Genetic regulation of volatile production in two melon introgression line collections with contrasting ripening behavior.

The importance of melon aroma in determining fruit quality has been highlighted in recent years. The fruit volatile profile is influenced by the type of fruit ripening. Non-climacteric fruits contain predominantly aldehydes, while climacteric fruits mainly produce esters. Several genes have been described to participate in volatile organic compounds (VOCs) biosynthesis pathways, but knowledge in this area is still incomplete. In this work we analysed the volatile profile of two reciprocal Introgression Line (IL) collections generated from a cross between 'Piel de Sapo' (PS) and 'Védrantais' (VED) melons, differing in their aroma profile and ripening behaviour. SPME GC-MS was performed to identify genes responsible for VOCs formation. More than 1000 QTLs for many volatiles were detected taken together both populations. Introgressions on chromosomes 3, 5, 6, 7 and 8 modified ester-aldehyde balance and were correlated to ripening changes in both genetic backgrounds. Some previously identified QTLs for fruit ripening might be involved in these phenotypes, such as ETHQV8.1 on chromosome 8 and ETHQV6.3 on chromosome 6. PS alleles on chromosomes 2, 6, 10 and 11 were found to increase ester content when introgressed in VED melons. Terpenes showed to be affected by several genomic regions not related to ripening. In addition, several candidate genes have been hypothesized to be responsible for some of the QTLs detected. The analysis of volatile compounds in two reciprocal IL collections has increased our understanding of the relationship between ripening and aroma and offers valuable plant material to improve food quality in melon breeding programs.

The haplotype-resolved T2T reference genome highlights structural variation underlying agronomic traits of melon.

Melon (Cucumis melo L.) is an important vegetable crop that has an extensive history of cultivation. However, the genome of wild and semi-wild melon types that can be used for the analysis of agronomic traits is not yet available. Here we report a chromosome-level T2T genome assembly for 821 (C. melo ssp. agrestis var. acidulus), a semi-wild melon with two haplotypes of ~373 Mb and ~364 Mb, respectively. Comparative genome analysis discovered a significant number of structural variants (SVs) between melo (C. melo ssp. melo) and agrestis (C. melo ssp. agrestis) genomes, including a copy number variation located in the ToLCNDV resistance locus on chromosome 11. Genome-wide association studies detected a significant signal associated with climacteric ripening and identified one candidate gene CM_ac12g14720.1 (CmABA2), encoding a cytoplasmic short chain dehydrogenase/reductase, which controls the biosynthesis of abscisic acid. This study provides valuable genetic resources for future research on melon breeding.

Regulation of climacteric fruit ripening in melon: recent advances and future challenges.

Fruit ripening is a complex and highly regulated process where tomato and strawberry have been the model species classically used for studying climacteric and non-climacteric fleshy fruit ripening types, respectively. Melon has emerged as an alternative ripening model because climacteric and non-climacteric cultivars exist, which makes it possible to dissect the regulation of ripening using a genetic approach. Several quantitative trait loci that regulate climacteric fruit ripening have been identified to date, and their combination in both climacteric and non-climacteric genetic backgrounds resulted in lines with different ripening behaviors, demonstrating that the climacteric intensity can be genetically modulated. This review discusses our current knowledge of the physiological changes observed during melon climacteric fruit ripening such as ethylene production, fruit abscission, chlorophyll degradation, firmness, and aroma, as well as their complex genetic control. From pioneer experiments in which ethylene biosynthesis was silenced, to the recent genetic edition of ripening regulators, current data suggest that the climacteric response is determined by the interaction of several loci under quantitative inheritance. The exploitation of the rich genetic diversity of melon will enable the discovery of additional genes involved in the regulation of the climacteric response, ultimately leading to breeding aromatic melon fruits with extended shelf life.

Thorough Characterization of ETHQB3.5, a QTL Involved in Melon Fruit Climacteric Behavior and Aroma Volatile Composition.

The effect of the QTL involved in climacteric ripening ETHQB3.5 on the fruit VOC composition was studied using a set of Near-Isogenic Lines (NILs) containing overlapping introgressions from the Korean accession PI 16375 on the chromosome 3 in the climacteric 'Piel de Sapo' (PS) genetic background. ETHQB3.5 was mapped in an interval of 1.24 Mb that contained a NAC transcription factor. NIL fruits also showed differences in VOC composition belonging to acetate esters, non-acetate esters, and sulfur-derived families. Cosegregation of VOC composition (23 out of 48 total QTLs were mapped) and climacteric ripening was observed, suggesting a pleiotropic effect of ETHQB3.5. On the other hand, other VOCs (mainly alkanes, aldehydes, and ketones) showed a pattern of variation independent of ETHQB3.5 effects, indicating the presence of other genes controlling non-climacteric ripening VOCs. Network correlation analysis and hierarchical clustering found groups of highly correlated compounds and confirmed the involvement of the climacteric differences in compound classes and VOC differences. The modification of melon VOCs may be achieved with or without interfering with its physiological behavior, but it is likely that high relative concentrations of some type of ethylene-dependent esters could be achieved in climacteric cultivars.

Fruit Morphology and Ripening-Related QTLs in a Newly Developed Introgression Line Collection of the Elite Varieties 'Védrantais' and 'Piel de Sapo'.

Melon is an economically important crop with widely diverse fruit morphology and ripening characteristics. Its diploid sequenced genome and multiple genomic tools make this species suitable to study the genetic architecture of fruit traits. With the development of this introgression line population of the elite varieties 'Piel de Sapo' and 'Védrantais', we present a powerful tool to study fruit morphology and ripening traits that can also facilitate characterization or pyramidation of QTLs in inodorous melon types. The population consists of 36 lines covering almost 98% of the melon genome, with an average of three introgressions per chromosome and segregating for multiple fruit traits: morphology, ripening and quality. High variability in fruit morphology was found within the population, with 24 QTLs affecting six different traits, confirming previously reported QTLs and two newly detected QTLs, FLQW5.1 and FWQW7.1. We detected 20 QTLs affecting fruit ripening traits, six of them reported for the first time, two affecting the timing of yellowing of the rind (EYELLQW1.1 and EYELLQW8.1) and four at the end of chromosome 8 affecting aroma, abscission and harvest date (EAROQW8.3, EALFQW8.3, ABSQW8.3 and HARQW8.3). We also confirmed the location of several QTLs, such as fruit-quality-related QTLs affecting rind and flesh appearance and flesh firmness.

Parvicella tangerina gen. nov., sp. nov. (Parvicellaceae fam. nov., Flavobacteriales), first cultured representative of the marine clade UBA10066, and Lysobacter luteus sp. nov., from activated sludge of a seawater-processing wastewater treatment plant.

Two strains isolated from a sample of activated sludge that was obtained from a seawater-based wastewater treatment plant on the southeastern Mediterranean coast of Spain have been characterized to achieve their taxonomic classification, since preliminary data suggested they could represent novel taxa. Given the uniqueness of this habitat, as this sort of plants are rare in the world and this one used seawater to process an influent containing intermediate products from amoxicillin synthesis, we also explored their ecology and the annotations of their genomic sequences. Analysis of their 16S rRNA gene sequences revealed that one of them, which was orange-pigmented, was distantly related to Vicingus serpentipes (family Vicingaceae) and to other representatives of neighbouring families in the order Flavobacteriales (class Flavobacteriia) by 88-89 % similarities; while the other strain, which was yellow-pigmented, was a putative new species of Lysobacter (family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria) with Lysobacter arseniciresistens as closest relative (97.3 % 16S rRNA sequence similarity to its type strain). Following a polyphasic taxonomic approach, including a genome-based phylogenetic analysis and a thorough phenotypic characterization, we propose the following novel taxa: Parvicella tangerina gen. nov., sp. nov. (whose type strain is AS29M-1T=CECT 30217T=LMG 32344T), Parvicellaceae fam. nov. (whose type genus is Parvicella), and Lysobacter luteus sp. nov. (whose type strain is AS29MT=CECT 30171T=LMG 32343T).

Modulating climacteric intensity in melon through QTL stacking.

Fruit ripening is one of the main processes affecting fruit quality and shelf life. In melon there are both climacteric and non-climacteric genotypes, making it a suitable species to study fruit ripening. In the current study, in order to fine tune ripening, we have pyramided three climacteric QTLs in the non-climacteric genotype "Piel de Sapo": ETHQB3.5, ETHQV6.3 and ETHQV8.1. The results showed that the three QTLs interact epistatically, affecting ethylene production and ripening-related traits such as aroma profile. Each individual QTL has a specific role in the ethylene production profile. ETHQB3.5 accelerates the ethylene peak, ETHQV6.3 advances the ethylene production and ETHQV8.1 enhances the effect of the other two QTLs. Regarding aroma, the three QTLs independently activated the production of esters changing the aroma profile of the fruits, with no significant effects in fruit firmness, soluble solid content and fruit size. Understanding the interaction and the effect of different ripening QTLs offers a powerful knowledge for candidate gene identification as well as for melon breeding programs, where fruit ripening is one of the main objectives.

Knock-Out of CmNAC-NOR Affects Melon Climacteric Fruit Ripening.

Fruit ripening is an important process that affects fruit quality. A QTL in melon, ETHQV6.3, involved in climacteric ripening regulation, has been found to be encoded by CmNAC-NOR, a homologue of the tomato NOR gene. To further investigate CmNAC-NOR function, we obtained two CRISPR/Cas9-mediated mutants (nor-3 and nor-1) in the climacteric Védrantais background. nor-3, containing a 3-bp deletion altering the NAC domain A, resulted in ~8 days delay in ripening without affecting fruit quality. In contrast, the 1-bp deletion in nor-1 resulted in a fully disrupted NAC domain, which completely blocked climacteric ripening. The nor-1 fruits did not produce ethylene, no abscission layer was formed and there was no external color change. Additionally, volatile components were dramatically altered, seeds were not well developed and flesh firmness was also altered. There was a delay in fruit ripening with the nor-1 allele in heterozygosis of ~20 days. Our results provide new information regarding the function of CmNAC-NOR in melon fruit ripening, suggesting that it is a potential target for modulating shelf life in commercial climacteric melon varieties.

Advances in bacterial concentration methods and their integration in portable detection platforms: A review.

Early detection and identification of microbial contaminants is crucial in many sectors, including clinical diagnostics, food quality control and environmental monitoring. Biosensors have recently gained attention among other bacterial detection technologies due to their simplicity, rapid response, selectivity, and integration/miniaturization potential in portable microfluidic platforms. However, biosensors are limited to the analysis of small sample volumes, and pre-concentration steps are necessary to reach the low sensitivity levels of few bacteria per mL required in the analysis of real clinical, industrial or environmental samples. Many platforms already exist where bacterial detection and separation/accumulation systems are integrated in a single platform, but they have not been compiled and critically analysed. This review reports on most recent advances in bacterial concentration/detection platforms with emphasis on the concentration strategy. Systems based on five concentration strategies, i.e. centrifugation, filtration, magnetic separation, electric separation or acoustophoresis, are here presented and compared in terms of processed sample volume, concentration efficiency, concentration time, ability to work with different types of samples, and integration potential, among others. The critical evaluation presented in the review is envision to facilitate the development of future platforms for fast, sensitive and in situ bacterial detection in real sample.

CRISPR/Cas9 gene editing uncovers the roles of CONSTITUTIVE TRIPLE RESPONSE 1 and REPRESSOR OF SILENCING 1 in melon fruit ripening and epigenetic regulation.

Melon (Cucumis melo) has emerged as an alternative model to tomato for studying fruit ripening due to the coexistence of climacteric and non-climacteric varieties. Previous characterization of a major quantitative trait locus (QTL), ETHQV8.1, that is able to trigger climacteric ripening in a non-climacteric background resulted in the identification of a negative regulator of ripening CTR1-like (MELO3C024518) and a putative DNA demethylase ROS1 (MELO3C024516) that is the orthologue of DML2, a DNA demethylase that regulates fruit ripening in tomato. To understand the role of these genes in climacteric ripening, in this study we generated homozygous CRISPR knockout mutants of CTR1-like and ROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two growing seasons with an earlier ethylene production profile being observed compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single-cytosine methylome analyses of the ROS1-knockout mutant revealed changes in DNA methylation in the promoter regions of the key ripening genes such as ACS1, ETR1, and ACO1, and in transcription factors associated with ripening including NAC-NOR, RIN, and CNR, suggesting the importance of ROS1-mediated DNA demethylation for triggering fruit ripening in melon.

A cryptic variation in a member of the Ovate Family Proteins is underlying the melon fruit shape QTL fsqs8.1.

KEY MESSAGE: The gene underlying the melon fruit shape QTL fsqs8.1 is a member of the Ovate Family Proteins. Variation in fruit morphology is caused by changes in gene expression likely due to a cryptic structural variation in this locus. Melon cultivars have a wide range of fruit morphologies. Quantitative trait loci (QTL) have been identified underlying such diversity. This research focuses on the fruit shape QTL fsqs8.1, previously detected in a cross between the accession PI 124112 (CALC, producing elongated fruit) and the cultivar 'Piel de Sapo' (PS, producing oval fruit). The CALC fsqs8.1 allele induced round fruit shape, being responsible for the transgressive segregation for this trait observed in that population. In fact, the introgression line CALC8-1, carrying the fsqs8.1 locus from CALC into the PS genetic background, produced perfect round fruit. Following a map-based cloning approach, we found that the gene underlying fsqs8.1 is a member of the Ovate Family Proteins (OFP), CmOFP13, likely a homologue of AtOFP1 and SlOFP20 from Arabidopsis thaliana and tomato, respectively. The induction of the round shape was due to the higher expression of the CALC allele at the early ovary development stage. The fsqs8.1 locus showed an important structural variation, being CmOFP13 surrounded by two deletions in the CALC genome. The deletions are present at very low frequency in melon germplasm. Deletions and single nucleotide polymorphisms in the fsqs8.1 locus could not be not associated with variation in fruit shape among different melon accessions, what indicates that other genetic factors should be involved to induce the CALC fsqs8.1 allele effects. Therefore, fsqs8.1 is an example of a cryptic variation that alters gene expression, likely due to structural variation, resulting in phenotypic changes in melon fruit morphology.

Bacteria Detection at a Single-Cell Level through a Cyanotype-Based Photochemical Reaction.

The detection of living organisms at very low concentrations is necessary for the early diagnosis of bacterial infections, but it is still challenging as there is a need for signal amplification. Cell culture, nucleic acid amplification, or nanostructure-based signal enhancement are the most common amplification methods, relying on long, tedious, complex, or expensive procedures. Here, we present a cyanotype-based photochemical amplification reaction enabling the detection of low bacterial concentrations up to a single-cell level. Photocatalysis is induced with visible light and requires bacterial metabolism of iron-based compounds to produce Prussian Blue. Bacterial activity is thus detected through the formation of an observable blue precipitate within 3 h of the reaction, which corresponds to the concentration of living organisms. The short time-to-result and simplicity of the reaction are expected to strongly impact the clinical diagnosis of infectious diseases.

The ß-galactosidase assay in perspective: Critical thoughts for biosensor development.

Recently, the ß-galactosidase assay has become a key component in the development of assays and biosensors for the detection of enterobacteria and E. coli in water quality monitoring. The assay has often performed below its maximum potential, mainly due to a poor choice of conditions. In this study we establish a set of optimal conditions and provide a rough estimate of how departure from optimal values reduces the output of the assay potentially decreasing its sensitivity. We have established that maximum response for detecting low cell concentrations requires an induction of the samples using IPTG at a concentration of 0.2 mM during 180 min. Permeabilization of the samples is mandatory as lack of it results in an almost 60% reduction in assay output. The choice of enzyme substrate is critical as different substrates yield products with different extinction coefficients or fluorescence yields. The concentration of substrate used must be high enough (around 3 to 4 times Km) to ensure that the activity measured is not substrate limited. Finally, as the color/fluorescence of the reaction products is highly dependent on pH, care must be taken to ensure that pH at the time of reading is high enough to provide maximum signal.

A novel introgression line collection to unravel the genetics of climacteric ripening and fruit quality in melon.

Introgression lines are valuable germplasm for scientists and breeders, since they ease genetic studies such as QTL interactions and positional cloning as well as the introduction of favorable alleles into elite varieties. We developed a novel introgression line collection in melon using two commercial European varieties with different ripening behavior, the climacteric cantalupensis 'Védrantais' as recurrent parent and the non-climacteric inodorus 'Piel de Sapo' as donor parent. The collection contains 34 introgression lines, covering 99% of the donor genome. The mean introgression size is 18.16 Mb and ~ 3 lines were obtained per chromosome, on average. The high segregation of these lines for multiple fruit quality traits allowed us to identify 27 QTLs that modified sugar content, altered fruit morphology or were involved in climacteric ripening. In addition, we confirmed the genomic location of five major genes previously described, which control mainly fruit appearance, such as mottled rind and external color. Most of the QTLs had been reported before in other populations sharing parental lines, while three QTLs (EAROQP11.3, ECDQP11.2 and FIRQP4.1) were newly detected in our work. These introgression lines would be useful to perform additional genetic studies, as fine mapping and gene pyramiding, especially for important complex traits such as fruit weight and climacteric ripening.

Genetic dissection of aroma biosynthesis in melon and its relationship with climacteric ripening.

Aroma is an essential trait in melon fruit quality, but its complexity and genetic basis are still poorly understood. The aim of this study was the identification of quantitative trait loci (QTLs) underlying volatile organic compounds (VOCs) biosynthesis in melon rind and flesh, using a Recombinant Inbred Line (RIL) population from the cross 'Piel de Sapo' (PS) × 'Védrantais' (VED), two commercial varieties segregating for ripening behavior. A total of 82 VOCs were detected by gas chromatography-mass spectrometry (GC-MS), and 166 QTLs were identified. The main QTL cluster was on chromosome 8, collocating with the previously described ripening-related QTL ETHQV8.1, with an important role in VOCs biosynthesis. QTL clusters involved in esters, lipid-derived volatiles and apocarotenoids were also identified, and candidate genes have been proposed for ethyl 3-(methylthio)propanoate and benzaldehyde biosynthesis. Our results provide genetic insights for deciphering fruit aroma in melon and offer new tools for flavor breeding.

QTLs and candidate genes analyses for fruit size under domestication and differentiation in melon (Cucumis melo L.) based on high resolution maps.

BACKGROUND: Melon is a very important horticultural crop produced worldwide with high phenotypic diversity. Fruit size is among the most important domestication and differentiation traits in melon. The molecular mechanisms of fruit size in melon are largely unknown. RESULTS: Two high-density genetic maps were constructed by whole-genome resequencing with two F2 segregating populations (WAP and MAP) derived from two crosses (cultivated agrestis × wild agrestis and cultivated melo × cultivated agrestis). We obtained 1,871,671 and 1,976,589 high quality SNPs that show differences between parents in WAP and MAP. A total of 5138 and 5839 recombination events generated 954 bins in WAP and 1027 bins in MAP with the average size of 321.3 Kb and 301.4 Kb respectively. All bins were mapped onto 12 linkage groups in WAP and MAP. The total lengths of two linkage maps were 904.4 cM (WAP) and 874.5 cM (MAP), covering 86.6% and 87.4% of the melon genome. Two loci for fruit size were identified on chromosome 11 in WAP and chromosome 5 in MAP, respectively. An auxin response factor and a YABBY transcription factor were inferred to be the candidate genes for both loci. CONCLUSION: The high-resolution genetic maps and QTLs analyses for fruit size described here will provide a better understanding the genetic basis of domestication and differentiation, and provide a valuable tool for map-based cloning and molecular marker assisted breeding.

Sonochemical coating of Prussian Blue for the production of smart bacterial-sensing hospital textiles.

In healthcare facilities, environmental microbes are responsible for numerous infections leading to patient's health complications and even death. The detection of the pathogens present on contaminated surfaces is crucial, although not always possible with current microbial detection technologies requiring sample collection and transfer to the laboratory. Based on a simple sonochemical coating process, smart hospital fabrics with the capacity to detect live bacteria by a simple change of colour are presented here. Prussian Blue nanoparticles (PB-NPs) are sonochemically coated on polyester-cotton textiles in a single-step requiring 15 min. The presence of PB-NPs confers the textile with an intensive blue colour and with bacterial-sensing capacity. Live bacteria in the textile metabolize PB-NPs and reduce them to colourless Prussian White (PW), enabling in situ detection of bacterial presence in less than 6 h with the bare eye (complete colour change requires 40 h). The smart textile is sensitive to both Gram-positive and Gram-negative bacteria, responsible for most nosocomial infections. The redox reaction is completely reversible and the textile recovers its initial blue colour by re-oxidation with environmental oxygen, enabling its re-use. Due to its simplicity and versatility, the current technology can be employed in different types of materials for control and prevention of microbial infections in hospitals, industries, schools and at home.

Integrated Photonic System for Early Warning of Cyanobacterial Blooms in Aquaponics.

Cyanobacterial blooms produce hazardous toxins, deplete oxygen, and secrete compounds that confer undesirable organoleptic properties to water. To prevent bloom appearance, the World Health Organization has established an alert level between 500 and 2000 cells·mL-1, beyond the capabilities of most optical sensors detecting the cyanobacteria fluorescent pigments. Flow cytometry, cell culturing, and microscopy may reach these detection limits, but they involve both bulky and expensive laboratory equipment or long and tedious protocols. Thus, no current technology allows fast, sensitive, and in situ detection of cyanobacteria. Here, we present a simple, user-friendly, low-cost, and portable photonic system for in situ detection of low cyanobacterial concentrations in water samples. The system integrates high-performance preconcentration elements and optical components for fluorescence measurement of specific cyanobacterial pigments, that is, phycocyanin. Phycocyanin has demonstrated to be more selective to cyanobacteria than other pigments, such as chlorophyll-a, and to present an excellent linear correlation with bacterial concentration from 102 to 104 cell·mL-1 (R2 = 0.99). Additionally, the high performance of the preconcentration system leads to detection limits below 435 cells·mL-1 after 10 min in aquaponic water samples. Due to its simplicity, compactness, and sensitivity, we envision the current technology as a powerful tool for early warning and detection of low pathogen concentrations in water samples.

Genetic dissection of climacteric fruit ripening in a melon population segregating for ripening behavior.

Melon is as an alternative model to understand fruit ripening due to the coexistence of climacteric and non-climacteric varieties within the same species, allowing the study of the processes that regulate this complex trait with genetic approaches. We phenotyped a population of recombinant inbred lines (RILs), obtained by crossing a climacteric (Védrantais, cantalupensis type) and a non-climcteric variety (Piel de Sapo T111, inodorus type), for traits related to climacteric maturation and ethylene production. Individuals in the RIL population exhibited various combinations of phenotypes that differed in the amount of ethylene produced, the early onset of ethylene production, and other phenotypes associated with ripening. We characterized a major QTL on chromosome 8, ETHQV8.1, which is sufficient to activate climacteric ripening, and other minor QTLs that may modulate the climacteric response. The ETHQV8.1 allele was validated by using two reciprocal introgression line populations generated by crossing Védrantais and Piel de Sapo and analyzing the ETHQV8.1 region in each of the genetic backgrounds. A Genome-wide association study (GWAS) using 211 accessions of the ssp. melo further identified two regions on chromosome 8 associated with the production of aromas, one of these regions overlapping with the 154.1 kb interval containing ETHQV8.1. The ETHQV8.1 region contains several candidate genes that may be related to fruit ripening. This work sheds light into the regulation mechanisms of a complex trait such as fruit ripening.