Genetic analysis and historical perspective of cashew (Anacardium occidentale L.) introduction into India.

Cashew (Anacardium occidentale L.), introduced into India about 400 years ago, is distributed widely in the coastal regions as spontaneous populations as well as in cultivation. Despite the plant's commercial exploitation, little is known about its actual introduction and diversification. We attempted to construct these events by investigating the level of genetic variation and genetic structure of cashew populations collected from different geographical regions of India. A total of 91 individuals from four populations were analysed using AFLP markers and morphometric data. AFLP analysis based on 354 polymorphic loci revealed Indian cashew to have low but relatively substantial genetic diversity for an introduced species (H(E) = 0.262 and I(S) = 0.404). Twenty-seven qualitative and quantitative traits also revealed the existence of considerable morphometric variation (24% around the mean values). Bayesian cluster analysis based on AFLP data did not indicate the existence of definite population differentiation. Morphometric analysis allocated 12% variation among all four populations, whereas AFLP variation observed in cashew individuals was entirely within populations. The results, supporting the possibility of cashew having been introduced into India repeatedly over a period of time but at a single location (west coast), are discussed and their implications for germplasm management are described.

Ana o 3, an important cashew nut (Anacardium occidentale L.) allergen of the 2S albumin family.

BACKGROUND: Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. We have previously cloned and characterized major cashew allergens belonging to the vicilin and legumin families of seed storage proteins. OBJECTIVE: Here we set out to describe a third major cashew allergen, a 2S albumin. METHODS: The recombinant cashew 2S albumin was amplified from a cDNA library by means of PCR, sequenced, and expressed in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition immunoblotting was used to identify the corresponding native cashew nut proteins. The mass of affinity-purified native allergen was determined by means of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectroscopy. Patients' sera were used to probe solid-phase 2S albumin peptides to identify linear epitopes. RESULTS: The cloned allergen, designated Ana o 3, was identified as 2S albumin. MALDI-TOF mass spectroscopy of native Ana o 3 yielded a molecular mass of 12,598 d. Immunoblot analysis showed 21 (81%) of 26 sera from patients with cashew allergy were reactive. Three native Ana o 3 large-subunit isoforms with molecular weights ranging from approximately 6 to 10 kd were identified. Probing of overlapping synthetic Ana o 3 peptides with patients' sera identified 16 reactive peptides, 4 of which gave strong signals and one of which positionally overlaps linear epitopes in mustard and walnut allergenic 2S albumins. The overlapping cashew and walnut epitopes also share considerable homology. CONCLUSIONS: We conclude that this 2S albumin protein is a major allergen in cashew nut and demonstrates a possible basis for cross-reactivity with walnut 2S albumin.

Ty1-copia retrotransposon-based SSAP marker development in cashew (Anacardium occidentale L.).

The most popular retrotransposon-based molecular marker system in use at the present time is the sequence-specific amplification polymorphism (SSAP) system . This system exploits the insertional polymorphism of long terminal repeat (LTR) retrotransposons around the genome. Because the LTR sequence is used to design primers for this method, its successful application requires sequence information from the terminal region of the mobile elements . In this study, two LTR sequences were isolated from the cashew genome and used successfully to develop SSAP marker systems. These were shown to have higher levels of polymorphism than amplified fragment length polymorphic markers for this species.

Molecular detection of cashew husk (Anacardium occidentale) adulteration in market samples of dry tea (Camellia sinensis).

Species-specific PCR primers were developed from intergenic spacer regions of 5S ribosomal RNA genes and used successfully in the detection of adulteration of cashew husk (Anacardium occidentale L.) in tea [Camellia sinensis (L.) O. Kuntze] samples. This is the first report of detecting adulteration in tea using molecular tools. Application of this approach in detecting adulteration of other biological materials in tea, medicinal herbs and the composition of admixtures of ayurvedic herbs has been discussed.

Comparative assessment of DNA fingerprinting techniques (RAPD, ISSR and AFLP) for genetic analysis of cashew (Anacardium occidentale L.) accessions of India.

Nineteen cashew accessions were analysed with 50 random primers, 12 ISSR primers and 6 AFLP primer pairs to compare the efficiency and utility of these techniques for detecting variation in cashew germplasm. Each marker system could discriminate between all of the accessions, albeit with varied efficiency of polymorphism detection. AFLP exhibited maximum discrimination efficiency with a genotype index of 1. The utility of each molecular marker technique, expressed as marker index, was estimated as a function of average band informativeness and effective multiplex ratio. Marker index was calculated to be more than 10 times higher in AFLP than in RAPD and ISSR. Similarity matrices were determined based on the data generated by molecular and morphometric analyses, and compared for congruency. AFLP displayed no correspondence with RAPD and ISSR. Correlation between ISSR and RAPD similarity matrices was low but significant (r = 0.63; p < 0.005). The similarity matrix based on morphometric markers exhibited no correlation with any of the molecular markers. AFLP, with its superior marker utility, was concluded to be the marker of choice for cashew genetic analysis.