Diagnostic KASP markers differentiate Vanilla planifolia, V. odorata, V. pompona, and their hybrids using leaf or cured pod tissues.

BACKGROUND: Vanilla is a globally important spice crop used in a variety of food, cosmetic, and pharmaceutical products. V. planifolia is the primary commercial species with V. x tahitensis also permissible for food use. Other aromatic species, including V. pompona, are used for food throughout Central and South America. Supply chain complexity hinders the vanilla bean industry and can lead to false claims of genetic and geographical origins to obtain higher prices. Beans of some species can be differentiated by experienced buyers, but hybrids and morphological differences caused by environmental variability or disease would best be resolved by diagnostic tests. METHODS AND RESULTS: Kompetitive Allele Specific Polymerase Chain Reaction is a widely used molecular marker that can genotype single nucleotide polymorphisms efficiently and inexpensively. Assays were designed to differentiate V. planifolia, V. x tahitensis, and V. pompona using publicly available vanilla genomics data. Ten KASP assays on chromosomes 1 through 7, the ITS region, and plastid-encoded rbcL gene successfully differentiated V. planifolia, V. odorata, and V. x tahitensis. Additional KASP assays on chromosomes 1 through 4, the ITS region, and rbcL gene successfully differentiated V. planifolia and V. pompona. Further, a method for extracting KASP-quality DNA from cured vanilla bean seeds was developed and successfully differentiated V. planifolia, V. odorata, V. x tahitensis, V. pompona, and their hybrids. CONCLUSION: The methods and results from this study can be used to identify interspecific hybrids, ensure the authenticity of cured vanilla beans, and reduce abuse within the vanilla supply chain.

The 'Tommy Atkins' mango genome reveals candidate genes for fruit quality.

BACKGROUND: Mango, Mangifera indica L., an important tropical fruit crop, is grown for its sweet and aromatic fruits. Past improvement of this species has predominantly relied on chance seedlings derived from over 1000 cultivars in the Indian sub-continent with a large variation for fruit size, yield, biotic and abiotic stress resistance, and fruit quality among other traits. Historically, mango has been an orphan crop with very limited molecular information. Only recently have molecular and genomics-based analyses enabled the creation of linkage maps, transcriptomes, and diversity analysis of large collections. Additionally, the combined analysis of genomic and phenotypic information is poised to improve mango breeding efficiency. RESULTS: This study sequenced, de novo assembled, analyzed, and annotated the genome of the monoembryonic mango cultivar 'Tommy Atkins'. The draft genome sequence was generated using NRGene de-novo Magic on high molecular weight DNA of 'Tommy Atkins', supplemented by 10X Genomics long read sequencing to improve the initial assembly. A hybrid population between 'Tommy Atkins' x 'Kensington Pride' was used to generate phased haplotype chromosomes and a highly resolved phased SNP map. The final 'Tommy Atkins' genome assembly was a consensus sequence that included 20 pseudomolecules representing the 20 chromosomes of mango and included ~ 86% of the ~ 439 Mb haploid mango genome. Skim sequencing identified ~ 3.3 M SNPs using the 'Tommy Atkins' x 'Kensington Pride' mapping population. Repeat masking identified 26,616 genes with a median length of 3348 bp. A whole genome duplication analysis revealed an ancestral 65 MYA polyploidization event shared with Anacardium occidentale. Two regions, one on LG4 and one on LG7 containing 28 candidate genes, were associated with the commercially important fruit size characteristic in the mapping population. CONCLUSIONS: The availability of the complete 'Tommy Atkins' mango genome will aid global initiatives to study mango genetics.

Development of species-specific molecular markers in Vanilla for seedling selection of hybrids.

Vanilla planifolia is the primary botanical source of vanilla extract used globally in various foods and beverages. V. planifolia has a global distribution based on a few foundational clones and therefore has limited genetic diversity. Many Vanilla species easily hybridize with V. planifolia and could be a source of valuable genetic traits like increased vanillin content, disease resistance, or early flowering. While breeding Vanilla hybrids may improve plant performance, basic molecular tools for this species are lacking. DNA-based molecular markers are the most efficient method to validate hybrid progeny, detect hybrids in commercial plantings, and identify unknown accessions. This study used publicly available sequence data to develop species-specific, qRT-PCR-based molecular markers for Vanilla. Over 580,000 assembled sequence fragments were filtered for species specificity and twenty-two targets were selected for qRT-PCR screening. Ten targets differentially amplified among V. planifolia, V. pompona, V. phaeantha, and V. palmarum with ΔCT values as high as 17.58 between species. The ten targets were used to validate the parentage of hybrid progeny from controlled crosses with most hybrid progeny showing amplification patterns similar to both parents. The ten targets were also used to screen sixteen Vanilla species for specificity, and supported species assignments for unknown accessions including the detection of putative hybrids. This is the first report using species-specific, qRT-PCR-based molecular markers in Vanilla. These markers are inexpensive, simple to develop, and can rapidly screen large populations. These methods will enable the further development of species-specific molecular markers when creating Vanilla interspecific hybrid populations.

Identification of a methyltransferase catalyzing the final step of methyl anthranilate synthesis in cultivated strawberry.

BACKGROUND: Methyl anthranilate (MA) contributes an attractive fruity note to the complex flavor and aroma of strawberry (Fragaria spp.), yet it is rare in modern cultivars. The genetic basis for its biosynthesis has not been elucidated. Understanding the specific genes required for its synthesis could allow  the development of gene/allele-specific molecular markers to speed breeding of flavorful strawberries. RESULTS: Ripe fruits from individuals in an F1 population resulting from a cross between a MA producer and a non-producer were examined using a bulk-segregant transcriptome approach. MA producer and non-producer transcriptomes were compared, revealing five candidate transcripts that strictly co-segregated with MA production. One candidate encodes an annotated methyltransferase. MA levels are lower when this transcript is suppressed with RNAi, and bacterial cultures expressing the protein produced MA in the presence of anthranilic acid. Frozen fruit powders reconstituted with anthranilic acid and a methyl donor produced MA only if the transcript was detected in the fruit powder. A DNA-based molecular marker was developed that segregates with the MA-producing gene variant. CONCLUSIONS: These analyses indicate that the methyltransferase, now noted ANTHRANILIC ACID METHYL TRANSFERASE (FanAAMT), mediates the ultimate step of MA production in cultivated strawberry. Identification of this gene and its associated molecular marker may hasten breeding efforts to introduce this important volatile into modern cultivars.

Identification of candidate flavonoid pathway genes using transcriptome correlation network analysis in ripe strawberry (Fragaria × ananassa) fruits.

New modulators of the strawberry flavonoid pathway were identified through correlation network analysis. The transcriptomes of red, ripe fruit from two parental lines and 14 of their progeny were compared, and uncharacterized transcripts matching the expression patterns of known flavonoid-pathway genes were identified. Fifteen transcripts corresponded with putative transcription factors, and several of these were examined experimentally using transient expression in developing fruits. The results suggest that two of the newly-identified regulators likely contribute to discrete nodes of the flavonoid pathway. One increases only LEUCOANTHOCYANIDIN REDUCTASE (LAR) and FLAVONOL 3'-HYDROXYLASE (F3'H) transcript accumulation upon overexpression. Another affects LAR and FLAVONOL SYNTHASE (FLS) after overexpression. The third putative transcription factor appears to be a universal regulator of flavonoid-pathway genes, as many pathway transcripts decrease in abundance when this gene is silenced. This report demonstrates that such systems-level approaches may be especially powerful when connected to an effective transient expression system, helping to provide rapid and strong evidence of gene function in key fruit-ripening processes.

Iridium-catalyzed reductive nitro-Mannich cyclization.

A new chemoselective reductive nitro-Mannich cyclization reaction sequence of nitroalkyl-tethered lactams has been developed. Relying on the rapid and chemoselective iridium(I)-catalyzed reduction of lactams to the corresponding enamine, subsequent nitro-Mannich cyclization of tethered nitroalkyl functionality provides direct access to important alkaloid natural-product-like structures in yields up to 81 % and in diastereoselectivities that are typically good to excellent. An in-depth understanding of the reaction mechanism has been gained through NMR studies and characterization of reaction intermediates. The new methodology has been applied to the total synthesis of (±)-epi-epiquinamide in four steps.

Identification of a strawberry flavor gene candidate using an integrated genetic-genomic-analytical chemistry approach.

BACKGROUND: There is interest in improving the flavor of commercial strawberry (Fragaria × ananassa) varieties. Fruit flavor is shaped by combinations of sugars, acids and volatile compounds. Many efforts seek to use genomics-based strategies to identify genes controlling flavor, and then designing durable molecular markers to follow these genes in breeding populations. In this report, fruit from two cultivars, varying for presence-absence of volatile compounds, along with segregating progeny, were analyzed using GC/MS and RNAseq. Expression data were bulked in silico according to presence/absence of a given volatile compound, in this case γ-decalactone, a compound conferring a peach flavor note to fruits. RESULTS: Computationally sorting reads in segregating progeny based on γ-decalactone presence eliminated transcripts not directly relevant to the volatile, revealing transcripts possibly imparting quantitative contributions. One candidate encodes an omega-6 fatty acid desaturase, an enzyme known to participate in lactone production in fungi, noted here as FaFAD1. This candidate was induced by ripening, was detected in certain harvests, and correlated with γ-decalactone presence. The FaFAD1 gene is present in every genotype where γ-decalactone has been detected, and it was invariably missing in non-producers. A functional, PCR-based molecular marker was developed that cosegregates with the phenotype in F1 and BC1 populations, as well as in many other cultivars and wild Fragaria accessions. CONCLUSIONS: Genetic, genomic and analytical chemistry techniques were combined to identify FaFAD1, a gene likely controlling a key flavor volatile in strawberry. The same data may now be re-sorted based on presence/absence of any other volatile to identify other flavor-affecting candidates, leading to rapid generation of gene-specific markers.

Pathway-Based Metabolomics Analysis Reveals Biosynthesis of Key Flavor Compounds in Mango.

Mango is a tropical fruit with global demand as a result of its high sensory quality and nutritional attributes. Improving fruit quality at the consumer level could increase demand, but fruit quality is a complex trait requiring a deep understanding of flavor development to uncover key pathways that could become targets for improving sensory quality. Here, a pathway-based metabolomics (untargeted and targeted) approach was used to explore biosynthetic mechanisms of key flavor compounds with five core metabolic pathways (butanoate metabolism, phenylalanine biosynthesis and metabolism, terpenoid backbone biosynthesis, linoleic and linolenic acid pathway, and carbon fixation and sucrose metabolism) in three mango cultivars. The relationships between flavor precursors and flavor compounds were identified using correlation analysis. With these novel strategies, differentially regulated metabolic flux through the pathways was first elucidated, demonstrating possible mechanisms of key flavor formation and regulation in mango fruits.

Functional Characteristics of Aldehyde Dehydrogenase and Its Involvement in Aromatic Volatile Biosynthesis in Postharvest Banana Ripening.

Butanol vapor feeding to ripe banana pulp slices produced abundant butyl butanoate, indicating that a portion of butanol molecules was converted to butanoate/butanoyl-CoA via butanal, and further biosynthesized to ester. A similar phenomenon was observed when feeding propanol and pentanol, but was less pronounced when feeding hexanol, 2-methylpropanol and 3-methylbutanol. Enzymes which catalyze the cascade reactions, such as alcohol dehydrogenase (ADH), acetyl-CoA synthetase, and alcohol acetyl transferase, have been well documented. Aldehyde dehydrogenase (ALDH), which is presumed to play a key role in the pathway to convert aldehydes to carboxylic acids, has not been reported yet. The conversion is an oxygen-independent metabolic pathway and is enzyme-catalyzed with nicotinamide adenine dinucleotide (NAD+) as the cofactor. Crude ALDH was extracted from ripe banana pulps, and the interference from ADH was removed by two procedures: (1) washing off elutable proteins which contain 95% of ADH, but only about 40% of ALDH activity, with the remaining ALDH extracted from the pellet residues at the crude ALDH extraction stage; (2) adding an ADH inhibitor in the reaction mixture. The optimum pH of the ALDH was 8.8, and optimum phosphate buffer concentration was higher than 100 mM. High affinity of the enzyme was a straight chain of lower aldehydes except ethanal, while poor affinity was branched chain aldehydes.

Rapid Structure-Based Annotation and Profiling of Dihydrochalcones in Star Fruit (Averrhoa carambola) Using UHPLC/Q-Orbitrap-MS and Molecular Networking.

Dihydrochalcones are a subclass of flavonoids. There has been growing interest in dihydrochalcones for their health benefits and potential to modulate flavor, but their comprehensive profile in diverse plant species is lacking. Star fruit is a tropical fruit rich in dihydrochalcones. In this study, a systematic annotation using UHPLC/Q-Orbitrap-MS and molecular networking was established to rapidly identify dihydrochalcones in 12 star fruit cultivars. A total of 53 dihydrochalcones were characterized within a short retention time including one novel compound (phloretin-3'-C-(2-O-trans-p-coumaroyl)-ß-d-fucopyranoside) and 23 compounds identified from the Averrhoa genus for the first time. 3-Hydroxyphloretin was the most abundant dihydrochalcone in star fruit. All the identified dihydrochalcones had a higher abundance in leaves compared to fruits. This is the first report that systematically investigates dihydrochalcones in star fruit of multiple cultivars, and the results could provide a useful reference for the future development and utilization of plant genetic resources.

Genotyping-By-Sequencing diversity analysis of international Vanilla collections uncovers hidden diversity and enables plant improvement.

Genomics-based diversity analysis of natural vanilla populations is important in order to guide conservation efforts and genetic improvement through plant breeding. Vanilla is a cultivated, undomesticated spice that originated in Mesoamerica prior to spreading globally through vegetative cuttings. Vanilla extract from the commercial species, mainly V. planifolia and V. × tahitensis, is used around the world as an ingredient in foods, beverages, cosmetics, and pharmaceuticals. The global reliance on descendants of a few foundational clones in commercial production has resulted in an industry at heightened risk of catastrophic failure due to extremely narrow genetic diversity. Conversely, national and institutional collections including those near the center of cultivation contain previously undiscovered diversity that could bolster the genetic improvement of vanilla and guide conservation efforts. Towards this goal, an international vanilla genotyping effort generated and analyzed 431,204 single nucleotide polymorphisms among 412 accessions and 27 species from eight collections. Phylogenetic and STRUCTURE analysis sorted vanilla by species and identified hybrid accessions. Principal Component Analysis and the Fixation Index (FST) were used to refine relationships among accessions and showed differentiation among species. Analysis of the commercial species split V. planifolia into three types with all V. × tahitensis accessions being most similar to V. planifolia type 2. Finally, an in-depth analysis of V. × tahitensis identified seven V. planifolia and six V. odorata accessions as most similar to the estimated parental genotypes providing additional data in support of the current hybrid theory. The prevalence of probable V. × tahitensis parental accessions from Belize suggests that V. × tahitensis could have originated from this area and highlights the need for vanilla conservation throughout Central and South America. The genetic groupings among accessions, particularly for V. planifolia, can now be used to focus breeding efforts on fewer accessions that capture the greatest diversity.

Streptomyces scabies 87-22 contains a coronafacic acid-like biosynthetic cluster that contributes to plant-microbe interactions.

Plant-pathogenic Streptomyces spp. cause scab disease on economically important root and tuber crops, the most important of which is potato. Key virulence determinants produced by these species include the cellulose synthesis inhibitor, thaxtomin A, and the secreted Nec1 protein that is required for colonization of the plant host. Recently, the genome sequence of Streptomyces scabies 87-22 was completed, and a biosynthetic cluster was identified that is predicted to synthesize a novel compound similar to coronafacic acid (CFA), a component of the virulence-associated coronatine phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Southern analysis indicated that the cfa-like cluster in S. scabies 87-22 is likely conserved in other strains of S. scabies but is absent from two other pathogenic streptomycetes, S. turgidiscabies and S. acidiscabies. Transcriptional analyses demonstrated that the cluster is expressed during plant-microbe interactions and that expression requires a transcriptional regulator embedded in the cluster as well as the bldA tRNA. A knockout strain of the biosynthetic cluster displayed a reduced virulence phenotype on tobacco seedlings compared with the wild-type strain. Thus, the cfa-like biosynthetic cluster is a newly discovered locus in S. scabies that contributes to host-pathogen interactions.

Recombineering using RecTE from Pseudomonas syringae.

In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.

A phased Vanilla planifolia genome enables genetic improvement of flavour and production.

The global supply of vanilla extract is primarily sourced from the cured beans of the tropical orchid species Vanilla planifolia. Vanilla plants were collected from Mesoamerica, clonally propagated and globally distributed as part of the early spice trade. Today, the global food and beverage industry depends on descendants of these original plants that have not generally benefited from genetic improvement. As a result, vanilla growers and processors struggle to meet global demand for vanilla extract and are challenged by inefficient and unsustainable production practices. Here, we report a chromosome-scale, phased V. planifolia genome, which reveals sequence variants for genes that may impact the vanillin pathway and therefore influence bean quality. Resequencing of related vanilla species, including the minor commercial species Vanilla × tahitensis, identified genes that could impact productivity and post-harvest losses through pod dehiscence, flower anatomy and disease resistance. The vanilla genome reported in this study may enable accelerated breeding of vanilla to improve high-value traits.

A Highly Sensitive Method to Detect Avocado Sunblotch Viroid for the Maintenance of Infection-Free Avocado Germplasm Collections.

The United States Department of Agriculture (USDA) Agricultural Research Service (ARS) Subtropical Horticulture Research Station (SHRS) in Miami, FL holds a large germplasm collection of avocado (Persea americana). The recent threat of infection by laurel wilt has encouraged the creation of a backup collection at a disease-free site. Creating the backup collection is complicated by infection of some trees in the germplasm collection with avocado sunblotch viroid (ASBVd). Infected trees are frequently asymptomatic, necessitating the use of a molecular diagnostic assay. Although a reverse-transcription based assay already exists and has been used to assay all germplasm at the station, some trees showed inconsistent results. We have developed a more sensitive and specific assay involving pre-amplification of the entire viroid cDNA followed by detection using real-time PCR and a TaqMan assay. A second screening of all germplasm identified additional ASBVd -infected trees and allowed us to confidently remove these trees from the station. This method enables avocado germplasm curators to proceed with the creation of a viroid-free backup collection.

Relationship between Sensory Attributes and Chemical Composition of Different Mango Cultivars.

The present study investigated the relationship between the chemical composition and sensory quality of different mango ( Mangifera indica L.) cultivars by multivariate statistical analysis. The results showed that the high hedonic rating of mangoes was due in part to its flavor profile such as fruity, pineapple, and coconut with sweetness. High hedonic liking and positive flavors of mangoes could be responsible for the volatile compounds including fruity esters, 1-octanol, ( E, Z)-2,6-nonadienal, and γ-octalactone with high contents of sugars. On the other hand, turpentine-like and green flavors of mangoes are attributed to the relatively low hedonic liking of mangoes, which correlated with high contents of amino acids and terpenes. These findings demonstrated that interaction between individual chemical compounds within mangoes could be responsible for the specific sensory qualities of mango cultivars and provided insight into a paradigm for the selection and development of new and more desirable mango cultivars in the future.

Genomics-based diversity analysis of Vanilla species using a Vanilla planifolia draft genome and Genotyping-By-Sequencing.

Demand for all-natural vanilla flavor is increasing, but its botanical source, Vanilla planifolia, faces critical challenges arising from a narrow germplasm base and supply limitations. Genomics tools are the key to overcoming these limitations by enabling advanced genetics and plant breeding for new cultivars with improved yield and quality. The objective of this work was to establish the genomic resources needed to facilitate analysis of diversity among Vanilla accessions and to provide a resource to analyze other Vanilla collections. A V. planifolia draft genome was assembled and used to identify 521,732 single nucleotide polymorphism (SNP) markers using Genotyping-By-Sequencing (GBS). The draft genome had a size of 2.20 Gb representing 97% of the estimated genome size. A filtered set of 5,082 SNPs was used to genotype a living collection of 112 Vanilla accessions from 23 species including native Florida species. Principal component analysis of the genetic distances, population structure, and the maternally inherited rbcL gene identified putative hybrids, misidentified accessions, significant diversity within V. planifolia, and evidence for 12 clusters that separate accessions by species. These results validate the efficiency of genomics-based tools to characterize and identify genetic diversity in Vanilla and provide a significant tool for genomics-assisted plant breeding.

The effect of wet granulation on the erosion behaviour of an HPMC-lactose tablet, used as a rate-controlling component in a pulsatile drug delivery capsule formulation.

The purpose of this study was to investigate the variability in the performance of a pulsatile capsule delivery system induced by wet granulation of an erodible HPMC tablet, used to seal the contents within an insoluble capsule body. Erodible tablets containing HPMC and lactose were prepared by direct compression (DC) and wet granulation (WG) techniques and used to seal the model drug propranolol inside an insoluble capsule body. Dissolution testing of capsules was performed. Physical characterisation of the tablets and powder blends used to form the tablets was undertaken using a range of experimental techniques. The wet granulations were also examined using the novel technique of microwave dielectric analysis (MDA). WG tablets eroded slower and produced longer lag-times than those prepared by DC, the greatest difference was observed with low concentrations of HPMC. No anomalous physical characteristics were detected with either the tablets or powder blends. MDA indicated water-dipole relaxation times of 2.9, 5.4 and 7.7x10(-8)ms for 15, 24 and 30% HPMC concentrations, respectively, confirming that less free water was available for chain disentanglement at high concentrations. In conclusion, at low HPMC concentrations water mobility is at its greatest during the granulation process, such formulations are therefore more sensitive to processing techniques. Microwave dielectric analysis can be used to predict the degree of polymer spreading in an aqueous system, by determination of the water-dipole relaxation time.

Methyl anthranilate and γ-decalactone inhibit strawberry pathogen growth and achene Germination.

Plant volatile compounds have been shown to affect microbial growth and seed germination. Here two fruity volatiles found in strawberry ( Fragaria × ananassa ), γ-decalactone ("peachlike" aroma) and methyl anthranilate ("grapelike" aroma), were tested for effects on relevant pathogens and seedling emergence. Significant growth reduction was observed for Botrytis cinerea , Colletotrichum gloeosporioides , Colletotrichum acutatum , Phomopsis obscurans , and Gnomonia fragariae at 1 mM γ-decalactone or methyl anthranilate, and 5 mM γ-decalactone or methyl anthranilate supplemented medium resulted in complete cessation of fungal growth. Phytophthora cactorum was especially sensitive to 1 mM γ-decalactone, showing complete growth inhibition. Bacteriostatic effects were observed in Xanthamonas cultures. Postharvest infestations on store-bought strawberries were inhibited with volatile treatment. The γ-decalactone volatile inhibited strawberry and Arabidopsis thaliana germination. These findings show that two compounds contributing to strawberry flavor may also contribute to shelf life and suggest that γ-decalactone may play an ecological role by preventing premature germination.