Fine Mapping of Lycopene Content and Flesh Color Related Gene and Development of Molecular Marker-Assisted Selection for Flesh Color in Watermelon (Citrullus lanatus).

Lycopene content and flesh color are important traits determined by a network of carotenoid metabolic pathways in watermelon. Based on our previous study of genetic inheritance and initial mapping using F2 populations of LSW-177 (red flesh) × cream of Saskatchewan (pale yellow flesh), red flesh color was controlled by one recessive gene regulating red and pale yellow pigmentation, and a candidate region related to lycopene content was detected spanning a 392,077-bp region on chromosome 4. To obtain a more precise result for further study, three genetic populations and a natural panel of 81 watermelon accessions with different flesh colors were used in this research. Herein, we narrowed the preliminary mapping region to 41,233 bp with the linkage map generated from F2 populations of LSW-177 (red flesh) × cream of Saskatchewan (pale yellow flesh) with 1,202 individuals. Two candidate genes, Cla005011 and Cla005012, were found in the fine mapping region; therein Cla005011 was a key locus annotated as a lycopene ß-cyclase gene. Phylogenetic tree analysis showed that Cla005011 was the closest relative gene in gourd. LSW-177 × PI 186490 (white flesh) and another BC1 population derived from garden female (red flesh) × PI 186490 were generated to verify the accuracy of the red flesh candidate gene region. By analyzing the expression levels of candidate genes in different developmental stages of different color watermelon varieties, Cla005011 for the expression differences was not the main reason for the flesh color variation between COS and LSW-177. This indicated that the LCYB gene might regulate fruit color changes at the protein level. A new marker-assisted selection system to identify red and yellow flesh colors in watermelon was developed with flesh color-specific CAPS markers and tested in 81 watermelon accessions.

Comparative transcriptome analysis of two contrasting watermelon genotypes during fruit development and ripening.

BACKGROUND: Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is an economically important crop with an attractive ripe fruit that has colorful flesh. Fruit ripening is a complex, genetically programmed process. RESULTS: In this study, a comparative transcriptome analysis was performed to identify the regulators and pathways that are involved in the fruit ripening of pale-yellow-flesh cultivated watermelon (COS) and red-flesh cultivated watermelon (LSW177). We first identified 797 novel genes to extend the available reference gene set. Second, 3958 genes in COS and 3503 genes in LSW177 showed at least two-fold variation in expression, and a large number of these differentially expressed genes (DEGs) during fruit ripening were related to carotenoid biosynthesis, plant hormone pathways, and sugar and cell wall metabolism. Third, we noted a correlation between ripening-associated transcripts and metabolites and the key function of these metabolic pathways during fruit ripening. CONCLUSION: The results revealed several ripening-associated actions and provide novel insights into the molecular mechanisms underlying the regulation of watermelon fruit ripening.

Development of cleaved amplified polymorphic sequence markers and a CAPS-based genetic linkage map in watermelon (Citrullus lanatus [Thunb.] Matsum. and Nakai) constructed using whole-genome re-sequencing data.

Cleaved amplified polymorphic sequence (CAPS) markers are useful tools for detecting single nucleotide polymorphisms (SNPs). This study detected and converted SNP sites into CAPS markers based on high-throughput re-sequencing data in watermelon, for linkage map construction and quantitative trait locus (QTL) analysis. Two inbred lines, Cream of Saskatchewan (COS) and LSW-177 had been re-sequenced and analyzed by Perl self-compiled script for CAPS marker development. 88.7% and 78.5% of the assembled sequences of the two parental materials could map to the reference watermelon genome, respectively. Comparative assembled genome data analysis provided 225,693 and 19,268 SNPs and indels between the two materials. 532 pairs of CAPS markers were designed with 16 restriction enzymes, among which 271 pairs of primers gave distinct bands of the expected length and polymorphic bands, via PCR and enzyme digestion, with a polymorphic rate of 50.94%. Using the new CAPS markers, an initial CAPS-based genetic linkage map was constructed with the F2 population, spanning 1836.51 cM with 11 linkage groups and 301 markers. 12 QTLs were detected related to fruit flesh color, length, width, shape index, and brix content. These newly CAPS markers will be a valuable resource for breeding programs and genetic studies of watermelon.

Stability of fruit quality traits in diverse watermelon cultivars tested in multiple environments.

Lycopene is a naturally occurring red carotenoid compound that is found in watermelon. Lycopene has antioxidant properties. Lycopene content, sugar content and hollowheart resistance are subject to significant genotype×environment interaction (G×E), which makes breeding for these fruit quality traits difficult. The objectives of this study were to (i) evaluate the influence of years and locations on lycopene content, sugar content and hollowheart resistance for a set of watermelon genotypes, and (ii) identify genotypes with high stability for lycopene, sugar, and hollowheart resistance. A diverse set of 40 genotypes was tested over 3 years and 8 locations across the southern United States in replicated, multi-harvest trials. Lycopene was tested in a subset of 10 genotypes. Data were analyzed using univariate and multivariate stability statistics (BLUP-GGE biplot) using SASGxE and RGxE programs. There were strong effects of environment as well as G×E interaction on watermelon quality traits. On the basis of stability measures, genotypes were classified as stable or unstable for each quality trait. 'Crimson Sweet' is an inbred line with high quality trait performance as well as trait stability. 'Stone Mountain', 'Tom Watson', 'Crimson Sweet' and 'Minilee' were among the best genotypes for lycopene content, sugar content and hollowheart resistance. We developed a stability chart based on marketable yield and average ranking generated from different stability measures for yield attributes and quality traits. The chart will assist in choosing parents for improvement of watermelon cultivars. See http://cuke.hort.ncsu.edu/cucurbit/wmelon/wmelonmain.html.

Synergism of human amnion-derived multipotent progenitor (AMP) cells and a collagen scaffold in promoting brain wound recovery: pre-clinical studies in an experimental model of penetrating ballistic-like brain injury.

One of the histopathological consequences of a penetrating ballistic brain injury is the formation of a permanent cavity. In a previous study using the penetrating ballistic-like brain injury (PBBI) model, engrafted human amnion-derived multipotent progenitor (AMP) cells failed to survive when injected directly in the injury tract, suggesting that the cell survival requires a supportive matrix. In this study, we seated AMP cells in a collagen-based scaffold, injected into the injury core, and investigated cell survival and neuroprotection following PBBI. AMP cells suspended in AMP cell conditioned medium (ACCS) or in a liquefied collagen matrix were injected immediately after a PBBI along the penetrating injury tract. Injured control rats received only liquefied collagen matrix. All animals were allowed to survive two weeks. Consistent with our previous results, AMP cells suspended in ACCS failed to survive; likewise, no collagen was identified at the injury site when injected alone. In contrast, both AMP cells and the collagen were preserved in the injury cavity when injected together. In addition, AMP cells/collagen treatment preserved some apparent brain tissue in the injury cavity, and there was measurable infiltration of endogenous neural progenitor cells and astrocytes into the preserved brain tissue. AMP cells were also found to have migrated into the subventricular zone and the corpus callosum. Moreover, the AMP cell/collagen treatment significantly attenuated the PBBI-induced axonal degeneration in the corpus callosum and ipsilateral thalamus and improved motor impairment on rotarod performance. Overall, collagen-based scaffold provided a supportive matrix for AMP cell survival, migration, and neuroprotection.

A comparison of two cognitive test paradigms in a penetrating brain injury model.

A rat model of penetrating ballistic-like brain injury (PBBI) was recently established to study military-relevant severe traumatic brain injury (TBI). The purpose of this study was to conduct a side-by-side evaluation of two well-established cognitive testing paradigms: the novel object recognition (NOR) task and the Morris water maze (MWM) task. Accordingly, male Sprague-Dawley rats were subjected to PBBI and their cognitive abilities were assessed at 7 and 21 days post-PBBI. Although PBBI animals had more difficulty completing both tasks compared to sham animals, their performance on the NOR task was confounded by a high degree of within-group variability that was likely due to attention deficits produced by the injury. In contrast, PBBI produced consistent, significant spatial learning deficits in the MWM task. Overall, these results suggest that the MWM task provides a more appropriate cognitive test for the PBBI model that would be useful for testing promising neuroprotective therapeutics.

Gene expression in developing watermelon fruit.

BACKGROUND: Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. RESULTS: High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar phenotype, i.e. seeded, bright red flesh, dark green rind, etc., determined that ethylene levels were highest during the green fruit stage followed by a decrease during the white and pink fruit stages. Additionally, quantitative Real-Time PCR was used to validate modulation of 127 ESTs that were differentially expressed in developing and ripening fruits based on array analysis. CONCLUSION: This study identified numerous ESTs with putative involvement in the watermelon fruit developmental and ripening process, in particular the involvement of the vascular system and ethylene. The production of ethylene during fruit development in watermelon gives further support to the role of ethylene in fruit development in non-climacteric fruits.

FasL, Fas, and death-inducing signaling complex (DISC) proteins are recruited to membrane rafts after spinal cord injury.

The Fas/CD95 receptor-ligand system plays an essential role in apoptosis that contributes to secondary damage after spinal cord injury (SCI), but the mechanism regulating the efficiency of FasL/Fas signaling in the central nervous system (CNS) is unknown. Here, FasL/Fas signaling complexes in membrane rafts were investigated in the spinal cord of adult female Fischer rats subjected to moderate cervical SCI and sham operation controls. In sham-operated animals, a portion of FasL, but not Fas was present in membrane rafts. SCI resulted in FasL and Fas translocation into membrane raft microdomains where Fas associates with the adaptor proteins Fas-associated death domain (FADD), caspase-8, cellular FLIP long form (cFLIPL ), and caspase-3, forming a death-inducing signaling complex (DISC). Moreover, SCI induced expression of Fas in clusters around the nucleus in both neurons and astrocytes. The formation of the DISC signaling platform leads to rapid activation of initiator caspase-8 and effector caspase-3, and the modification of signaling intermediates such as FADD and cFLIP(L) . Thus, FasL/Fas-mediated signaling after SCI is similar to Fas expressing Type I cell apoptosis.

Consumer acceptability of low-sugar watermelon sweetened with non-calorie sweetener by a Native American community.

Watermelons are a good source of lycopene, a carotenoid that exhibits antioxidant activity and may protect against some cancers. However, intake of watermelon may be restricted for individuals who have diabetes or those who limit carbohydrate intake. A low-sugar watermelon was developed at Lane, Oklahoma using traditional plant breeding techniques. The objective of this study was to determine whether the artificially sweetened low-sugar watermelon was acceptable with Native Americans, a group with a high incidence of diabetes. The red flesh from a low-sugar watermelon and a commercial variety of watermelon was removed and cut into cubes. Low and high levels of artificial sweetener were added to the low-sugar watermelon. Students at a Native American school (Grades 1-12) and adults at a Native American Feeding Center were asked to rate how much they liked or disliked the watermelon using a seven-point hedonic scale. Sugar composition, pH, lycopene and other carotenoids were analyzed from samples using established methods. The pH, lycopene, beta-carotene and total carotenoid levels were similar among fruit. Artificially sweetened fruit were rated slightly more acceptable in taste than the commercial control watermelons by both age groups. The low-sugar watermelons were lower in sugar composition but were comparable with conventional melons in all other quality factors and were found acceptable in taste by a broad age group of Native American consumers.

Inflammatory and apoptotic signaling after spinal cord injury.

Central nervous system (CNS) destruction in spinal cord injury (SCI) is caused by a complex series of cellular and molecular events. Recent studies have concentrated on signaling by receptors in the tumor necrosis factor receptor (TNFR) superfamily that mediate diverse biological outcomes ranging from inflammation to apoptosis. From the perspective of basic science research, understanding how receptor signaling mediates these divergent responses is critical in clarifying events underlying irreversible cell injury in clinically relevant models of SCI. From a clinical perspective, this work also provides novel targets for the development of therapeutic agents that have the potential to protect the spinal cord from irreversible damage and promote functional recovery. In this review, we discuss how the formation of alternate signaling complexes and receptor membrane localization after SCI can influence life and death decisions of cells stimulated through two members of the TNFR superfamily, Fas/CD95 and TNFR1.

Carotenoid content of 50 watermelon cultivars.

The lycopene content of 50 commercial cultivars of seeded and seedless red-fleshed watermelons was determined. Scanning colorimetric and spectrophotometric assays of total lycopene were used to separate watermelon cultivars into low (<50 mg/kg fw), average (50-70 mg/kg fw), high (70-90 mg/kg fw), and very high (>90 mg/kg fw). Cultivars varied greatly in lycopene content, ranging from 33 to 100 mg/kg. Most of the seeded hybrid cultivars had average lycopene contents. Sixteen of the 33 seedless types had lycopene contents in the high and very high ranges. All-trans-lycopene was the predominant carotenoid (84-97%) in all watermelon cultivars measured by high-performance liquid chromatography, but the germplasm differed in the relative amounts of cis-lycopene, beta-carotene, and phytofluene. Red-fleshed watermelon genotypes vary extensively in carotenoid content and offer opportunities for developing watermelons with specifically enhanced carotenoids.

The purification, physical/chemical characterization, and cDNA sequence of cantaloupe fruit polygalacturonase-inhibiting protein.

ABSTRACT Polygalacturonase-inhibiting proteins (PGIPs) are believed to aid in plant defense against fungal pathogens by inhibiting polygalacturonases (PGs) secreted by the invading organism. In an effort to better understand this type of plant-pathogen interaction in cucurbits, we have isolated a cantaloupe PGIP (CmPGIP) from 5 to 15 day postanthesis cantaloupe fruit. CmPGIP inhibited crude extracts of PG from two of four fungal pathogens of cantaloupe that were tested. Results from assays for PG activity that utilized rate of substrate viscosity reduction or rate of reducing group formation were consistent with CmPGIP inhibition of endo-PG activity. The M(r) of CmPGIP by sedimentation equilibrium or MALDITOF MS was 38,500. The pI of CmPGIP was approximately 8.2, and its absorptivity at 280 nm was 0.93 ml/mg. The circular dichroism spectrum of native CmPGIP exhibited strong negative ellipticity in the near UV and possessed a far UV spectrum indicative of beta-sheet periodic structure. Amino acid sequences of the N terminus and a cyanogen bromide peptide were used to construct oligonucleotide primers for polymerase chain reaction sequencing. The sequenced open reading frame predicts a mature protein of 307 amino acids with up to 68% identity to other PGIP molecules. Northern blot analysis revealed differential expression during fruit development. The isolation and structural information obtained for CmPGIP by this investigation provide a foundation for the development of molecular strategies for pre- and postharvest crop protection.

The effects of frozen storage conditions on lycopene stability in watermelon tissue.

The purpose of this investigation was to evaluate the rate of deterioration of lycopene in watermelon tissue during frozen storage, because little is known about the stability of watermelon tissue lycopene under cold storage conditions. Heart tissue from each of nine individual watermelons was stored at -20 or -80 degrees C as either small chunks or puree and periodically sampled over a year's time. Initial freeze-thaw experiments indicated that a small percentage of lycopene, approximately 4-6%, degraded during an initial freeze-thaw. Analyses of the samples showed a loss of approximately 30-40% lycopene over a year's storage at -20 degrees C and a loss of approximately 5-10% over the same period at -80 degrees C. Lycopene was slightly more stable in pureed compared with diced watermelon tissue at -20 degrees C, but not at -80 degrees C. The kinetic data were best fitted by application of two simultaneous, first-order decay processes. HPLC analysis of the samples after a year's storage suggested that beta-carotene was more stable during storage at -20 degrees C than was lycopene.